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Current and Future Innovations in Stem Cell Technologies – Labmate Online

By daniellenierenberg

Stem Cells 101

Every cell type in the body that makes up organs and tissues arose from a more primitive cell type called a stem cell. Stem cells are the foundation of living organisms, with the unique ability to self-renew and differentiate into specialised cell types. There are three different types of stem cell, classified by the number of specialised cell types they can produce: i) pluripotent stem cells (e.g. embryonic stem cells) can generate any specialised cell type; ii) multipotent stem cells (e.g. mesenchymal stem cells) are able to generate multiple, but not all, specialised cell types; and, iii) unipotent stem cells (e.g. epidermal stem cells that produce skin) give rise to only one cell type. It was long believed that stem cell differentiation into specialised cell types only occurs in one direction. There have been many exciting advances in stem cell biology, most notable the discovery of induced pluripotent stem cells (iPSCs) that demonstrated a mature differentiated specialised cell can be reverted to a primitive pluripotent stem cell (Takahashi K, 2006). This discovery transformed our understanding of stem cell biology enabling exciting and substantial advances in stem cell tools, technologies and applications. This article focuses on pluripotent stem cells, as they offer the most promising future applications.

To harness the power of stem cells, they must first be maintained in vitro tissue culture. Culture expansion of stem cells is tricky because they must be maintained in an undifferentiated state and not permitted to differentiate into other cell types until desired. In short, if stem cells are not dividing in log phase growth, they are differentiating. Historically, pluripotent stem cells were notoriously difficult to work with in the lab largely because of the of inherent variability of reagents derived from animal tissues.

An important concept affecting current and future innovations in stem cell technologies is Good Manufacturing Practice (GMP). This is governed by formal regulations administered by drug regulatory agencies (for example the FDA) that control the manufacture processes of medicines. The use of stem cells as therapeutic agents has necessitated specialised drug regulations known as Advanced Therapeutic Medicinal Products (ATMPs). Unlike chemically synthesised medicines where the final product can be defined through chemical analysis, ATMPs are complex biological living entities whereby the entire manufacturing process defines the final product. In simple terms, every reagent that touches the stem cells in the manufacturing process throughout the entire lifetime of the stem cell becomes a component of the final product. As such, in the real world the quality and consistency of the reagents used in a stem cell manufacturing process is paramount for downstream clinical applications, even if the project is still in the R&D or preclinical phase. Once reserved for clinical applications, GMP has become a dominating concept that affects all aspects of stem cell research and applications. Researchers and clinical developers benefit alike from GMP-focused innovations in stem cell technologies that deliver consistent growth properties and high-quality results.

Significant advances that overcome the challenges of the past have been made in all aspects of in vitro stem cell culture. These include advances in tissue culture medium, extracellular matrix, 3D synthetic cell culture plastic, growth factors, dissociation enzymes, cryopreservation agents and differentiation technologies. The workflow to culture stem cells in vitro is not a linear process but rather a continuous circle that can be broken down into 6 steps: 1) Extracellular Matrix coating of tissue culture plasticware; 2) Revival/seeding of tissue culture flasks; 3) Expansion of the cell culture in an incubator; 4) Culture medium change; 5) Subculture or passaging one flask to many; and 6) Cryopreservation of the stem cell culture. The stem cell workflow is shown in Figure 1.

The art of culturing stem cells is a lot easier today than in the past. Stem cells grow as adherent cultures on the surface of tissue culture flasks or dishes (image shown in Figure 1, Step 3). For the stem cells to adhere to the surface it must be coated with extracellular matrix. In the early days, it was an effort to maintain stem cells in culture because the cultures needed to be grown on a feeder layer of fibroblast cells. The requirement for a second cell culture combined with the stem cell culture is laborious to set up and severely limited experiments and applications (due to the contaminating fibroblasts mixed with the stem cells). Extracellular matrix isolated from mouse tumours removed the need for feeder layer cultures but can be variable in consistency and contain contaminants. Today, researchers benefit from recombinantly expressed extracellular matrix containing laminin-511 fragments that provides highly efficient adherence of a broad range of cell types and is easy to use (with only 1 hour coating time required that saves time and cost). Exceptional pluripotent stem cell adherence is achieved with laminin-511 fragments. The recombinant extracellular matrix laminin-511 is expressed in mammalian cell culture (e.g. CHO cells) or insect culture (e.g. silkworm) that eliminates the need for animal derived products in the extracellular matrix. Alternatively, synthetic 3D plastic scaffolds (e.g. Alvetex) are also available that offer a rigid defined matrix that is non-biological.

Early stem cell culture media required the medium to be replenished daily. This means 7 days a week in the lab tending to the stem cell cultures. Optimisation of tissue culture medium composition enables cultures to be maintained over the weekend without a medium change, enabling feeder-free, weekend-free stem cell culture. This may sound insignificant but does have a huge impact on the lifestyle of researchers working with stem cells. Unlike early tissue culture media, the composition of the culture media are fully defined and contain no animal derived products. Removal of animal-derived products offers important advantages by removing variability inherent in animal-derived products and guaranteeing consistent cell growth. Furthermore, animal-free formulations eleminate the risk of infection arising from the animal product (e.g. TSE risk). Growth factors are a critical component of the culture medium to maintain the stem cells in an undifferentiated state. Products available on the market contain growth factors that are expressed and isolated from barley.

Stem cells undergo cellular division in the culture vessel. As they expand, they will eventually outgrow their home and must be subcultured to separate flasks to provide space for further growth. Common practice is to use a digestive enzyme to free the stem cells from the culture surface. Trypsin isolated from bovine is commonplace in the tissue culture laboratory. Advances in the products available today use trypsin expressed in maize that is stable at room temperature in solution. Collagenase is an alternative dissociation reagent that is gentle and efficient on a wide range of cells and is available both animal-free and GMP grade - again enabling robust consistent culture conditions, and removing the dependence on animal derived products that are inherently variable.

The stem cells harvested from cultures can be frozen and stored (or cryopreserved) safely for several decades. When required, the cryopreserved stem cells may be defrosted, revived and expanded in culture providing a renewable source of stem cells. During cryopreservation of stem cells, it is critical to prevent cell death and changes in genotype/phenotype. Todays cryopreservation media can maintain consistent high cell viability after thawing; maintaining cell pluripotency, normal karyotype and proliferation even after long term cell storage. Traditionally, the cryopreservation process involved a rate-controlled freezer or a specialised container to freeze the cells at -1C/min. Advances in cryopreservation agents have removed the need for rate-controlled freezing. The process is now simple - you just place the stem cell suspension into a -80C freezer. Moreover, cryopreservation agents are available in GMP grade and with no animal-derived ingredients.

The power of stem cells lies in their ability both to self-renew and to differentiate into specialised cell types. The process of differentiation removes the stem cells from the workflow towards applications. Directed differentiation of stem cells into specific cell types enables the number of applications to grow. A typical differentiation protocol uses stepwise changes in culture medium, cytokines, growth factors and extracellular matrix over several weeks to direct the stem cells into a particular lineage and fate. Today, innovative technologies use genetic reprogramming factors that rapidly (< 1 week) differentiate stem cells into mature cell phenotypes. This advance significantly reduces time to experiment and increases manufacturing capacity for differentiated cell types.

Table 1. Advances in Stem Cell Technologies.Description Area of Innovation Examples of Innovative ProductsExtracellular Matrix Recombinant Laminin Expressed in CHO and Silkworm iMatrix-511Culture Medium No medium change required over the weekend, GMP grade, animal free StemFit MediumGrowth Factors Recombinant, GMP grade, animal free StemFit PuroteinDissociation Reagents Trypsin enzyme recombinantly expressed in maize. Collagenase & Neutral Protease expressed in Clostridium histolyticum TrypLECollagenase NBNeutral Protease NBCryopreservation Rate-controlled freezing not required. GMP grade, animal free and available for clinical use. Suitable for all cell types. STEM-CELLBANKERDifferentiation Rapid directed differentiation through genetic reprogramming Quick-Skeletal MuscleQuick-EndotheliumQuick-Neuron

There are unlimited applications that arise from a renewable source of mature cell types. One exciting area of innovation using differentiated stem cells is in disease modelling. Studying a disease state in an organ or tissue has in the past been limited to using in vivo animal models; whereas, differentiated stem cells opened the opportunity to create disease states in specific cell types in vitro. In addition, current technologies enable organoids or mini organs to be generated in the laboratory. Disease specific induced pluripotent stem cells can also be used to create disease models in vitro that are valuable tools for the study of disease and drug development without the need for in vivo animal models. In theory, any tissue is possible to create in vitro. In an exciting example of stem cell disease modelling, Dr Takayama from the CiRA in Kyoto, Japan has successfully modelled the life cycle of SARS-CoV-2 in both organoids and undifferentiated pluripotent stem cells (Takayama, 2020) (Sano, 2021) (Figure 2). In another example, the Skeletal Muscle Differentiation Kit was used to produce skeletal muscle myotubes from stem cells to create an in vitro disease model (Figure 3). In a direct application, pluripotent stem cell models of skeletal muscle have also been successfully used to develop a novel treatment for Duchenne muscular dystrophy (Moretti, 2020).

Promising progress is being made to create meat in the laboratory or what is commonly called cultured meat. Environmental concerns are driving the need for more sustainable meat production over traditional farming methods. Stem cell research in itself is reducing the need for the use of animals across multiple aspects as highlighted here. Producing cultured meat is straightforward in principle but faces many challenges in practice, for example maintaining the correct environment and stimuli for cultured cells to produce meat with the correct consistency and characteristics of the animal derived product. Stem cell cultures are expanded at scale in bioreactors and differentiated into skeletal muscle cells. These can be structured, using an edible scaffold for example, or used unstructured as the raw material to produce meat products (Figure 4). Tools and technologies are readily available to achieve this goal: expansion and differentiation of stem cells is highly efficient. However, a key consideration is the cost of goods. Current technologies are too costly but these are pioneering times and research is moving at an exciting pace.

The promise and potential of stem technologies to advance biology, medicine and food production can only be fulfilled if stem cell culture conditions are consistent, and accessible to research scientists and commercial operations alike. Exciting advances across multiple aspects of the stem cell workflow have streamlined processes to deliver products that are fully defined and animal-free. Furthermore, clinical translation of stem cell therapies and drug discovery are accelerated by the availability of GMP compliant reagents. The foundations are set for a bright future of discoveries and applications emerging from stem cell technologies.

Dr William Hadlington-Booth is the business unit manager for stem cell technologies and the extracellular matrix at AMSBIO. Erik Miljan, PhD, is a pioneer in the development of cellular therapies for a range of degenerative and disease conditions. He holds a PhD in biochemistry from Hong Kong University. For further information please contact:William@amsbio.com

Moretti, A. F., et al. (2020). Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nature Medicine, 26, 207214.Takahashi K., et al. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. . Cell, 126, 663-676.Takayama, K. (2020). In Vitro and Animal Models for SARS-CoV-2 research. Trends in Pharmacological Sciences, 41. 513-517.Sano, E., et al. (2021). Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells. Iscience, 24(5), 102428.

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Current and Future Innovations in Stem Cell Technologies - Labmate Online

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‘Ghost heart’: Built from the scaffolding of a pig and the patient’s cells, this cardiac breakthrough may soon be ready for transplant into humans -…

By daniellenierenberg

"It actually changed my life," said Taylor, who directed regenerative medicine research at Texas Heart Institute in Houston until 2020. "I said to myself, 'Oh my gosh, that's life.' I wanted to figure out the how and why, and re-create that to save lives."

That goal has become reality. On Wednesday at the Life Itself conference, a health and wellness event presented in partnership with CNN, Taylor showed the audience the scaffolding of a pig's heart infused with human stem cells -- creating a viable, beating human heart the body will not reject. Why? Because it's made from that person's own tissues.

"Now we can truly imagine building a personalized human heart, taking heart transplants from an emergency procedure where you're so sick, to a planned procedure," Taylor told the audience.

"That reduces your risk by eliminating the need for (antirejection) drugs, by using your own cells to build that heart it reduces the cost ... and you aren't in the hospital as often so it improves your quality of life," she said.

Debuting on stage with her was BAB, a robot Taylor painstakingly taught to inject stem cells into the chambers of ghost hearts inside a sterile environment. As the audience at Life Itself watched BAB functioning in a sterile environment, Taylor showed videos of the pearly white mass called a "ghost heart" begin to pinken.

"It's the first shot at truly curing the number one killer of men, women and children worldwide -- heart disease. And then I want to make it available to everyone," said Taylor to audience applause.

"She never gave up," said Michael Golway, lead inventor of BAB and president and CEO of Advanced Solutions, which designs and creates platforms for building human tissues.

"At any point, Dr. Taylor could have easily said 'I'm done, this just isn't going to work. But she persisted for years, fighting setbacks to find the right type of cells in the right quantities and right conditions to enable those cells to be happy and grow."

Giving birth to a heart

"We were putting cells into damaged or scarred regions of the heart and hoping that would overcome the existing damage," she told CNN. "I started thinking: What if we could get rid of that bad environment and rebuild the house?"

Soon, she graduated to using pig's hearts, due to their anatomical similarity to human hearts.

"We took a pig's heart, and we washed out all the cells with a gentle baby shampoo," she said. "What was left was an extracellular matrix, a transparent framework we called the 'ghost heart.'

"Then we infused blood vessel cells and let them grow on the matrix for a couple of weeks," Taylor said. "That built a way to feed the cells we were going to add because we'd reestablished the blood vessels to the heart."

The next step was to begin injecting the immature stem cells into the different regions of the scaffold, "and then we had to teach the cells how to grow up."

"We must electrically stimulate them, like a pacemaker, but very gently at first, until they get stronger and stronger. First, cells in one spot will twitch, then cells in another spot twitch, but they aren't together," Taylor said. "Over time they start connecting to each other in the matrix and by about a month, they start beating together as a heart. And let me tell you, it's a 'wow' moment!"

But that's not the end of the "mothering" Taylor and her team had to do. Now she must nurture the emerging heart by giving it a blood pressure and teaching it to pump.

"We fill the heart chambers with artificial blood and let the heart cells squeeze against it. But we must help them with electrical pumps, or they will die," she explained.

The cells are also fed oxygen from artificial lungs. In the early days all of these steps had to be monitored and coordinated by hand 24 hours a day, 7 days a week, Taylor said.

"The heart has to eat every day, and until we built the pieces that made it possible to electronically monitor the hearts someone had to do it person -- and it didn't matter if it was Christmas or New Year's Day or your birthday," she said. "It's taken extraordinary groups of people who have worked with me over the years to make this happen."

But once Taylor and her team saw the results of their parenting, any sacrifices they made became insignificant, "because then the beauty happens, the magic," she said.

"We've injected the same type of cells everywhere in the heart, so they all started off alike," Taylor said. "But now when we look in the left ventricle, we find left ventricle heart cells. If we look in the atrium, they look like atrial heart cells, and if we look in the right ventricle, they are right ventricle heart cells," she said.

"So over time they've developed based on where they find themselves and grown up to work together and become a heart. Nature is amazing, isn't she?"

Billions and billions of stem cells

As her creation came to life, Taylor began to dream about a day when her prototypical hearts could be mass produced for the thousands of people on transplant lists, many of whom die while waiting. But how do you scale a heart?

"I realized that for every gram of heart tissue we built, we needed a billion heart cells," Taylor said. "That meant for an adult-sized human heart we would need up to 400 billion individual cells. Now, most labs work with a million or so cells, and heart cells don't divide, which left us with the dilemma: Where will these cells come from?"

"Now for the first time we could take blood, bone marrow or skin from a person and grow cells from that individual that could turn into heart cells," Taylor said. "But the scale was still huge: We needed tens of billions of cells. It took us another 10 years to develop the techniques to do that."

The solution? A bee-like honeycomb of fiber, with thousands of microscopic holes where the cells could attach and be nourished.

"The fiber soaks up the nutrients just like a coffee filter, the cells have access to food all around them and that lets them grow in much larger numbers. We can go from about 50 million cells to a billion cells in a week," Taylor said. "But we need 40 billion or 50 billion or 100 billion, so part of our science over the last few years has been scaling up the number of cells we can grow."

Another issue: Each heart needed a pristine environment free of contaminants for each step of the process. Every time an intervention had to be done, she and her team ran the risk of opening the heart up to infection -- and death.

"Do you know how long it takes to inject 350 billion cells by hand?" Taylor asked the Life Itself audience. "What if you touch something? You just contaminated the whole heart."

Once her lab suffered an electrical malfunction and all of the hearts died. Taylor and her team were nearly inconsolable.

"When something happens to one of these hearts, it's devastating to all of us," Taylor said. "And this is going to sound weird coming from a scientist, but I had to learn to bolster my own heart emotionally, mentally, spiritually and physically to get through this process."

Enter BAB, short for BioAssemblyBot, and an "uber-sterile" cradle created by Advance Solutions that could hold the heart and transport it between each step of the process while preserving a germ-free environment. Taylor has now taught BAB the specific process of injecting the cells she has painstakingly developed over the last decade.

"When Dr. Taylor is injecting cells, it has taken her years to figure out where to inject, how much pressure to put on the syringe, and the best speed and pace to add the cells," said BAB's creator Golway.

"A robot can do that quickly and precisely. And as we know, no two hearts are the same, so BAB can use ultrasound to see inside the vascular pathway of that specific heart, where Dr. Taylor is working blind, so to speak," Golway added. "It's exhilarating to watch -- there are times where the hair on the back of my neck literally stands up."

Taylor left academia in 2020 and is currently working with private investors to bring her creation to the masses. If transplants into humans in upcoming clinical trials are successful, Taylor's personalized hybrid hearts could be used to save thousands of lives around the world.

In the US alone, some 3,500 people were on the heart transplant waiting list in 2021.

"That's not counting the people who never make it on the list, due to their age or heath," Taylor said. "If you're a small woman, if you're an underrepresented minority, if you're a child, the chances of getting an organ that matches your body are low.

If you do get a heart, many people get sick or otherwise lose their new heart within a decade. We can reduce cost, we can increase access, and we can decrease side effects. It's a win-win-win."

Taylor can even envision a day when people bank their own stem cells at a young age, taking them out of storage when needed to grow a heart -- and one day even a lung, liver or kidney.

"Say they have heart disease in their family," she said. "We can plan ahead: Grow their cells to the numbers we need and freeze them, then when they are diagnosed with heart failure pull a scaffold off the shelf and build the heart within two months.

"I'm just humbled and privileged to do this work, and proud of where we are," she added. "The technology is ready. I hope everyone is going to be along with us for the ride because this is game-changing."

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'Ghost heart': Built from the scaffolding of a pig and the patient's cells, this cardiac breakthrough may soon be ready for transplant into humans -...

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Stem Cells: A Case for the Use of Human Embryos in Scientific Research

By daniellenierenberg

ABSTRACT

Embryonic stem cells have immense medical potential. While both their acquisition for and use in research are fraught with controversy, arguments against their usage are rebutted by showing that embryonic stem cells are not equivalent to human lives. It is then argued that not using human embryos is unethical. Finally, an alternative to embryonic stem cells is presented.

Embryonic stem cells have the potential to cure nearly every disease and condition known to humanity. Stem cells are natures Transformers. They are small cells that can regenerate indefinitely, waiting to transform into a specialized cell type such as a brain cell, heart cell or blood cell [1]. Most stem cells form during the earliest stages of human development, immediately when an embryo is formed. These cells, known as embryonic stem cells (ESCs), eventually develop into every single type of cell in the body. As the embryo develops, adult stem cells (ASCs) replace these all-powerful embryonic stem cells. ASCs can only become a number of different cells within their potency. This limited application means an adult mesenchymal stem cell cannot become a neural cell.

By harnessing the unique ability of embryonic stem cells to transform into functional cells, scientists can develop treatments for a number of diseases and injuries, according to the California Institute for Regenerative Medicine, a private organization which awards grants for stem cell research [1]. For example, scientists at the Cleveland Clinic converted ESCs into heart muscle cells and injected them into patients who suffered from heart attacks. The cells continued to grow and helped the patients hearts recover [2].

With this enormous potential to cure devastating diseases, including heart failure, spinal cord injuries and Alzheimers disease, governments and research organizations have the moral imperative to support and encourage embryonic stem cell research. President Barack Obama signed an executive order in 2009 loosening federal funding restrictions on stem cell research, saying, We will aim for America to lead the world in the discoveries it one day may yield. [3]. The National Institute of Health and seven state governments, including California, Maryland and New York, followed Obamas lead by creating programs that offered over $5 billion in funding and other incentives to scientists and research institutions for stem cell research [4].

Scientists believe that harnessing the capability of embryonic stem cells will unlock the cure for countless diseases. I am very excited about embryonic stem cells, said Dr. Dieter Egli, professor of developmental cell biology at Columbia University. They will lead to unprecedented discoveries that will transform life. I have no doubt about it. [5]. The results thus far are inspiring. In 2016, Kris Boesen, a 21-year-old college student from Bakersfield, California, suffered a severe spinal cord injury in a car accident that left him paralyzed from the neck down. In a clinical trial conducted by Dr. Charles Liu at the University of Southern California Keck School of Medicine, Boesen was injected with 10 million embryonic stem cells that transformed into nerve cells [6]. Three months after the treatment, Boesen regained the use of his arms and hands. He could brush his teeth, operate a motorized wheelchair, and live more independently. All Ive wanted from the beginning was a fighting chance, he said. The power of stem cells made his wish possible [6].

Embryonic stem cell treatments may also cure type 1 diabetes. Type 1 diabetes, which affects 42 million worldwide, is an autoimmune disorder that results in the destruction of insulin-producing beta cells found in the pancreas [7]. ViaCyte, a company in San Diego, California, is developing an implant that contains replacement beta cells originating from embryonic stem cells [7]. The implant will preserve or replace the original beta cells to protect them from the patients immune system [7]. The company believes that if successful, this strategy will effectively cure type 1 diabetes. Patients with the disease will no longer have to closely monitor their blood sugar levels and inject insulin [7]. ViaCyte projects that an experimental version of this implant will become available by 2020 [7].

Ultimately, scientists believe they will grow complex organs using stem cells within the next decade [8]. Over 115,000 people in the United States need a life-saving organ donation, and an average of 20 people die every day due to the lack of available organs for transplant, according to the American Transplant Foundation [9]. Three-dimensional printing of entire organs derived from stem cells holds the most promise for solving the organ shortage crisis [8]. Researchers at the University of California, San Diego have successfully printed part of a functional liver [8]. While the printed liver is not ready for transplant, it still performs the functions of a normal liver. This has helped scientists reduce the need for often cruel and unethical animal testing. The scientists expose drugs to the printed liver and observe how it reacts. The livers response closely mimics that of a human beings and no living animals are harmed in the process [8].

Research using embryonic stems cells provides an unprecedented understanding of human development and the potential to cure devastating diseases. However, stem cell research has generated controversy among religious organizations such as the Catholic Church as well as the pro-life movement [3]. That is because scientists harvest stem cells from embryos donated by fertility clinics. Opponents of embryonicstem cell research equate the destruction of an embryo to the murder of an innocent human being [10]. Pope Benedict XVI said that harvesting stem cells is not only devoid of the light of God but is also devoid of humanity [3]. However, this view does not reflect a reasonable understanding and interpretation of basic biology. Researchers typically harvest embryonic stem cells from an embryo five days after fertilization [1]. At this stage, the entire embryo consists of less than 250 cells, smaller than the tip of a pin. Of these cells, only 30 are embryonic stem cells, which cannot perform any human function [11]. For comparison, an adult has more than 72 trillion cells, each with a specialized function [3]. Therefore, this microscopic blob of cells in no way represents human life.

With no functional cells, there exist no characteristics of a human being. Fundamentalist Christians believe that the presence or absence of a heartbeat signifies the beginning and end of a human life [10]. However, at this stage there is no heart, not even a single heart cell [10]. Some contend that brain activity, or the ability to feel, defines a human being. Michael Gazzaniga, president of the Cognitive Neuroscience Institute at the University of California, Santa Barbara, explains in his book,The Ethical Brain,that the fertilized egg is a clump of cells with no brain. [12]. There is no brain nor nerve cells that could allow this cellular object to interact with its environment [12]. The only uniquely human feature of embryonic cells at this stage is that they contain human DNA. This means that a 5-day-old human embryo is effectively no different than the Petri dishes of human cells that have grown in laboratories for decades with no controversy or opposition. Therefore, if the cluster of cells in the earliest stage of a human embryo is considered a human life, a growing plate of skin cells must also be considered human life. Few would claim that a Petri dish of human cells is morally equivalent to a living human or any other animal. Why, then, would a microscopic collection of embryonic cells have the same moral status as an adult human?

The status of the human embryo comes from itspotentialto turn into a fully grown human being. However, the potential of this entity to become an individual does not logically mean that it has the same status as an individual who can think and feel. If this were true, virtually every cell grown in a laboratory would be subject to the same controversy. This is because scientists have developed technology to convert an ordinary cell such as a skin cell into an embryo [10]. Although this requires a laboratory with special conditions, the normal development of a human being also requires special conditions in the womb of the mother. Therefore, almost any cell could be considered a potential individual, so it is illogical to conclude that a cluster of embryonic cells deserves a higher moral status.

Hundreds of thousands of embryos are destroyed each year in a process known as in vitro fertilization (IVF), a popular procedure that helps couples have children [13]. Society has an ethical obligation to use these discarded embryos to make medical advancements rather than simply throw them in the trash for misguided ideological and religious reasons as opponents of embryonic stem cell research desire.

With IVF, a fertility clinician harvests sperm and egg cells from the parents and creates an embryo in a laboratory before implanting it in the womans womb. However, creating and implanting a single embryo is expensive and often leads to unsuccessful implantation. Instead, the clinician typically creates an average of seven embryos and selects the healthiest few to implant [13].

This leaves several unused embryos for every one implanted. The couple can pay a fee to preserve the unused embryos by freezing them or can donate them to another family. Otherwise, they are slated for destruction [14]. A 2011 study in the Journal of the American Society for Reproductive Medicine found that 19 percent of the unused embryos are discarded and only 3 percent are donated for scientific research [14]. Many of these embryos could never grow into a living person given the chance because they are not healthy enough to survive past early stages of development [14]. If a human embryo is already destined for destruction or has no chance of survival, scientists have the ethical imperative to use these embryos to research and develop medical treatments that could save lives. The modern version of the Hippocratic oath states, I will apply, for the benefit of the sick, all measures which are required [to heal] [10]. Republican Senator Orrin Hatch of Utah supports the pro-life movement, which recognizes early embryos as human individuals. However, even he favors using the leftover embryos for the greater good. The morality of the situation dictates that these embryos, which are routinely discarded, be used to improve and save lives. The tragedy would be in not using these embryos to save lives when the alternative is that they would be discarded. [3]

Although scientists have used embryonic stem cells (ESCs) for promising treatments, they are not ideal, and scientists hope to eliminate the need for them. Primarily, ESCs come from an embryo with different DNA than the patient who will receive the treatment, meaning they are not autologous. ESCs are not necessarily compatible with everyone and could cause the immune system to reject the treatment [11]. The most promising alternative to ESCs are known as induced pluripotent stem cells. In 2008, scientists discovered a way to reprogram human skin cells to embryonic stem cells [15]. Scientists easily obtained these cells from a patients skin, converted them into the desired cell type, then transplanted them into the diseased organ without risk of immune rejection [15]. This eliminates any ethical concerns because no embryos are harvested or destroyed in the process. However, induced stem cells have their own risks. Recent studies have shown that they can begin growing out of control and turn into cancer [3]. Several of the first clinical trials with induced stem cells, including one aimed at curing blindness by regenerating a patients retinal cells, were halted because potentially cancerous mutations were detected [3].

Scientists believe that induced stem cells created in a laboratory will one day completely replace embryonic stem cells harvested from human embryos. However, the only way to create perfect replicas of ESCs is to thoroughly understand their structure and function. Scientists still do not completely understand how ESCs work. Why does a stem cell sometimes become a nerve cell, sometimes become a heart cell and other times regenerate to produce another stem cell? How can we tell a stem cell what type of cell to become? To develop a viable alternative to ESCs, scientists must first answer these questions with experiments on ESCs from human embryos. Therefore, extensive embryonic stem cell research today will eliminate the need for embryonic stem cells in the future.

The Biomedical Engineering Society Code of Ethics calls upon engineers to use their knowledge, skills, and abilities to enhance the safety, health and welfare of the public. [16] Stem cell research epitomizes this. Stem cells hold the cure for numerous diseases ranging from spinal cord injuries to organ failure and have the potential to transform modern medicine. Therefore, the donation of human embryos to scientific research falls within most conventional ethical frameworks and should be allowed with minimal restriction.

Because of widespread ignorance about the science behind stem cells, ill-informed opposition has prevented scientists from receiving the funding and support they need to save millions of lives. For example, George W. Bushs religious opposition to stem cell research resulted in a 2001 law severely limiting government funding for such research [3]. Although most opponents of stem cell research compare the destruction of a human embryo to the death of a living human, the biology of these early embryos is no more human than a plate of skin cells in a laboratory. Additionally, all embryos sacrificed for scientific research would otherwise be discarded and provide no benefit to society. If society better understood the process and potential of embryonic stem cell research, more people would surely support it.

Within the next decade, stem cells will likely provide simple cures for diseases that are currently untreatable, such as Alzheimers disease and organ failure [1]. As long as scientists receive support for embryonic stem cell research, stem cell therapies will become commonplace in clinics and hospitals around the world. Ultimately, the fate of this new medical technology lies in the hands of the public, who must support propositions that will continue to allow and expand the impact of embryonic stem cell research.

By Jonathan Sussman, Viterbi School of Engineering, University of Southern California

At the time of writing this paper, Jonathan Sussman was a senior at the University of Southern California studying biomedical engineering with an emphasis in biochemistry. He was an undergraduate research assistant in the Graham Lab investigating proteomics of cancer cells and was planning to attend an MD/PhD program.

[1] Stem Cell Information,Stem Cell Basics, 2016. [Online]. Available at:https://stemcells.nih.gov/info/basics/3.htm%5BAccessed 11 Oct. 2018].

[2] Cleveland Clinic, Stem Cell Therapy for Heart Disease | Cleveland Clinic, 2017. [Online]. Available at:https://my.clevelandclinic.org/health/diseases/17508-stem-cell-therapy-for-heart-disease%5BAccessed 14 Oct. 2018].

[3] B. Lo and L. Parham, Ethical Issues in Stem Cell Research,Endocrine Reviews, 30(3), pp.204-213, 2009.

[4] G. Gugliotta,Why Many States Now Have Stem Cell Research Programs, 2015. [Online]. Available at:http://www.governing.com/topics/health-human-services/last-decades-culture-wars-drove-some-states-to-fund-stem-cell-research.html%5BAccessed 14 Oct. 2018].

[5] D. Cyranoski,How human embryonic stem cells sparked a revolution,Nature Journal, 2018. [Online]. Available at:https://www.nature.com/articles/d41586-018-03268-4%5BAccessed 11 Oct. 2018].

[6] K. McCormack,Young man with spinal cord injury regains use of hands and arms after stem cell therapy, The Stem Cellar, 2016. [Online]. Available at:https://blog.cirm.ca.gov/2016/09/07/young-man-with-spinal-cord-injury-regains-use-of-hands-and-arms-after-stem-cell-therapy/%5BAccessed 11 Oct. 2018].

[7] A. Coghlan,First implants derived from stem cells to cure type 1 diabetes,New Scientist, 2017. [Online]. Available at:https://www.newscientist.com/article/2142976-first-implants-derived-from-stem-cells-to-cure-type-1-diabetes/%5BAccessed 11 Oct. 2018].

[8] C. Scott,University of California San Diegos 3D Printed Liver Tissue May Be the Closest Weve Gotten to a Real Printed Liver,3DPrint.com | The Voice of 3D Printing / Additive Manufacturing, 2018. [Online]. Available at:https://3dprint.com/118932/uc-san-diego-3d-printed-liver/%5BAccessed 11 Oct. 2018].

[9] American Transplant Foundation,Facts and Myths about Transplant. [Online]. Available at:https://www.americantransplantfoundation.org/about-transplant/facts-and-myths/%5BAccessed 11 Oct. 2018].

[10] A. Siegel, Ethics of Stem Cell Research,Stanford Encyclopedia of Philosophy, 2013. [Online]. Available at:https://plato.stanford.edu/entries/stem-cells/%5BAccessed 11 Oct. 2018].

[11] I. Hyun,Stem Cells The Hastings Center,The Hastings Center, 2018. [Online]. Available at:https://www.thehastingscenter.org/briefingbook/stem-cells/%5BAccessed 11 Oct. 2018].

[12] M. Gazzaniga,The Ethical Brain,New York: Harper Perennial, 2006.

[13] M. Bilger,Shocking Report Shows 2.5 Million Human Beings Created for IVF Have Been Killed | LifeNews.com,LifeNews, 2016. [Online]. Available at:https://www.lifenews.com/2016/12/06/shocking-report-shows-2-5-million-human-beings-created-for-ivf-have-been-killed/%5BAccessed 11 Oct. 2018].

[14] Harvard Gazette, Stem cell lines created from discarded IVF embryos, 2008. [Online]. Available at:https://news.harvard.edu/gazette/story/2008/01/stem-cell-lines-created-from-discarded-ivf-embryos/%5BAccessed 11 Oct. 2018].

[15] K. Murray,Could we make babies from only skin cells?, CNN, 2017. [Online]. Available at:https://www.cnn.com/2017/02/09/health/embryo-skin-cell-ivg/index.html%5BAccessed 11 Oct. 2018].

[16] Biomedical Engineering Society,Biomedical Engineering Society Code of Ethics, 2004. [Online]. Available at:https://www.bmes.org/files/CodeEthics04.pdf%5BAccessed 11 Oct. 2018].

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New Technique Turns Back the Aging Clock by 30 Years – Gilmore Health News

By daniellenierenberg

Researchers from the Babraham Institute in Cambridge, United Kingdom have revealed a new method that can make it possible to reverse aging considerably.

This novel technique can time jump skin cells by around 30 years, according to the research team. The number of years is notably longer than what earlier reprogramming techniques had managed.

Read Also: A Study of Naked Mole-Rats Gives New Insights on the Aging Process

Findings from this study have the potential of transforming regenerative medicine, which aims to fix or replace old or worn-out cells. They could promote a more focused approach to fighting aging.

The research appeared in eLife, a peer-reviewed biomedical and life sciences journal.

Stem cells are at the core of regenerative medicine, which is also sometimes called stem cell therapy. They help in repairing or replacing injured, dysfunctional, or diseased cells or tissue. They can transform into any specialized cells.

Regenerative medicine researchers have also been exploring for years how to reserve the process that is, converting specialized cells to stem cells. They have developed ways to create what are called induced stem cells, key tools in regenerative biology.

Read Also: Anti-Aging Research: Researchers Identify the Regulators of Skin Aging

While helpful for many things, stem cells can also cause problems. They could, for instance, lead to cancers through wild cell multiplication. It is, therefore, valuable to be able to reprogram induced stem cells back to the specialized cells they are from.

However, scientists have found it difficult to re-differentiate stem cells back into specialized cells. The new method in the current study helps to overcome the existing challenge.

The technique, which derives from the work of Professor Shinya Yamanaka, does not totally get rid of cell identity. It stops halfway through the process of reprogramming. This, thus, enabled cells to become younger and regain their youthful function.

Yamanaka, who got the 2012 Nobel Prize in Physiology or Medicine, discovered in 2007 a method for turning normal cells into unspecialized stem cells. The process involves four specific molecules known as the Yamanaka factors and takes about 50 days to complete.

By contrast, this new technique referred to as maturation phase transient reprogramming exposes skin cells to those molecules for only 13 days. The cells temporarily lost their identity after that. However, the partly reprogrammed cells appeared to regain markers of skin cells when allowed to grow under usual conditions.

Read Also: Study: Rapamycin May Help You Fight Skin Sagging and Wrinkles

Researchers examined measures of cellular age to confirm the rejuvenation of the cells. They looked at both the epigenetic clock and the transcriptome. Those measures indicated that the reprogrammed cells were comparable to cells that were around 30 years younger.

However, it was not just about appearance. The cells also regained youthful function.

Rejuvenated fibroblasts (skin cells) produced more collagen proteins, which provide structure to tissues and help to heal wounds. The cells also moved into areas in need of repair faster, compared to older cells. This indicates they have the potential of being used to make cells that promote more rapid wound healing.

The scientists noted that the new technique produced an effect on other genes connected to age-related disorders and symptoms. For instance, the APBA2 gene (linked to Alzheimers disease) and the MAF gene (associated with cataracts) displayed changes in youthful transcription levels.

Read Also: HGH Benefits: A Comprehensive List of Research-Backed Benefits You Could Expect from Using Growth Hormone

Future research may, therefore, open up more curative possibilities, going by these findings.

Our results represent a big step forward in our understanding of cell reprogramming, said Dr. Diljeet Gill, study co-author and a postdoc in Professor Wolf Reiks lab. We have proved that cells can be rejuvenated without losing their function and that rejuvenation looks to restore some function to old cells. The fact that we also saw a reverse of ageing indicators in genes associated with diseases is particularly promising for the future of this work.

The research team next plans to try and figure out the mechanism that underlies the successful cell reprogramming. This, scientists hope, could make it possible to promote rejuvenation without needing to reprogram but relying only on underlying regulators.

Multi-omic rejuvenation of human cells by maturation phase transient reprogramming

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Is a Bioengineered Heart From Recipient Tissues the Answer to the Shortage of Donors in Heart Transplantation? – Cureus

By daniellenierenberg

According to reports, currently, 64.34 million people suffer from heart failure worldwide[1]. Furthermore, the number of patients with end-organ heart failure is rising, leading to an all-time high in the number of people waiting for an organ transplant[2]. Several strategies have been devised to increase this strained supply of heart for transplantation, including expanding donor criteria[3], use of advanced perfusion machines such as organ care systems (OCS) to improve viability[4], use of normothermic regional perfusion (NRP) in donor from cardiac death (DCD) hearts, and xenotransplantation. Recently, the focus has shifted to new procedures using regenerative cells, angiogenesis factors, biological matrices, biocompatible synthetic polymers, and online registry systems that utilize bioimplants. These advanced technologies are collectively referred to as tissue engineering[5-8]. Ultimately, the goal is to grow a heart de novo. In addition to the unlimited organ supply, the new organ would be antigenically identical to the recipient as the recipients cells would be used, eliminating the need for immunosuppressive agents.

Even though bioengineering a fully functioning heart is in its infancy, huge strides have been made in achieving this goal. Scientists have been able to bioengineer models of the heart, lungs, pancreas, liver, and kidney. An important strategy for supporting the recipients cells and creating an autologous tissue/organ is to create a mechanical, geometrical, and biological environment that closely mimics the native organs properties. The breakthrough in growing an artificial heart was the invention of the decellularization of extracellular matrix (ECM), which maintains the native vascular network[9]. Numerous tissues and organs have been engineered using decellularization, including livers [10], lungs[11], kidneys[12], corneas[13], bladders[14], vasculature[15], articular cartilage[16], intestines[17], and hearts[18]. There has been some success in engineering a heart in the lab. Although technological innovations and biological model systems have resulted in great progress, constructing such complicated tissue structures effortlessly remains a challenge. This review aims to outline the techniques involved in bioengineering a heart in the lab and the challenges involved in developing it into a viable organ for transplantation (Figure 1).

The human heart comprises various cells, each specialized to perform a specific task. A human heart contains roughly 2-3 billion cardiomyocytes, making up only about one-third of its total cells [19]. Additionally, other cells include endothelial cells, fibroblasts, and specialized conducting cells like Purkinje fibers. On top of that, structural scaffolds support the functions of cells arranged into structures, such as vessels, muscles, and nerves. These scaffolds mainly consist of polysaccharides and proteoglycans embedded in complex sugars and chemokines matrix, allowing the heart to coordinate its mechanical and electrical functions [20,21]. Sprawled around this is a collection of protein fibers such as collagen and elastin, which confers mechanical strength to the heart and allow for the constant loading and unloading forces[22,23]. Thus, it is necessary to construct a scaffold around which the specialized cells can grow and maintain vitality through blood perfusion to recreate a functioning heart in a laboratory [24] (Figure 2).

Extracellular matrix (ECM) and cells in an organ display a dynamic reciprocity, whereby the ECM constantly adapts to the demands of the cells[25], and selecting the appropriate scaffold is the key component for growing a viable organ in the lab. Researchers have also studied various synthetic scaffolds as potential surrogates for the ECM, but none can replicate its intricacy or structure compared to native ECM. It is possible to vascularize synthetic materials such as polylactic acid (PLLA) and polylactic glycolic acid (PLGA) and to produce them consistently[26,27]. The significant advantage of synthetic ECM is its production scalability as it does not require to be harvested from living tissue, but these do not match the native myocardiums tensile strength. Hydrogels have also been studied extensively and even accepted by the Food and Drug Administration for drug delivery and adjunct for cell therapy. Hydrogels consist of a cross-linked hydrophilic polymer matrix with over 30% water content [28]. However, they have poor cell retention [29] or poor tensile strength [30]; hence, they are not feasible as a primary scaffold for constructing an organ. Decellularizing the whole heart and leaving the ECM serves as a potential solution to this problem with the particular advantage of having a balanced composition of all the proteins present physiologically [31].

The Badylak laboratory developed the first technique for decellularizing tissue[32]. This process involved the removal of the cell, leaving only the ECM, which retained its composition, architecture, and mechanical properties. There are several methods for removing cells from the ECM. These methods include physical methods (e.g., freeze/thaw cycles), enzymatic degradation (e.g., trypsin), and removal by using chemicals (e.g., sodium dodecyl sulfate)[33]. Ott et al. noted that decellularization could be achieved with different detergent solutions. Comparative studies on decellularization methods have mixed results regarding the superiority of different techniques [34-37]. Based on the results, the sodium dodecyl sulfate (SDS) solution was found to be the best [18]. However, a few studies have suggested that SDS treatment causes degradation of the ECM with a reduction in elastin, collagen, and glycosaminoglycans (GAG) content [34]. The decellularization process utilizes 1% SDS perfused through the coronary circulation, followed by washing it with de-ionized water and subsequently 1% Triton-X-100 (Sigma). Finally, the organ remnant is washed with phosphate-buffered saline (PBS) wash buffer, antibiotic, and protease, leaving a decellularized ECM[38,39]. Using this technique, they decellularized the heart, reseeded it with neonatal cardiac cells, and grew the first beating rodent heart in the lab [18]. Decellularized tissue provides a dynamic environment for the orientation and coupling of cells and facilitates the exchange of nutrients and oxygen throughout the depth of the tissue. Moreover, this process efficiently removes both allogeneic and xenogeneic antigens, possibly preventing the need for immunosuppressants [33], which is especially important as one of the causes of heart failure in transplanted hearts is myocardial fibrosis from chronic rejection [40]. This process can be potentially avoided by using a decellularized heart to generate an ECM scaffold which can then be repopulated using the recipients cells.

Researchers have used animal heart ECM and human heart ECM scaffolds to provide this decellularized ECM scaffold. The porcine heart has often been deemed suitable for its similarity with the human heart [41]. As decellularization removes most of the cells, much of the antigen load is removed. However, the porcine heart ECM contains -1,3-galactose epitope (-gal), which can stimulate an immune response [42,43]. One way to circumvent this is to use pigs lacking -gal epitope, but this technique needs further research. Another possible problem with using a porcine heart is the possible risk of horizontal transmission of porcine viruses like the porcine endogenous retrovirus, cytomegalovirus, HSB, circovirus, etc. [44,45]. Although a few tests can detect the presence of these viruses, they have poor sensitivity, and hence further work has to be done [46].

A cadaveric heart that is unfit for transplant can also be used to harvest an ECM scaffold [47]. The only drawback to this is that it may not always be possible to achieve the desired level of tissue engineering fidelity with these matrices because they may be damaged or diseased. Moreover, there is an assumption that they are superior for the growth and differentiation of human cells, but there is no robust evaluation to support this assumption. The method for decellularization of the cadaveric human heart is similar to that of other animals, utilizing 1% SDS and 1% Triton X-100, with the only difference being a longer perfusion time for these chemicals [48,49].

These cells are highly specialized and terminally differentiated, and hence, they do not proliferate normally. Therefore, to repopulate a human-sized scaffold, autologous human cardioblasts must be isolated or expanded in large quantities. Hence, for the recellularization of ECM, a method of inducing progenitor cells had to be devised. Thus, the discovery of methods to reprogram or induce adult cells into pluripotent stem cells was a significant milestone in stem cell biology and tissue bioengineering[50-52].

Once we have the cells for repopulation of ECM, recellularization is required to achieve a functional organ product for implantation. For recellularization to be achieved, choosing appropriate cell sources, seeding cells optimally, and cultivating them using organ-specific cultures are needed [24]. Cells from fetuses and adults, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) have all been used[24]. Obtained with ease and ethically, stem cells from bone marrow stroma or adipose tissue (MSC) have shown promise as the ideal cells for recellularization [53]. In addition, human somatic cells can be reprogrammed to produce iPSCs, and they exhibit properties similar to ESCs [54].

A potential solution to the problem of getting a large number of human cells for tissue engineering or other regenerative medicine approaches is the ability to produce iPSCs from readily available autologous cells such as fibroblasts or blood cells[55,56]. The only drawback to using iPSCs is the possibility of teratoma formation due to its pluripotent nature [48,57]. However, the potential solution to this problem is to allow controlled differentiation toward a cardiac lineage before implantation into the ECM [58]. Although previously any attempts to produce iPSCs would result in karyotype instability [59], recent advances have been made with iPSCs maintaining chromosomal integrity [60]. These advances have ushered astep forward in the pursuit of creating viable organs in the lab.

Cell seeding techniques depend on the type of organ being engineered, and, for the heart, it usually involves seeding by perfusion through the vascular tree [24]. This step is called re-endothelization and is usually the first step to recellularization. A dynamic communication between endothelial cells and cardiomyocyte populations occurs via direct cell interactions and the secretion of various factors[61,62]. It is evident from multiple reports that seeding endothelial cell populations and cardiomyocyte populations simultaneously provides functional benefits that aid in maintaining the recellularization process [63]. Interestingly, endothelial cells have also demonstrated the ability to differentiate into cardiomyocytes in other cardiomyocyte cells [64], which may aid in more efficient recellularization. Moreover, besides the advantage, the recellularization of both the vascular tree and the heart parenchyma must be uniform to prevent two key issues in the heart, namely, thrombogenesis[65] and arrhythmogenesis[66].

Improved cell concentration and diffusion over the scaffold can be achieved by optimizing the mechanical environment, scaffold coating, and cell perfusion systems by using multiple perfusion routes simultaneously, which for the heart involves both direct intramyocardial injections and perfusion of the vascular tree [67]. However, the potential problem with intramyocardial injections is that even though the injection site shows dense cellularity, the cells are generally poorly distributed throughout the scaffold [58]. Moreover, sequential injections of cardiac cells will likely be required to rebuild the chamber parenchyma, which may compromise matrix integrity [48]. Nevertheless, given that cardiac cells include fibroblasts, in which ECM is produced and secreted, there is a possibility that endogenous matrix repair may occur after cell seeding to help resolve this issue [62].

While sourcing cells for recellularization using stem cells is a work in progress, multiple studies have explored ways to develop mature cardiomyocytes derived from iPSCs that are more physiologically similar to native cardiomyocytes [68,69]. One of the most recent cardiac constructs was engineered using PSC-derived cardiac cells in a ratio of equal cardiomyocyte and noncardiomyocyte cells, cultured in serum-free media [70]. Cardiomyocytes cultivated in this method were elongated, had organized sarcomeres and distinguished bands, and exhibited increased contractility [70]. It is encouraging to see these results that stem cells can be used to produce cardiomyocytes similar to native mature cells, reinforcing the notion that stem cells can be a cardiac cell source.

After enough cells have been seeded onto an organ scaffold, cell culture is required. A bioreactor is required for perfusion and provides a nutrient-rich environment that encourages organ-specific cell growth [24]. Bioreactors should allow nutrient-rich oxygen to be pumped with adjustable rates of flow and pressure and monitor and control the pH and temperature of the media. Moreover, mechanical stimulation is also an essential component for engineering organs of the musculoskeletal and cardiovascular systems [71]. A wide range of mechanical properties is employed in the design of bioreactors, including substrate stiffness and dynamic changes in stiffness throughout culture, pulsatile flow, and providing stretch to enhance cell maturation, alignment, and generation of force in engineered constructs [72]. Presently, there are several types of bioreactors available, with Radnoti [73] and BIOSTAT B-DCU II [74], to name a few. In addition, there has been an increase in bioreactor designs incorporating real-time monitoring to assess the status of engineered tissues. These designs may incorporate biochemical probes to assess transmural pressure changes or sampling ports to test cells viability and biochemical composition after recellularization [75,76]. The incorporation of sampling methods within bioreactor designs will keep constructs sterile, allowing for modifications in stimuli to be made while maintaining a closed system, and providing researchers with valuable feedback on cell responses throughout bioengineering. Further research is being conducted to make bioreactors that can be used to maintain the perfect milieu for growing these bioengineered tissues and organs.

For an organ to be viable for transplant, three things must be ensured: sterility of the process, structural integrity, and, lastly, patency for surgical anastomosis. Biological tissues are sterilized by gamma radiations or peracetic acid at low concentrations before the ECM is repopulated with cells[77]. Once the cells are added, antibacterial, antifungals, and other antibiotic drugs can be utilized. It is re-evaluated for integrity before the ECM is recellularized and only gets the green light for cell seeding if structural integrity is maintained. Interestingly, with the aid of endoscopy, decellularized constructs can be easily manipulated and visualized for macro and microstructure defects at the level of chambers, papillary muscle, and valves[47]. One of the most important aspects of evaluating the integrity of ECM is to check for intact coronary vasculature, which can be done by micro-optical coherence tomography [48].

Heart constructs engineered in the lab have been demonstrated to undergo cyclical muscular contraction but also have been shown to respond to drugs and exhibit electrical activity. However, electrocardiography analysis of the bioengineered hearts has shown irregular wave morphology due to loss of coupling between cardiomyocytes [78]. Therefore, it will be crucial to develop continuous monitoring of cardiac electrophysiology, function, and even vascular patency if these artificial constructs can be transplanted into patients.

Over the past decade, research in regenerative medicine has enabled us to understand better the challenges associated with developing a bioartificial heart. The first challenge was creating a biocompatible scaffold which has already been resolved with the development of various decellularization techniques, making it possible to generate an anatomically accurate and vascularized heart scaffold. With the advent of newer techniques for iPSC generation of stable karyotype, cell generation is also potentially resolved. Presently, research has to be aimed to address the challenges in reseeding the ECM scaffold. A potential solution might be the advancement in 3D-printed matrixes with embedded cells. However, decellularized ECM remains the gold standard for now as 3D-printed matrixes cannot replicate the complexity and structural integrity of the natural component of ECM.

Another potential problem is the creation of a bioreactor that can efficiently maintain the environment required for the growth of cardiac and other differentiated cells around the decellularized ECM scaffold. Constructing organs is no easy feat and involves much technical expertise. Hence, many resources are required in every step of artificially reproducing tissues and organs. Thus, even if bioengineering a heart is a possibility in the near future, it may not be financially feasible to use them for transplantation until the cost of making such constructs is lowered. Additionally, we do not know the long-term viability of such constructs. These constructs use chemicals to decellularize ECM as well as induce the conversion of adult cells into pluripotent cells. Some questions arise on how the complex network of cells and ECM would interact over the long run. The heart is a complex organ that requires a highly specialized conduction system to ensure efficient, coordinated, and purposeful contraction of the heart chambers. Any deviance may lead to fatal arrhythmia or thrombus formation. We are yet to reproduce a perfect conduction system in the lab, let alone test its long-term functionality. Furthermore, the use of induced pluripotent cells also raises the prospect of long-term tumorigenesis and malignancy. Despite rapid advances in bioengineering and artificial hearts, research and clinical trials must be conducted to determine the long-term feasibility of using these organs.

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Researchers find new function performed by almost half of brain cells – Medical News Today

By daniellenierenberg

Astrocytes make up almost half of the mammalian brain cells. They are called glial cells because scientists originally thought that these starlight-shaped structures serve as nerve glue.

Research suggests that these cells control the growth of axons, or the neuronal projections that carry electrical impulses.

However, scientists still considered astrocytes to be supporting actors behind neurons, which are the primary cells of the brain and nervous system.

Now, scientists at Tufts University in Massachusetts and other institutions realize that astrocytes may execute a significantly greater performance in brain activity.

Dr. Moritz Armbruster, a research assistant professor of neuroscience at Tufts, led a team of researchers in harnessing novel technology to study astrocyte-neuron exchanges.

To their surprise, the scientists observed electrical activity in astrocyte processes within mouse brain tissue. They reported: This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyteneuron interaction.

Dr. Armbruster and his fellow authors published their findings in Nature Neuroscience.

Using innovative tools, the Tufts team developed a technique to detect and observe electrical activity in brain cell interactions. These properties could not be seen before now.

Dr. Chris Dulla, corresponding author of the study, is an associate professor of neuroscience at the Tufts University School of Medicine and Graduate School of Biomedical Sciences. He explained that he and his colleagues []use viruses to express fluorescent proteins in the mouse brain, and thats what lets us measure this activity.

In an interview with Medical News Today, he elaborated:

[W]e had other experiments that made us think that this new type of activity must be happening in astrocytes. We just didnt have a way to show it[] So, we developed these new techniques to image the activity of the astrocytes and, using them, we showed that this thing that we thought must be happening actually was happening.

Neurotransmitters are chemical messengers that facilitate the transfer of electrical signals between neurons and support the blood-brain barrier. Scientists have long understood that astrocytes control these substances to support neuronal health.

This study breaks ground in showing that neurons release potassium ions, which change the astrocytes electrical activity. This modulation affects how the astrocytes control neurotransmitters.

Until now, scientists could not image potassium activity in the brain.

Neurons and astrocytes talk with each other in a way that has not been known about before, Dr. Dulla said.

Dr. Dulla maintains that human brain cells work the same way as mouse tissue. He said that mouse and human brain cells use the same proteins and molecules involved in brain activity.

Besides, using human tissue samples presents ethical challenges, Dr. Dulla noted: [We] have to be really careful and judicious [] with the experiments we design, and [we] dont get a chance to see [human tissue] samples like [we] can do with mice.

However, the professor shared that extensive databases give [scientists] a chance to just access human brain tissue without doing an experiment [themselves], but just getting the data that someone else has already done.

This wealth of information further demonstrates similarities between human and mouse cells and lets researchers deduce that the same processes are happening in each. The main difference is that human cells are larger and more abundant.

He also pointed out that the study highlights a bidirectional relationship between these brain cells, as astrocytes influence the neurons as well.

These findings about astrocyte-neuron interactions open a new world of questions regarding brain pathology, memory, and learning.

MNT also discussed this study with Dr. Santosh Kesari, who was not involved in this research. He is a neurologist at Providence Saint Johns Health Center in Santa Monica, CA, and regional medical director for the Research Clinical Institute of Providence Southern California.

Dr. Kesari said that this study confirms earlier research.

[T]his is one of many studies thats showing increasingly, how astrocytes and neurons interact, how they affect each other and then connecting the dots to how that affects brain function behavior, memory, seizures, dementia, and even in the context of brain tumors, all these cells interact. Dr. Santosh Kesari

Most medication development for brain disorders currently targets neurons. Dr. Kesari agreed that this study might shine light on a new path.

Maybe we should really be understanding the astrocyte side of things to develop drugs that may impact brain health by looking at that astrocytic role in brain disorders, he said.

The ability to image cell processes, as in this study, makes it possible to explore other activities within the brain as well.

The researchers are also screening existing drugs in hopes of manipulating astrocyte-neuron processes. Scientists could come close to repairing brain injuries or helping people increase their learning capacity if this proves successful.

They are also making their tools available to other labs to explore more areas of interest, such as breathing, headache, and many other neurological disorders.

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Researchers find new function performed by almost half of brain cells - Medical News Today

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Sugared proteins called proteoglycans start to give up their secrets – EurekAlert

By daniellenierenberg

image:A team at Scripps Research demonstrated how protein-sugar clusters called proteoglycans can guide processes like cell maturation and neuronal synapse formation, among other functions. As one example, pictured, semi-synthetic syndecan-1 proteoglycan rescues the maturation of mouse embryonic stem cells into neural precursor cells (red and green). view more

Credit: Meg Critcher, Scripps Research

LA JOLLA, CAScientists at Scripps Research have developed a set of methods for the closer study of one of the least-accessible, least-understood players in biology: protein-sugar conjugates called proteoglycans.

These molecules are often thickly present on the surfaces of cells and are known to have a broad set of functions in the body, though how they work and how their dysfunctions contribute to diseases are largely mysteries.

The scientists, who report their work in Nature Chemical Biology on May 12, 2022, devised synthetic proteoglycans that closely mimic real ones but have convenient chemical handles for modifying them. These and other aspects of their research platform enable the systematic study of how proteoglycans structure affects their functions in health and disease. The scientists demonstrated the effectiveness of their platform by using it to make new discoveries about proteoglycans roles in early cell development and in cancer cell spreading.

Were essentially unpacking the complexity of these molecules by constructing them in a modular way ourselves, and studying them in a tightly controlled environment, says study senior author Mia Huang, PhD, associate professor in the Department of Molecular Medicine at Scripps Research.

A proteoglycan starts as just a proteinthe so-called core proteinbut this protein contains special sites where any of a variety of sugar-related molecular chains called glycosaminoglycans (GAGs) can be linked. Within the cell where the protein originates, enzymes catalyze the attachment of GAGs to it, and this newborn proteoglycan normally is further decorated with clusters of sulfur and oxygen atoms called sulfates. The finished proteoglycan may be anchored into the cell membrane, its GAG chains waving in the extracellular fluid like seagrass, or it may be secreted from the cell to perform other functions.

With such complexity, it is no surprise that proteoglycans have versatile functionsthey are present in virtually all tissues, including cartilage, collagen, bone, skin, blood vessels, brain cells and mucosal surfaces. They help steer processes such as cell maturation, cell adhesion, cell migration, and neuronal synapse formation; serve as receptors for protein signaling partners; and are even used by some viruses and bacteria to latch onto cells. But proteoglycans complexity also means that how they do what they do, and with what partners, remains largely undiscovered. Scientists arent even certain how many proteoglycans there are in human and other mammalian cellsalthough there are at least dozens.

Huang and her team, including first authors Timothy OLeary, PhD and Meg Critcher, respectively a postdoctoral researcher and doctoral candidate in the Huang Lab during the study, constructed proteoglycan core proteins that are almost identical to known core proteins, but contain special molecular handles enabling the researchers to change the numbers and locations and types of GAG chains that bind to them. This allows the researchers to study systematically how the function of a proteoglycan changes as its GAG arrangement changes.

The researchers also developed techniques allowing them to anchor their proteoglycans in cell membranes or to let them float freely, to see how this affects proteoglycans functions in different circumstances.

Using their synthetic versions of common proteoglycans called syndecans, the scientists were able to study the respective contributions of GAG chains and core proteins. Specifically, they looked at two key biological processes mediated by syndecans: the maturing of stem cells, and the spreading of breast cancer cells on an extracellular matrix.

We learned from these experiments that not only the GAG chains but also the core proteins contribute to proteoglycan function, says Critcher. Notably, we also found that proteoglycans role in cancer cell spreading depends heavily on whether they are anchored to the cell membrane or free-floating.

The team also incorporated a method called proximity tagging to help them identify proteoglycans interaction partners. Huang and colleagues are now using this, and their modular construction technique, to study the interactions of syndecans and other proteoglycans in different contextsand with different GAG arrangementsand otherwise to explore their structures and functions.

Chemical editing of proteoglycan architecture was co-authored by Timothy OLeary, Meg Critcher, Tesia Stephenson, Xueyi Yang, Abdullah Hassan, Noah Bartfield, Richard Hawkins, and Mia Huang.

Funding for the research was provided by the National Institutes of Health (R00HD090292, R35GM142462).

About Scripps Research

Scripps Research is an independent, nonprofit biomedical institute ranked the most influential in the world for its impact on innovation by Nature Index. We are advancing human health through profound discoveries that address pressing medical concerns around the globe. Our drug discovery and development division, Calibr, works hand-in-hand with scientists across disciplines to bring new medicines to patients as quickly and efficiently as possible, while teams at Scripps Research Translational Institute harness genomics, digital medicine and cutting-edge informatics to understand individual health and render more effective healthcare. Scripps Research also trains the next generation of leading scientists at our Skaggs Graduate School, consistently named among the top 10 US programs for chemistry and biological sciences. Learn more atwww.scripps.edu.

Nature Chemical Biology

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Sugared proteins called proteoglycans start to give up their secrets - EurekAlert

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Other Notable Health Studies & Research From May 11, 2022 – Study Finds

By daniellenierenberg

Worlds 1st Focused Ultrasound Cancer Immunotherapy Center LaunchedUVA Health and the Charlottesville-based Focused Ultrasound Center today announced the launch of theFocused Ultrasound Cancer Immunotherapy Center, the worlds first center dedicated specifically to advancing a focused ultrasound and cancer immunotherapy treatment approach that could revolutionize 21st-century cancer care.

A Study by the Gwangju Institute of Science and Technology Investigates Mercury Contamination in Freshwater Lakes in KoreaDuring the 1950s and 1960s, Minamata Bay in Japan was the site of widespread mercury poisoning caused by the consumption of fish containing methylmercurya toxic form of mercury that is synthesized when bacteria react with mercury released in water.

Researchers identify possible new target to treat newborns suffering from lack of oxygen or blood flow in the brainThe condition, known as hypoxic-ischemic encephalopathy (HIE), can result in severe brain damage, which is why researchers at theCase Western Reserve University School of Medicineand UH Rainbow Babies & Childrens Hospital (UH Rainbow) are studying the condition to evaluate how HIE is treated and develop new, more effective options.

Should You Give Your Child Opioids for Post-Operative Pain Management?Routine head and neck procedures, such as removal of tonsils and adenoids and the placement of ear tubes, may cause moderate to severe pain in pediatric patients.

Two birds with one stone: a refined bioinformatic analysis can estimate gene copy-number variations from epigenetic dataA team led by Dr. Manel Esteller, Director of the Josep Carreras Leukaemia Research Institute, has improved the computational identification of potentially druggable gene amplifications in tumors, from epigenetic data.

Some Shunts Used After Epilepsy Surgery May Risk Chronic HeadachesSurgeons who observe persistent fluid buildup after disconnecting epileptic and healthy brain areas should think twice before installing low-pressure nonprogrammable drainage shunts, according to a study coauthored by Rutgers pediatric and epilepsy neurosurgeonYasunori Nagahamathat found chronic headaches could result from these procedures.

Re-defining the selection of surgical procedure in sufferers with tuberous sclerosis complicatedBy illustrating a number of instances of tuberous sclerosis in sufferers whove undergone surgical resection with seizure-free outcomes, researchers have recognized components that decide choice of sufferers for profitable surgical procedure.

Scientists study links between obesity, age and body chemistryA team of Clemson University scientists is making inroads in understanding the relationship between certain enzymes that are normally produced in the body and their role in regulating obesity and controlling liver diseases.

Clemson scientists discover new tools to fight potentially deadly protozoa that has pregnant women avoiding cat litter boxesNow, a group of researchers from Clemson University have discovered a promising therapy for those who suffer from toxoplasmosis, a disease caused by the microscopic protozoa Toxoplasma gondii.

Rising income inequality linked to Americans declining healthRising levels of income inequality in the United States may be one reason that the health of Americans has been declining in recent decades, new research suggests.

New research to understand how the brain handles optical illusions and makes predictionsNew research projects are underway at the Allen Institute to address these questions through OpenScope, the shared neuroscience observatory that allows scientists around the world to propose and direct experiments conducted on one of the Institutes high-throughput experimental platforms.

Robotic therapy: A new effective treatment for chronic stroke rehabilitationA study led by Dr. Takashi Takebayashi and published in the journal Stroke suggests continuing therapy for chronic stroke patients is still beneficial while suggesting a radical alternative.

Children with history of maltreatment could undergo an early maturation of the immune systemThe acute psychosocial stress states stimulate the secretion of an antibody type protein which is decisive in the first immune defence against infection, but only after puberty.

Toxoplasmosis: propagation of parasite in host cell stoppedA new method blocks the protein regulation of the parasite Toxoplasma gondii and causes it to die off inside the host cell.

Research shows the role empathy may play in musicCan people who understand the emotions of others better interpret emotions conveyed through music? A new study by an international team of researchers suggests the abilities are linked.

Effects of stress on adolescent brains triple networkA new studyinBiological Psychiatry: Cognitive Neuroscience and Neuroimaging, published by Elsevier, has used functional magnetic resonance imaging (fMRI) to examine the effects of acute stress and polyvicitimization, or repeated traumas, on three brain networks in adolescents.

Reform to Mental Health Act must prompt change in support for familiesFamily members of people with severe mental health challenges need greater support to navigate the UKs care system following changes announced in yesterdays Queens Speech, say the authors of a new study published in theBritish Journal of Social Work.

New knowledge about airborne virus particles could help hospitalsMeasurements taken by researchers at Lund University in Sweden of airborne virus in hospitals provide new knowledge about how best to adapt healthcare to reduce the risk of spread of infection.

Guidance developed for rare dancing eyes syndromeExperts from Evelina London Childrens Hospital developed the guidance in collaboration with a worldwide panel of experts and families of children with the condition.

Genetic study identifies migraine causes and promising therapeutic targetsQUT genetic researchers have found blood proteins that cause migraine and have a shared link with Alzheimers disease that could potentially be prevented by repurposing existing therapeutics.

How do genomes evolve between species? The key role of 3D structure in male germ cellsA study led by scientists at the UAB and University of Kent uncovers how the genome three-dimensional structure of male germ cells determines how genomes evolve over time.

Novel Supramolecular CRISPRCas9 Carrier Enables More Efficient Genome EditingRecently, a research team from Kumamoto University, Japan, have constructed a highly flexible CRISPR-Cas9 carrier using aminated polyrotaxane (PRX) that can not only bind with the unusual structure of Cas9 and carry it into cells, but can also protect it from intracellular degradation by endosomes.

Obesity, diabetes and high blood pressure increase mortality from COVID-19 especially among young and middle-aged peopleObesity, impaired blood glucose metabolism, and high blood pressure increase the risk of dying from COVID-19 in young and middle-aged people to a level mostly observed in people of advanced age.

Are most ORR electrocatalysts promising nanocatalytic medicines for tumor therapy?The current searches for medical catalysts mainly rely on trial-and-error protocols, due to the lack of theoretical guidance.

The combination makes the difference: New therapeutic approach against breast cancerResearchers at the University of Basel have now discovered an approach that involves a toxic combination with a second target gene in order to kill the abnormal cells.

Glatiramer acetate compatible with breastfeedingA study conducted by the neurology department of Ruhr-Universitt Bochum (RUB) at St. Josef Hospital on the drug glatiramer acetate can relieve mothers of this concern during the breastfeeding period.

A*STAR, NHCS, NUS And Novo Nordisk To Collaborate On Cardiovascular Disease ResearchThe Agency for Science, Technology and Researchs (A*STAR) Genome Institute of Singapore (GIS) and Bioinformatics Institute (BII), as well as the National Heart Centre Singapore (NHCS), National University of Singapore (NUS), and pharmaceutical company Novo Nordisk have signed an agreement to study the mechanisms underlying cardiovascular disease progressionespecially the condition called heart failure with preserved ejection fraction (HFpEF).

Taking ownership of your healthA study published this month inAge and Ageing by The Japan Collaborate Cohort (JACC) Study group at Osaka University assessed the impact of modifying lifestyle behaviors on life expectancy from middle age onwards.

Experimental evolution illustrates gene bypass process for mitosisResearchers from Nagoya University demonstrated gene bypass events for mitosis using evolutionary repair experiments.

Temporomandibular Disorder-Induced Pain Likely to Worsen in Late Menopause TransitionNew study evaluates the influence of menopause symptoms on the intensity of temporomandibular disorder-induced pain throughout the full menopause transition.

Breathtaking solution for a breathless problemA drop in oxygen levels, even when temporary, can be critical to brain cells. This explains why the brain is equipped with oxygen sensors. Researchers from Japan and the United States report finding a new oxygen sensor in the mouse brain.

How calming our spinal cords could provide relief from muscle spasmsAn Edith Cowan University (ECU) studyinvestigating motoneurons in the spine has revealed two methods can make our spinal cords less excitable and could potentially be usedto treat muscle spasms.

Analysis Finds Government Websites Downplay PFAS Health RisksState and federal public health agencies often understate the scientific evidence surrounding the toxicity of per- and polyfluoroalkyl substances (PFAS) in their public communications, according toan analysispublished today in the journalEnvironmental Health.

Multiple diagnoses are the norm with mental illness; new genetic study explains whyThe study, published this weekin the journalNature Genetics, found that while there is no gene or set of genes underlying risk for all of them, subsets of disordersincluding bipolar disorder and schizophrenia; anorexia nervosa and obsessive-compulsive disorder; and major depression and anxietydo share a common genetic architecture.

Drinkers sex plus brewing method may be key to coffees link to raised cholesterolThe sex of the drinker as well as the brewing method may be key to coffees link with raised cholesterol, a known risk factor for heart disease, suggests research published in the open access journalOpen Heart.

Artificial cell membrane channels composed of DNA can be opened and locked with a keyIn new research, Arizona State University professorHao Yan, along with ASU colleagues and international collaborators from University College London describe the design and construction of artificial membrane channels, engineered using short segments of DNA.

Single cell RNA sequencing uncovers new mechanisms of heart diseaseResearchers at the Hubrecht Institute have now successfully applied a new revolutionary technology (scRNA-seq) to uncover underlying disease mechanisms, including specifically those causing the swelling.

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Other Notable Health Studies & Research From May 11, 2022 - Study Finds

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Newsletter April 2022 – Progress in Cline’s cell lab and in the stem cell therapy field – Marketscreener.com

By daniellenierenberg

Spring has arrived in Gothenburg, and the Cline is excited to bring you some exciting news and updates from our team

The first stage of Ex-vivo testing completed

Early this month, Cline announced that the first stage of our ex-vivo experiments was carried out with encouraging performance. This newsletter will take a deeper look at what's happening in our labs and what these tests mean for StemCART.

These experiments, which began in January 2022, are an important milestone for the StemCART project and will push the project into the next development stage. In these tests, Cline has several aims; 1) demonstrate that the matrix developed by Cline successfully functions, 2) the successful differentiation of induced pluripotent stem cells (iPSCs) into functional chondrocytes (cartilage cells), and 3) to show induced healing of the injured cartilage tissue.

To achieve this, Cline has been collaborating with orthopedic surgeons and a hospital to collect cartilage tissue from patients undergoing surgery. Cline then takes this tissue from the hospital to our cell labs. At the lab we induce an artificial cartilage damage to mimic joint injuries before implanting the cells and matrix together at the injury site.

In this first stage of testing, the supporting matrix demonstrated the expected functionality in successfully fixing cells to the area of interest.

Read more about this in our latest press release or where Cline was recently featured on ORTHOWORLD.

Next steps for StemCART

The ex-vivo tests continues and Cline will carry out at least 24 further experiments in several stages. The results from these will be communicated after the completion of each stage. The upcoming stage of 10 experiments will test a higher cell concentration and focus on determining the functionality of the chondrocytes. Testing will also be expanded to include tissue of different cartilage origin, such as knee, shoulder, and hip.

StemCART's ultimate vision is as a cell-based Advanced Therapy Medical Product (ATMP) that will revolutionize the treatment of cartilage damage by providing patients with new functional cartilage and curing the condition, thus eliminating pain. StemCART provides several advantages over other therapy strategies such as autologous chondrocytes implantation and mesenchymal stem cells (MSCs) in that it provides reparative cartilage to the joint, and that an allogeneic cell source has much better scalability.

As part of the journey to this goal, Cline will continue preparing for in-human clinical trials, including scaling up production in a GMP facility together with partners, developing QA/QC methods, as well as the necessary safety testing and documentation for a clinical trial application. Cline has begun this work by evaluating different development and manufacturing options and engaging in regulatory pathway strategic planning activities.

Cline envisions out-licensing StemCART to a commercial partner following successful phase I trials. The process to identify and engage potential partners is ongoing, with the aim of generating interest in the commercialization of StemCART.

Exciting industry news and developments

2022 has already been an exciting year in the world of stem cell-based therapy and cartilage repair, showing the increasing interest and potential paradigm shift towards cell-based treatment. For example in the MSC segment, the Lund-based company Xintela recently began its first-in-human clinical trial for mesenchymal stem cells (MSC) in knee osteoarthritis (OA). Similarly, Cynata Therapeutics, working with iPSC-derived MSCs to treat knee OA, together with Fujifilm Cellular Dynamics, is currently conducting a large phase III trial. For more insights into the current landscape of cartilage repair treatments and current status of new cell-based treatments, you can read Cline Scientific's latest publication, "Insights into the present and future of cartilage regeneration and joint repair," available at https://www.mdpi.com/journal/ijms/special_issues/Cartilage_Repair.

Another leap forward for iPSC-derived tissue therapy is the conclusion of a world-first clinical trial, showing that implanting iPSC-derived corneal tissue into four nearly blind patients was safe and effective. The team from Osaka University used iPS cells to create the cornea tissue, which caused improvement of symptoms and eyesight and did not lead to any rejection or tumorigenicity.

Finally, in related orthopedic industry news, Bioventus acquired its partner CartiHeal for up to 450M USD. CartiHeal is an orthopedic device company that has developed the cartilage repair implant Agili-C, which was recently approved by the FDA. Agili-C is a cell-free scaffold implant for cartilage and osteochondral defects caused by either osteoarthritis or trauma.

We look forward to continuing to share Cline's journey in future newsletters!

Warmest regards,

The Cline Team

Click hereto subscribe to future newsletters and press releases.https://news.cision.com/cline/SubscriptionRegistrationDialog

Cline Scientific AB (publ) Telefon: 031-387 55 55Argongatan 2 C E-post: info@clinescientific.com431 53 MLNDAL Hemsida: http://www.clinescientific.com

About Cline ScientificCline Scientific develops advanced cancer diagnostics and regenerative medicine treatments. The company is working heavily with R&D through joint collaborations with pharmaceutical companies and academic researchers around the world. The focus is on projects in the cancer diagnostic and stem cell therapy fields since Clines nanotechnology here provides unmet solutions to critical challenges and functions. The unique patented surface nanotechnology is used in cell-based products and processes to drive projects within Life Science into and through the clinical phase.

https://news.cision.com/cline/r/newsletter-april-2022---progress-in-cline-s-cell-lab-and-in-the-stem-cell-therapy-field,c3555837

https://mb.cision.com/Main/12114/3555837/1571081.pdf

(c) 2022 Cision. All rights reserved., source Press Releases - English

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Newsletter April 2022 - Progress in Cline's cell lab and in the stem cell therapy field - Marketscreener.com

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Stem cell-based regenerative medicine – PMC

By daniellenierenberg

Stem Cell Investig. 2019; 6: 19.

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Contributions: (I) Conception and design: E Fathi, R Farahzadi; (II) Administrative support: E Fathi, R Farahzadi; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: R Farahzadi, N Rajabzadeh; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Received 2018 Nov 11; Accepted 2019 Mar 17.

Recent developments in the stem cell biology provided new hopes in treatment of diseases and disorders that yet cannot be treated. Stem cells have the potential to differentiate into various cell types in the body during age. These provide new cells for the body as it grows, and replace specialized cells that are damaged. Since mesenchymal stem cells (MSCs) can be easily harvested from the adipose tissue and can also be cultured and expanded in vitro they have become a good target for tissue regeneration. These cells have been widespread used for cell transplantation in animals and also for clinical trials in humans. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine as well as in regenerative medicine. Based on the studies in this field, MSCs found wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration etc.

Keywords: Mesenchymal stem cells (MSCs), animal model, cell-based therapy, regenerative medicine

Stem cells are one of the main cells of the human body that have ability to grow more than 200 types of body cells (1). Stem cells, as non-specialized cells, can be transformed into highly specialized cells in the body (2). In the other words, Stem cells are undifferentiated cells with self-renewal potential, differentiation into several types of cells and excessive proliferation (3). In the past, it was believed that stem cells can only differentiate into mature cells of the same organ. Today, there are many evidences to show that stem cells can differentiate into the other types of cell as well as ectoderm, mesoderm and endoderm. The numbers of stem cells are different in the tissues such as bone marrow, liver, heart, kidney, and etc. (3,4). Over the past 20 years, much attention has been paid to stem cell biology. Therefore, there was a profound increase in the understanding of its characteristics and the therapeutic potential for its application (5). Today, the utilization of these cells in experimental research and cell therapy represents in such disorders including hematological, skin regeneration and heart disease in both human and veterinary medicine (6).The history of stem cells dates back to the 1960s, when Friedenstein and colleagues isolated, cultured and differentiated to osteogenic cell lineage of bone marrow-derived cells from guinea pigs (7). This project created a new perspective on stem cell research. In the following, other researchers discovered that the bone marrow contains fibroblast-like cells with congenic potential in vitro, which were capable of forming colonies (CFU-F) (8). For over 60 years, transplantation of hematopoietic stem cells (HSCs) has been the major curative therapy for several genetic and hematological disorders (9). Almost in 1963, Till and McCulloch described a single progenitor cell type in the bone marrow which expand clonally and give rise to all lineages of hematopoietic cells. This research represented the first characterization of the HSCs (10). Also, the identification of mouse embryonic stem cells (ESCs) in 1981 revolutionized the study of developmental biology, and mice are now used extensively as one of the best option to study stem cell biology in mammals (11). Nevertheless, their application a model, have limitations in the regenerative medicine. But this model, relatively inexpensive and can be easily manipulated genetically (12). Failure to obtain a satisfactory result in the selection of many mouse models, to recapitulate particular human disease phenotypes, has forced researchers to investigate other animal species to be more probably predictive of humans (13). For this purpose, to study the genetic diseases, the pig has been currently determined as one the best option of a large animal model (14).

Stem cells, based on their differentiation ability, are classified into different cell types, including totipotent, pluripotent, multipotent, or unipotent. Also, another classification of these cells are based on the evolutionary stages, including embryonic, fetal, infant or umbilical cord blood and adult stem cells (15). shows an overview of stem cells classifications based on differentiation potency.

An overview of the stem cell classification. Totipotency: after fertilization, embryonic stem cells (ESCs) maintain the ability to form all three germ layers as well as extra-embryonic tissues or placental cells and are termed as totipotent. Pluripotency: these more specialized cells of the blastocyst stage maintain the ability to self-renew and differentiate into the three germ layers and down many lineages but do not form extra-embryonic tissues or placental cells. Multipotency: adult or somatic stem cells are undifferentiated cells found in postnatal tissues. These specialized cells are considered to be multipotent; with very limited ability to self-renew and are committed to lineage species.

Toti-potent cells have the potential for development to any type of cell found in the organism. In the other hand, the capacity of these cells to develop into the three primary germ cell layers of the embryo and into extra-embryonic tissues such as the placenta is remarkable (15).

The pluripotent stem cells are kind of stem cells with the potential for development to approximately all cell types. These cells contain ESCs and cells that are isolated from the mesoderm, endoderm and ectoderm germ layers that are organized in the beginning period of ESC differentiation (15).

The multipotent stem cells have less proliferative potential than the previous two groups and have ability to produce a variety of cells which limited to a germinal layer [such as mesenchymal stem cells (MSCs)] or just a specific cell line (such as HSCs). Adult stem cells are also often in this group. In the word, these cells have the ability to differentiate into a closely related family of cells (15).

Despite the increasing interest in totipotent and pluripotent stem cells, unipotent stem cells have not received the most attention in research. A unipotent stem cell is a cell that can create cells with only one lineage differentiation. Muscle stem cells are one of the example of this type of cell (15). The word uni is derivative from the Latin word unus meaning one. In adult tissues in comparison with other types of stem cells, these cells have the lowest differentiation potential. The unipotent stem cells could create one cell type, in the other word, these cells do not have the self-renewal property. Furthermore, despite their limited differentiation potential, these cells are still candidates for treatment of various diseases (16).

ESCs are self-renewing cells that derived from the inner cell mass of a blastocyst and give rise to all cells during human development. It is mentioned that these cells, including human embryonic cells, could be used as suitable, promising source for cell transplantation and regenerative medicine because of their unique ability to give rise to all somatic cell lineages (17). In the other words, ESCs, pluripotent cells that can differentiate to form the specialized of the various cell types of the body (18). Also, ESCs capture the imagination because they are immortal and have an almost unlimited developmental potential. Due to the ethical limitation on embryo sampling and culture, these cells are used less in research (19).

HSCs are multipotent cells that give rise to blood cells through the process of hematopoiesis (20). These cells reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the human and animal (21). Also, these cells can replenish missing or damaged components of the hematopoietic and immunologic system and can withstand freezing for many years (22).The mammalian hematopoietic system containing more than ten different mature cell types that HSCs are one of the most important members of this. The ability to self-renew and multi-potency is another specific feature of these cells (23).

Adult stem cells, as undifferentiated cells, are found in numerous tissues of the body after embryonic development. These cells multiple by cell division to regenerate damaged tissues (24). Recent studies have been shown that adult stem cells may have the ability to differentiate into cell types from various germ layers. For example, bone marrow stem cells which is derived from mesoderm, can differentiate into cell lineage derived mesoderm and endoderm such as into lung, liver, GI tract, skin, etc. (25). Another example of adult stem cells is neural stem cells (NSCs), which is derived from ectoderm and can be differentiate into another lineage such as mesoderm and endoderm (26). Therapeutic potential of adult stem cells in cell therapy and regenerative medicine has been proven (27).

For the first time in the late 1990s, CSCs were identified by John Dick in acute myeloid diseases. CSCs are cancerous cells that found within tumors or hematological cancers. Also, these cells have the characteristics of normal stem cells and can also give rise to all cell types found in a particular cancer sample (28). There is an increasing evidence supporting the CSCs hypothesis. Normal stem cells in an adult living creature are responsible for the repair and regeneration of damaged as well as aged tissues (29). Many investigations have reported that the capability of a tumor to propagate and proliferate relies on a small cellular subpopulation characterized by stem-like properties, named CSCs (30).

Embryonic connective tissue contains so-called mesenchymes, from which with very close interactions of endoderm and ectoderm all other connective and hematopoietic tissues originate, Whereas, MSCs do not differentiate into hematopoietic cell (31). In 1924, Alexander A. Maxi mow used comprehensive histological detection to identify a singular type of precursor cell within mesenchyme that develops into various types of blood cells (32). In general, MSCs are type of cells with potential of multi-lineage differentiation and self-renewal, which exist in many different kinds of tissues and organs such as adipose tissue, bone marrow, skin, peripheral blood, fallopian tube, cord blood, liver and lung et al. (4,5). Today, stem cells are used for different applications. In addition to using these cells in human therapy such as cell transplantation, cell engraftment etc. The use of stem cells in veterinary medicine has also been considered. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine.

The isolation method, maintenance and culture condition of MSCs differs from the different tissues, these methods as well as characterization of MSCs described as (36). MSCs could be isolated from the various tissues such as adipose tissue, bone marrow, umbilical cord, amniotic fluid etc. (37).

Diagram for adipose tissue-derived mesenchymal stem cell isolation (3).

Diagram for bone marrow-derived MSCs isolation (33). MSC, mesenchymal stem cell.

Diagram for umbilical cord-derived MSCs isolation (34). MSC, mesenchymal stem cell.

Diagram for isolation of amniotic fluid stem cells (AFSCs) (35).

Diagram for MSCs characterization (35). MSC, mesenchymal stem cell.

The diversity of stem cell or MSCs sources and a wide aspect of potential applications of these cells cause to challenge for selecting an appropriate cell type for cell therapy (38). Various diseases in animals have been treated by cell-based therapy. However, there are immunity concerns regarding cell therapy using stem cells. Improving animal models and selecting suitable methods for engraftment and transplantation could help address these subjects, facilitating eventual use of stem cells in the clinic. Therefore, for this purpose, in this section of this review, we provide an overview of the current as well as previous studies for future development of animal models to facilitate the utilization of stem cells in regenerative medicine (14). Significant progress has been made in stem cells-based regenerative medicine, which enables researchers to treat those diseases which cannot be cured by conventional medicines. The unlimited self-renewal and multi-lineage differentiation potential to other types of cells causes stem cells to be frontier in regenerative medicine (24). More researches in regenerative medicine have been focused on human cells including embryonic as well as adult stem cells or maybe somatic cells. Today there are versions of embryo-derived stem cells that have been reprogrammed from adult cells under the title of pluripotent cells (39). Stem cell therapy has been developed in the last decade. Nevertheless, obstacles including unwanted side effects due to the migration of transplanted cells as well as poor cell survival have remained unresolved. In order to overcome these problems, cell therapy has been introduced using biocompatible and biodegradable biomaterials to reduce cell loss and long-term in vitro retention of stem cells.

Currently in clinical trials, these biomaterials are widely used in drug and cell-delivery systems, regenerative medicine and tissue engineering in which to prevent the long-term survival of foreign substances in the body the release of cells are controlled (40).

Today, the incidence and prevalence of heart failure in human societies is a major and increasing problem that unfortunately has a poor prognosis. For decades, MSCs have been used for cardiovascular regenerative therapy as one of the potential therapeutic agents (41). Dhein et al. [2006] found that autologous bone marrow-derived mesenchymal stem cells (BMSCs) transplantation improves cardiac function in non-ischemic cardiomyopathy in a rabbit model. In one study, Davies et al. [2010] reported that transplantation of cord blood stem cells in ovine model of heart failure, enhanced the function of heart through improvement of right ventricular mass, both systolic and diastolic right heart function (42). In another study, Nagaya et al. [2005] found that MSCs dilated cardiomyopathy (DCM), possibly by inducing angiogenesis and preventing cardial fibrosis. MSCs have a tremendous beneficial effect in cell transplantation including in differentiating cardiomyocytes, vascular endothelial cells, and providing anti-apoptotic as well angiogenic mediators (43). Roura et al. [2015] shown that umbilical cord blood mesenchymal stem cells (UCBMSCs) are envisioned as attractive therapeutic candidates against human disorders progressing with vascular deficit (44). Ammar et al., [2015] compared BMSCs with adipose tissue-derived MSCs (ADSCs). It was demonstrated that both BMSCs and ADSCs were equally effective in mitigating doxorubicin-induced cardiac dysfunction through decreasing collagen deposition and promoting angiogenesis (45).

There are many advantages of small animal models usage in cardiovascular research compared with large animal models. Small model of animals has a short life span, which allow the researchers to follow the natural history of the disease at an accelerated pace. Some advantages and disadvantages are listed in (46).

Despite of the small animal model, large animal models are suitable models for studies of human diseases. Some advantages and disadvantages of using large animal models in a study protocol planning was elaborated in (47).

Chronic wound is one of the most common problem and causes significant distress to patients (48). Among the types of tissues that stem cells derived it, dental tissuederived MSCs provide good sources of cytokines and growth factors that promote wound healing. The results of previous studies showed that stem cells derived deciduous teeth of the horse might be a novel approach for wound care and might be applied in clinical treatment of non-healing wounds (49). However, the treatment with stem cells derived deciduous teeth needs more research to understand the underlying mechanisms of effective growth factors which contribute to the wound healing processes (50). This preliminary investigation suggests that deciduous teeth-derived stem cells have the potential to promote wound healing in rabbit excisional wound models (49). In the another study, Lin et al. [2013] worked on the mouse animal model and showed that ADSCs present a potentially viable matrix for full-thickness defect wound healing (51).

Many studies have been done on dental reconstruction with MSCs. In one study, Khorsand et al. [2013] reported that dental pulp-derived stem cells (DPSCs) could promote periodontal regeneration in canine model. Also, it was shown that canine DPSCs were successfully isolated and had the rapid proliferation and multi-lineage differentiation capacity (52). Other application of dental-derived stem cells is shown in .

Diagram for application of dental stem cell in dentistry/regenerative medicine (53).

As noted above, stem cells have different therapeutic applications and self-renewal capability. These cells can also differentiate into the different cell types. There is now a great hope that stem cells can be used to treat diseases such as Alzheimer, Parkinson and other serious diseases. In stem cell-based therapy, ESCs are essentially targeted to differentiate into functional neural cells. Today, a specific category of stem cells called induced pluripotent stem (iPS) cells are being used and tested to generate functional dopamine neurons for treating Parkinson's disease of a rat animal model. In addition, NSC as well as MSCs are being used in neurodegenerative disorder therapies for Alzheimers disease, Parkinsons disease, and stroke (54). Previous studies have shown that BMSCs could reduce brain amyloid deposition and accelerate the activation of microglia in an acutely induced Alzheimers disease in mouse animal model. Lee et al. [2009] reported that BMSCs can increase the number of activated microglia, which effective therapeutic vehicle to reduce A deposits in AD patients (55). In confirmation of previous study, Liu et al. [2015] showed that transplantation of BMSCs in brain of mouse model of Alzheimers disease cause to decrease in amyloid beta deposition, increase in brain-derived neurotrophic factor (BDNF) levels and improvements in social recognition (56). In addition of BMSCs, NSCs have been proposed as tools for treating neurodegeneration disease because of their capability to create an appropriate cell types which transplanted. kerud et al. [2001] demonstrated that NSCs efficiently express high level of glial cell line-derived neurotrophic factor (GDNF) in vivo, suggesting a use of these cells in the treatment of neurodegenerative disorders, including Parkinsons disease (57). In the following, Venkataramana et al. [2010] transplanted BMSCs into the sub lateral ventricular zones of seven Parkinsons disease patients and reported encouraging results (58).

The human body is fortified with specialized cells named MSCs, which has the ability to self-renew and differentiate into various cell types including, adipocyte, osteocyte, chondrocyte, neurons etc. In addition to mentioned properties, these cells can be easily isolated, safely transplanted to injured sites and have the immune regulatory properties. Numerous in vitro and in vivo studies in animal models have successfully demonstrated the potential of MSCs for various diseases; however, the clinical outcomes are not very encouraging. Based on the studies in the field of stem cells, MSCs find wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration and etc. In addition, these cells are particularly important in the treatment of the sub-branch neurodegenerative diseases like Alzheimer and Parkinson.

The authors wish to thank staff of the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Funding: The project described was supported by Grant Number IR.TBZMED.REC.1396.1218 from the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest: The authors have no conflicts of interest to declare.

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Stem cell-based regenerative medicine - PMC

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Global Stem Cell Market To Be Driven By Increasing Activities To Use Stem Cells In Regenerative Medicines In The Forecast Period Of 2022-2027 …

By daniellenierenberg

The new report by Expert Market Research titled, Global Stem Cell Market Report and Forecast 2022-2027, gives an in-depth analysis of the globalstem cell market, assessing the market based on its segments like types, treatment types, applications and major regions. The report tracks the latest trends in the industry and studies their impact on the overall market. It also assesses the market dynamics, covering the key demand and price indicators, along with analysing the market based on the SWOT and Porters Five Forces models.

Request a free sample copy in PDF or view the report summary@https://www.expertmarketresearch.com/reports/stem-cell-market/requestsample

The key highlights of the report include:

Market Overview (2017-2027)

The stem cell business is growing due to an increase in activities to use stem cells in regenerative treatments due to their medicinal qualities. The increasing use of human-induced pluripotent stem cells (iPSCs) for the treatment of hereditary cardiac difficulties, neurological illnesses, and genetic diseases such as recessive dystrophic epidermolysis bullosa (RBED) is driving the market forward.

Furthermore, because human-induced pluripotent stem cells (iPSCs) may reverse immunosuppression, they serve as a major source of cells for auto logic stem cell therapy, boosting the industrys expansion. Furthermore, the rising incentives provided by major businesses to deliver breakthrough stem cell therapies, as well as the increased use of modern resources and techniques in research and development activities (R&D), are propelling the stem cell market forward.

Because of increased research and development (R&D) in the United States and Canada, North America accounts for a significant portion of the overall stem cell business. Furthermore, the increased frequency of non-communicable chronic diseases such as cancer and Parkinsons disease, among others, is boosting the use of stem cell therapy, boosting the industrys growth. Furthermore, the regions stronghealthcaresector is improving access to innovative cell therapy treatments, assisting the regional stem cell industrys expansion. Aside from that, due to the rising use of regenerative treatments, the Asia Pacific area is predicted to rise rapidly. Furthermore, rising clinical trials are assisting market expansion due to low labour costs and the availability of raw materials in the region, contributing considerably to overall industry growth.

Industry Definition and Major Segments

A stem cell is a type of cell that has the ability to develop into a variety of cells, including brain cells and muscle cells. It can also help to repairtissuesthat have been injured. Because stem cells have the potential to treat a variety of non-communicable and chronic diseases, including Alzheimers and diabetes, theyre being used in medical and biotechnological research to repair tissue damage caused by diseases.

Explore the full report with the table of contents@https://www.expertmarketresearch.com/reports/stem-cell-market

The major product types of stem cell are:

The market can be broadly categorised on the basis of its treatment types into:

Based on applications, the market is divided into:

The EMR report looks into the regional markets of stem cell-like:

Market Trends

The market is expected to rise due to increased research activity in regenerative medicine and biotechnology to personalise stem cell therapy. The usage of stem cells is predicted to increase as the need for treatment of common disorders, such as age-related macular degeneration (AMD), grows among the growing geriatric population. Due to multiple error bars during research operations, it becomes extremely difficult to characterise cell products because each cell has unique properties. As a result, the integration of cutting-edge technologies such as artificial intelligence (AI), blockchain, and machine learning is accelerating. Artificial intelligence (AI) is being used to analyse images quickly, forecast cell functions, and classify tissues in order to identify cell products, which is expected to boost the market growth.

With the rising frequency of cancer and cancer-related research initiatives, blockchain technology is increasingly being used to collect and assimilate data in order to improve access to clinical outcomes and the latest advances. Blockchain can also help with data storage for patients while improving the cost-effectiveness of cord-blood banking for advanced research and development (R&D) purposes. In addition, the use of machine learning techniques to analyse photos and infer the relationship between cellular features is boosting the market growth. The increased interest in understanding cellular processes and identifying critical processes using deep learning is expected to move the stem cell business forward.

Latest News on Global Stem Cell Market@https://www.expertmarketresearch.com/pressrelease/global-stem-cell-market

Key Market Players

The major players in the market are Pluristem Therapeutics Inc., Thermo Fisher Scientific Inc., Cellular Engineering Technologies, Merck KGaA, Becton, Dickinson and Company, and STEMCELL Technologies Inc The report covers the market shares, capacities, plant turnarounds, expansions, investments and mergers and acquisitions, among other latest developments of these market players.

About Us:

Expert Market Research is a leading business intelligence firm, providing custom and syndicated market reports along with consultancy services for our clients. We serve a wide client base ranging from Fortune 1000 companies to small and medium enterprises. Our reports cover over 100 industries across established and emerging markets researched by our skilled analysts who track the latest economic, demographic, trade and market data globally.

At Expert Market Research, we tailor our approach according to our clients needs and preferences, providing them with valuable, actionable and up-to-date insights into the market, thus, helping them realize their optimum growth potential. We offer market intelligence across a range of industry verticals which include Pharmaceuticals, Food and Beverage, Technology, Retail, Chemical and Materials, Energy and Mining, Packaging and Agriculture.

Media Contact

Company Name: EMR Inc.Contact Person: Sofia Williams, Corporate Sales Specialist U.S.A.Email: sales@expertmarketresearch.comToll Free Number: +1-415-325-5166 | +44-702-402-5790Address: 30 North Gould Street, Sheridan, WY 82801, USACity: SheridanState: WyomingCountry: United StatesWebsite: https://www.expertmarketresearch.com

IntroducingProcurement ResourcesServices of EMR Inc.

*We at Expert Market Research always thrive to give you the latest information. The numbers in the article are only indicative and may be different from the actual report.

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Global Stem Cell Market To Be Driven By Increasing Activities To Use Stem Cells In Regenerative Medicines In The Forecast Period Of 2022-2027 ...

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Montefiore Einstein Cancer Center Finds CAR-T Therapy Effective in Black and Hispanic Patients – Newswise

By daniellenierenberg

Newswise April 28, 2022 (BRONX, NY)CAR-T therapy, a form of immunotherapy that revs up T-cells to recognize and destroy cancer cells, has revolutionized the treatment of blood cancers, including certain leukemias, lymphomas, and most recently, multiple myeloma. However, Black and Hispanic people were largely absent from the major clinical trials that led to the U.S. Food and Drug Administration approval of CAR-T cell therapies.

In a study published today in Bone Marrow Transplantation (BMT), investigators at the National Cancer Institute-designated Montefiore Einstein Cancer Center (MECC) report that Black and Hispanic patients had outcomes and side effects following CAR-T treatment that were comparable to their white and Asian counterparts.

Representation in cancer clinical trials is vital to ensuring that treatments are safe and effective for everyone, said Mendel Goldfinger, M.D., co-corresponding author of the paper, a medical oncologist at Montefiore Health System, assistant professor of medicine at Albert Einstein College of Medicine, and member of the MECC Cancer Therapeutics Program. We couldnt have been happier to learn that our patients who identify as Black and Hispanic have the same benefits from CAR-T therapy as white patients. We can only begin to say that a cancer treatment is transformational when these therapies benefit everyone who comes to us for care.

People who identify as Black and Hispanic often have tumor biology, immune system biology, and side effects that are distinct from white people. However, very few minorities were enrolled in the major trials that led the FDA to approve CAR-T cell therapy.

Parity for Black and Hispanic PatientsThe new BMT study evaluated outcomes for 46 participants treated at Montefiore between 2015 and 2021. Seventeen of the participants were Hispanic, 9 were African American, 15 were white, and 5 were Asian.

Among Black and Hispanic patients, 58% achieved a complete response after treatment and 19% achieved a partial response. For white and Asian patients, 70% achieved a complete response and 20% had a partial response, indicating no statistical differences among racial and ethnic backgrounds. Results were similar with respect to major side effects experienced: Approximately 95% of participants in each group had mild to moderate cytokine release syndrome, a common side effect to immunotherapy in which people experience fever and other flu-like symptoms.

Diversifying Cancer Clinical TrialsOur findings demonstrate that we are able to effectively treat people from historically marginalized groups using CAR-T; our hope is that more people from a diverse range of racial and ethnic backgrounds will be included in clinical trials, said co-author Amit Verma, M.B.B.S., associate director of translational science at MECC, director of the division of hemato-oncology at Montefiore and Einstein, and professor of medicine and of developmental and molecular biology at Einstein. Ira Braunschweig, M.D., associate professor of medicine at Einstein and director of Stem Cell Transplantation and Cellular Therapy and clinical program director, Hematologic Malignancies at Montefiore, is also co-corresponding author on the study.

At Montefiore, approximately 80% of clinical trial participants are minorities, compared with the nationwide figure of only 8%.

As an academic medical center, it is not enough to make novel therapies like CAR-T available, said Susan Green-Lorenzen, R.N. M.S.N., system senior vice president of operations at Montefiore and study co-author. We need to be at the forefront of ensuring that these treatments are effective for the communities we serve this research reflects this commitment.

The study is titled Efficacy and safety of CAR-T cell therapy in minorities. In addition to Drs. Goldfinger, Verma, and Braunschweig and Ms. Green-Lorenzen, other Einstein and Montefiore authors are Astha Thakkar, M.D., Michelly Abreu, N.P., Kith Pradhan, Ph.D., R. Alejandro Sica, M.D., Aditi Shastri, M.D., Noah Kornblum, M.D., Nishi Shah, M.D., M.P.H., Ioannis Mantzaris, M.D., M.S., Kira Gritsman, M.D., Ph.D., Eric Feldman, M.D., and Richard Elkind, P.A.-C.

***

About Albert Einstein College of MedicineAlbert Einstein College of Medicineis one of the nations premier centers for research, medical education and clinical investigation. During the 2021-22 academic year, Einstein is home to 732M.D.students, 190Ph.D.students, 120 students in thecombined M.D./Ph.D. program, and approximately 250postdoctoral research fellows. The College of Medicine has more than 1,900 full-time faculty members located on the main campus and at itsclinical affiliates. In 2021, Einstein received more than $185 million in awards from the National Institutes of Health. This includes the funding of majorresearch centersat Einstein in cancer, aging, intellectual development disorders, diabetes, clinical and translational research, liver disease, and AIDS. Other areas where the College of Medicine is concentrating its efforts include developmental brain research, neuroscience, cardiac disease, and initiatives to reduce and eliminate ethnic and racial health disparities. Its partnership withMontefiore, the University Hospital and academic medical center for Einstein, advances clinical and translational research to accelerate the pace at which new discoveries become the treatments and therapies that benefit patients. For more information, please visiteinsteinmed.org, read ourblog, followus onTwitter, like us onFacebook,and view us onYouTube.

About Montefiore Health SystemMontefiore Health System is one of New Yorks premier academic health systems and is a recognized leader in providing exceptional quality and personalized, accountable caretoapproximately three million people in communities across the Bronx, Westchester and the Hudson Valley. It is comprised of 10hospitals, including the Childrens Hospital at Montefiore, Burke Rehabilitation Hospital and more than 200 outpatient ambulatory care sites. The advanced clinical and translational research at its medical school, Albert Einstein College of Medicine, directly informs patient care and improves outcomes. From the Montefiore-Einstein Centers of Excellence in cancer, cardiology and vascular care, pediatrics, and transplantation,toits preeminent school-based health program, Montefiore is a fully integrated healthcare delivery system providing coordinated, comprehensive caretopatients and their families. For more information, please visitwww.montefiore.org. Followus onTwitter and Instagram and LinkedIn, or view us onFacebookandYouTube.

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Montefiore Einstein Cancer Center Finds CAR-T Therapy Effective in Black and Hispanic Patients - Newswise

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Interim Data Targeting CD117 Show Promising MRD Results and Safety in MDS/AML – Targeted Oncology

By daniellenierenberg

Early outcomes with the combination of JSP191, fludarabine, and low-dose total body radiation (TBI) demonstrated facilitation of full donor myeloid chimerism, clearing of minimal residual disease (MRD), and a well-tolerated safety profile in older patients with myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) receiving non-myeloablative (NMA) allogenic hematopoietic cell transplantation (AHCT).

Results from the phase 1 trial (NCT04429191) presented at the 2022 Transplantation & Cellular Therapy Meetings, showed there were no infusion toxicities or serious adverse events with JSP191, and no instances of primary graft failure in first 24 patients enrolled on the trial; only 1 patient had secondary graft failure and went on to have successful retransplant. Additionally, MRD clearance was observed in 12 patients, and JSP191 pharmacokinetics were shown to be predictable.

AHCT is the only curative treatment for many patients with MDS/AML, even though there have been advancements in therapy for these patients in recent years. While transplant has proven feasible for adults well into their 70s, the optimal conditioning regimen for older adults remains unknown as more intensive regimens tend to be associated with transplant-related mortality, while less intensive nonmyeloablative regimens have resulted historically in higher rates of disease relapse and progression, Lori Muffly, MD, MS, said in her presentation.

Therefore, a conditioning regimen that results in minimal toxicity but has enhanced disease control is needed in order to improve transplantation outcomes in this population, Muffly, associate professor of medicine (blood and marrow transplantation and cellular therapy) at Stanford Healthcare, continued.

JSP191 is a humanized monoclonal antibody meant to block stem cell factor binding site on CD117, which is necessary for hematopoietic stem cell (HSC) survival and HSC interactions in the bone marrow niche. After the bone marrow niche is emptied because of JSP191 binding to CD117, healthy donor cells are able to engraft. Preclinical models showed synergy between anti-CD117 monoclonal antibodies and low-dose TBI to help deplete HSC and facilitate donor cell engraftment.

For the first 24 patients with MDS (n = 13) or AML (n = 11), primary end points evaluated were safety, tolerability, and pharmacokinetics of the combination. Secondary end points included engraftment and donor chimerism, MRD clearance, relapse-free survival, graft-vs-host disease (GVHD), non-relapse mortality, and overall survival. Patients received AHCT, then 200 to 300 cGy of TBI, 30 mg/m2 of fludarabine for 3 days, and 0.6 mg/kg of intravenous JSP191.

To determine the starting date of fludarabine, real-time pharmacokinetic measurements and modeling were used after JSP191 was administered. For the first 7 patients, TBI was increased from 200 to 300 cGy to aid lymphoablation. Tacrolimus, sirolimus, and mycohphenolate motefil were used as GVHD prophylaxis.

Consistent pharmacokinetics and predictable clearance were observed with JSP191 over the 2 weeks after administration. All patients were able to receive donor cell infusion between 9 and 15 days following administration of the antibody. Interestingly, we did see in some patients very low levels of the antibody present on the day of donor cell infusion, and this did not appear to impact donor cell engraftment, Muffly said.

Bone marrow aspirations taken at screening and between administration of the antibody and fludarabine/TBI showed JSP191 depletes hemopoietic stem and progenitor cells (HSPC). In the CD34-positive, CD45RA-negative population, there was a 66% mean depletion of HSPC. The investigators do not believe this reflects the nadir of HSPC depletion, Muffly explained, and that the depletion continues until donor stem cell infusion.

All patients experienced neutropenia followed by neutrophil engraftment between TD+15 and TD+26. Primary engraftment was seen in all patients, with only 1 patient losing myeloid chimerism early, which was associated with disease progression. T cell chimerism improved when patients went up from 200 to 300 cGy.

Using flow cytometry, cytogenetics, and next-generation sequencing, investigators were able to track MRD in patients with de novo AML (n = 8) and AML from MDS (n = 3). Of the 9 patients with AML who were MRD positive at the time of enrollment, 6 were MRD negative at the time of follow-up. Eleven of 13 patients with MDS were MRD positive at enrollment, and 8 were MRD negative at the last follow-up.

After 6 months median follow-up (range, 2-12 months), there were no reports of classical grade II-IV acute GVHD. One case of late onset grade III-IV acute gastrointestinal GVHD was reported as of the latest follow-up, but this patient had non-relapse mortality. Any instances of chronic GVHD has yet to be reported due to insufficient median follow-up time. Morphologic relapse occurred in 4 patients, 3 with AML and 1 with MDS.

The median age for these patients was 70 years (range, 62-79), with a requirement of 60 years of age or older or an AHCT-comorbidity index of 3 or more to enroll in the trial. They could not have prior AHCT and needed a human leukocyte antigenmatched related or unrelated donor. Over half of patients received only a hypomethylating agent-containing regimens.

JSP191 in combination with fludarabine and low-dose TBI is a novel conditioning platform that appears safe, well tolerated, has demonstrated on-target effects of HSPC depletion, permits full donor myeloid chimerism, and results in promising early MRD clearance, Muffly concluded.

Reference:

Muffly L, Lee CJ, Gandhi A, et al. Preliminary data from a phase 1 study of JSP191, an anti-CD117 monoclonal antibody, in combination with low dose irradiation and fludarabine conditioning is well-tolerated, facilitates chimerism and clearance of minimal residual disease in older adults with MDS/AML undergoing allogeneic HCT. Presented at: 2022 Transplantation & Cellular Therapy Meetings; Salt Lake City, UT; April 23-26, 2022. Abstract LBA4. https://bit.ly/3xRTwee

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Interim Data Targeting CD117 Show Promising MRD Results and Safety in MDS/AML - Targeted Oncology

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FDA Grants Direct Biologics Regenerative Medicine Advanced Therapy (RMAT) Designation for the use of ExoFlo in COVID-19 Related ARDS USA – English -…

By daniellenierenberg

AUSTIN, Texas, April 13, 2022 /PRNewswire/ -- Direct Biologics, an innovative biotechnology company with a groundbreaking extracellular vesicle (EV) platform drug technology, announced that the U.S. Food and Drug Administration (FDA) has awarded their EV drug product ExoFlo with a Regenerative Medicine Advanced Therapy (RMAT) designation for the treatment of Acute Respiratory Distress Syndrome (ARDS) associated with COVID-19. The RMAT program is designed to expedite the approval of promising regenerative medical products in the US that demonstrate clinical evidence indicating the ability to address an unmet medical need for a serious life-threatening disease or condition. Under the RMAT designation, the FDA provides intensive guidance on drug development and post-market requirements through early and frequent interactions. Additionally, an RMAT confers eligibility for accelerated approval and priority review of biologics licensing applications (BLA).

"After intensively reviewing our preclinical data, manufacturing processes, and clinical data from our Phase II multicenter, double blinded, placebo controlled randomized clinical trial, the FDA has recognized ExoFlo as a lifesaving treatment for patients suffering from Acute Respiratory Distress Syndrome (ARDS) due to severe or critical COVID-19," said Mark Adams, Chief Executive Officer. "The additional attention, resources, and regulatory benefits provided by an RMAT designation demonstrate that the FDA views ExoFlo as a product that can significantly enhance the standard of care for the thousands still dying from ARDS every week in the US," he said.

"We are very pleased that the FDA has recognized the lifesaving potential of our platform drug technology ExoFlo. The RMAT has provided a pathway to expedite our drug development to achieve a BLA in the shortest possible time," said Joe Schmidt, President. "I am very proud of our team. Everyone has been working around the clock for years in our mission to save human lives taken by a disease that lacks treatment options, both in the US and abroad. We are grateful for the opportunity to accelerate development of ExoFlo under the RMAT designation as it leads us closer to our goal of bringing our life saving drug to patients who desperately need it."

ExoFlo is an acellular human bone marrow mesenchymal stem cell (MSC) derived extracellular vesicle (EV) product. These nanosized EVs deliver thousands of signals in the form of regulatory proteins, microRNA, and messenger RNA to cells in the body, harnessing the anti-inflammatory and regenerative properties of bone marrow MSCs without the cost, complexity and limitations of scalability associated with MSC transplantation. ExoFlo is produced using a proprietary EV platform technology by Direct Biologics, LLC.

Physicians can learn more and may request information on becoming a study site at clinicaltrials.gov. For more information on Direct Biologics and regenerative medicine, visit: https://directbiologics.com.

About Direct BiologicsDirect Biologics, LLC, is headquartered in Austin, Texas, with an R&D facility located at the University of California, and an Operations and Order Fulfillment Center located in San Antonio, Texas. Direct Biologics is a market-leading innovator and cGMP manufacturer of regenerative medical products, including a robust EV platform technology. Direct Biologics' management team holds extensive collective experience in biologics research, development, and commercialization, making the Company a leader in the evolving segment of next generation regenerative biotherapeutics. Direct Biologics has obtained and is pursuing multiple additional clinical indications for ExoFlo through the FDA's investigational new drug (IND) process. For more information visit http://www.directbiologics.com.

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Adding Bispecific Antibody to Natural Killer Cells May Be Effective in Heavily Pretreated Lymphoma – www.oncnursingnews.com/

By daniellenierenberg

The addition of the innate cell engager AMF13 to preactivated and expanded natural killer (NK) cells may represent an effective treatment for pretreated patients with advanced lymphoma, according to findings from a phase 1/2 study (NCT04074746) that were presented during the 2022 AACR Annual Meeting. 1

Results showed that patients experienced a median overall response rate (ORR) of 89.5% (n = 17/19). Overall, 10 patients experienced complete responses (CRs) and 7 experienced partial responses (PRs).2

Lead author Yago Nieto, MD, PhD, a professor of medicine in the Department of Stem Cell Transplantation and Cellular Therapy at the University of Texas MD Anderson Cancer Center, in Houston, discussed the findings during a press conference during the meeting. He said the study team was pleasantly surprised by the quality of tumor responses in patients with resistant lymphomas.

This is the first clinical trial using off the shelf cord blood-derived cytokine-induced memory-likeex vivoexpanded NK cells precomplexed with the innate cell engager AMF13 construct to treat patients with CD30-positive relapsed/refractory Hodgkin lymphoma, he said. We saw very encouraging activity in this population of very heavily pretreated patients.

The current standard of care for relapsed CD30-positive lymphomas is brentuximab vedotin (Adcetris), an antibody-drug conjugate that delivers a toxic cytoskeleton destabilizing agent to cells expressing CD30. However, not all these lymphomas respond to brentuximab vedotin. When that treatment fails, those tumors then become extremely resistant to killing and patients are left with very few effective therapeutic options.

To address the problem, investigators enrolled 22 patients with relapsed or refractory CD30+ lymphoma into this single-center phase 1/2 trial, 20 of whom were diagnosed with Hodgkin lymphoma (HL). All had active progressive disease at enrollment, and none received bridging therapy. Patients were heavily pretreated, with a median of 7 (range, 1-14) prior lines of therapy. Nine underwent autologous stem cell transplantation (SCT) and 5 received allogeneic SCT.

Eligible patients had relapsed/refractory CD30-positive classical HL, B-cell non-Hodgkin lymphoma, anaplastic large-cell lymphoma, or peripheral T-cell lymphoma that was refractory or intolerant to brentuximab vedotin. They needed to have an ECOG performance status of 2 or below, and adequate renal, hepatic, pulmonary, and cardiac function.

The median age was 40 years (range, 20-75). Most patients were white (68.2%) and male 68.1%).

Patients receive 2 cycles of fludarabine/cyclophosphamide, followed by AFM13-CB NK cells at 3 dose levelsDL1 (106NK/gg), DL2 (107NK/kg), and DL3 (108NK/kg)on day 0 plus 3 weekly intravenous infusions of 200 mg AFM13, a CD30/CD16A bispecific antibody. Nineteen patients completed both planned cycles of treatment.

Nieto and colleagues isolated NK cells from cord blood, then used a mixture of cytokines to activate the cells into a memory-like state, making them more persistent and effective. They then expanded the cells in culture and complexed them with AFM13.

At a median follow-up of 11 months, progression-free survival (PFS) and overall survival (OS) rates across all 3 dose levels were 52% and 81%, respectively. Across all dose levels, 53% of patients experienced CR and 37% had PR. Eleven percent had progressive disease.

Expansion of NK cells occurred immediately after infusion and persisted for 3 weeks.

Investigators established DL3 as the recommend phase 2 dose (RP2D). All 13 (100%) patients treated at this dose level responded to therapy, including eight CRs (62%).Five of those patients were in CR after cycle 1, and 3 additional patients converted from PR to CR after cycle 2, Nieto added.

The median PFS was 67% and the median OS was 93% in the RP2D population.

Investigators did not record any cytokine release syndrome or graft vs host disease (GVHD), or neurotoxicity. Our preliminary results show an excellent tolerability profile, Nieto said.

There was no instance of infusion-related reactions (IRRs) associated with AFM13-NK cells across 40 infusions. There was 1 instance of grade 3 IRR and 4 grade 2 IRRs in 108 infusions of AFM13 alone. Investigators observed no dose limiting toxicities.

Never before in mankind have we seen this approach, really leading to pretty staggering results, Timothy Yap, MBBS, PhD, FRCP, a medical oncologist and associate director of translational research in the Institute for Personalized Cancer Therapy at the University of Texas MD Anderson Cancer Center, said. Everyone can see for themselves how impressive these results are. In addition to that, the actual tolerability profile is truly excellent with no instances of cytokine release syndrome, no neurotoxicity, no GVHD. Truly, truly impressive.

References

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Adding Bispecific Antibody to Natural Killer Cells May Be Effective in Heavily Pretreated Lymphoma - http://www.oncnursingnews.com/

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Are COVID-19-Linked Arrhythmias Caused by Viral Damage to the Heart’s Pacemaker Cells? – Weill Cornell Medicine Newsroom

By daniellenierenberg

The SARS-CoV-2 virus can infect specialized pacemaker cells that maintain the hearts rhythmic beat, setting off a self-destruction process within the cells, according to a preclinical study co-led by researchers at Weill Cornell Medicine, NewYork-Presbyterian and NYU Grossman School of Medicine. The findings offer a possible explanation for the heart arrhythmias that are commonly observed in patients with SARS-CoV-2 infection.

In the study, reported March 8 in Circulation Research, the researchers used an animal model as well as human stem cell-derived pacemaker cells to show that SARS-CoV-2 can readily infect pacemaker cells and trigger a process called ferroptosis, in which the cells self-destruct but also produce reactive oxygen molecules that can impact nearby cells.

This is a surprising and apparently unique vulnerability of these cellswe looked at a variety of other human cell types that can be infected by SARS-CoV-2, including even heart muscle cells, but found signs of ferroptosis only in the pacemaker cells, said study co-senior author Dr. Shuibing Chen, the Kilts Family Professor of Surgery and a professor of chemical biology in surgery and of chemical biology in biochemistry at Weill Cornell Medicine.

Arrhythmias including too-quick (tachycardia) and too-slow (bradycardia) heart rhythms have been noted among many COVID-19 patients, and multiple studies have linked these abnormal rhythms to worse COVID-19 outcomes. How SARS-CoV-2 infection could cause such arrhythmias has been unclear, though.

In the new study, the researchers, including co-senior author Dr. Benjamin tenOever of NYU Grossman School of Medicine, examined golden hamstersone of the only lab animals that reliably develops COVID-19-like signs from SARS-CoV-2 infectionand found evidence that following nasal exposure the virus can infect the cells of the natural cardiac pacemaker unit, known as the sinoatrial node.

To study SARS-CoV-2s effects on pacemaker cells in more detail and with human cells, the researchers used advanced stem cell techniques to induce human embryonic stem cells to mature into cells closely resembling sinoatrial node cells. They showed that these induced human pacemaker cells express the receptor ACE2 and other factors SARS-CoV-2 uses to get into cells and are readily infected by SARS-CoV-2. The researchers also observed large increases in inflammatory immune gene activity in the infected cells.

The teams most surprising finding, however, was that the pacemaker cells, in response to the stress of infection, showed clear signs of a cellular self-destruct process called ferroptosis, which involves accumulation of iron and the runaway production of cell-destroying reactive oxygen molecules. The scientists were able to reverse these signs in the cells using compounds that are known to bind iron and inhibit ferroptosis.

This finding suggests that some of the cardiac arrhythmias detected in COVID-19 patients could be caused by ferroptosis damage to the sinoatrial node, said co-senior author Dr. Robert Schwartz, an associate professor of medicine in the Division of Gastroenterology and Hepatology at Weill Cornell Medicine and a hepatologist at NewYork-Presbyterian/Weill Cornell Medical Center.

Although in principle COVID-19 patients could be treated with ferroptosis inhibitors specifically to protect sinoatrial node cells, antiviral drugs that block the effects of SARS-CoV-2 infection in all cell types would be preferable, the researchers said.

The researchers plan to continue to use their cell and animal models to investigate sinoatrial node damage in COVID-19and beyond.

There are other human sinoatrial arrhythmia syndromes we could model with our platform, said co-senior author Dr. Todd Evans, the Peter I. Pressman M.D. Professor of Surgery and associate dean for research at Weill Cornell Medicine. And, although physicians currently can use an artificial electronic pacemaker to replace the function of a damaged sinoatrial node, theres the potential here to use sinoatrial cells such as weve developed as an alternative, cell-based pacemaker therapy.

Many Weill Cornell Medicine physicians and scientists maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosurespublic to ensure transparency. For this information, see profiles for Dr. Todd Evans, and Dr. Robert Schwartz.

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Are COVID-19-Linked Arrhythmias Caused by Viral Damage to the Heart's Pacemaker Cells? - Weill Cornell Medicine Newsroom

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Sailing the Genome in Search of Safe Harbors – Technology Networks

By daniellenierenberg

Cell and gene therapies are poised to have a major impact on the landscape of modern medicine, carrying the potential to treat an array of different diseases with unmet clinical need.

However, the number of approved, clinically adopted cell and gene therapies is mere compared to the amount that are currently in development. A major barrier for the translation of such therapies is the safe integration of therapeutic genes into the human genome. The insertion of therapeutic genes bears the risk of off target effects, or integration of the gene into an unintended location.

A number of different strategies have been proposed to mitigate this effect. The most recent body of work comes from a collaboration between Harvards Wyss Institute for Biologically Inspired Engineering, Harvard Medical School (HMS) and the ETH Zurich in Switzerland.

Published in Cell Report Methods, the research focused on identifying safe spots in the genome. These locations, known as genomic safe harbors (GSHs), are areas in the genome that meet the following criteria: they can be accessed easily by genome-editing strategies, are within a safe distance from genes that possess functional properties and permit expression of a therapeutic gene, only once it has landed in the harbor. A simple analogy is deciding which harbor to dock a boat there are many considerations, and these depend on the type of boat you are sailing, the weather conditions and ease of access.

The research team adopted computational strategies that enabled the identification of 2,000 predicted GSHs. From this initial identification, they successfully validated two of the sites both in vitro and in vivo using reporter proteins.

Technology Networks interviewed the studys first author, Dr. Erik Aznauryan, research fellow in the laboratory of Professor George Church at Harvard Medical School. Aznauryan dives into further detail on the history of GSH research, the methods adopted to validate the GSH sites and the potential applications of this research.

Molly Campbell (MC): Can you talk about the history of genomic safe harbor research, and how they were discovered?

Erik Aznauryan (EA): Three genomic sites were empirically identified in previous studies to support stable expression of genes of interest in human cells: AAVS1, CCR5 and hRosa26. All these examples were established without any a-priori safety assessment of the genomic loci they reside in.

Attempts have been made to identify human GSH sites that would satisfy various safety criteria, thus avoiding the disadvantages of existing sites. One approach developed by Sadelain and colleagues used lentiviral transduction of beta-globin and green fluorescence protein genes into induced pluripotent stem cells (iPSCs), followed by the assessment of the integration sites in terms of their linear distance from various coding and regulatory elements in the genome, such as cancer genes, miRNAs and ultraconserved regions.

They discovered one lentiviral integration site that satisfied all of the proposed criteria, demonstrating sustainable expression upon erythroid differentiation of iPSCs. However, global transcriptome profile alterations of cells with transgenes integrated into this site were not assessed. A similar approach by Weiss and colleagues used lentiviral integrations in Chinese hamster ovary (CHO) cells to identify sites supporting long-term protein expression for biotechnological applications (e.g., recombinant monoclonal antibody production). Although this study led to the evaluation of multiple sites for durable, high-level transgene expression in CHO cells, no extrapolation to human genomic sites was carried out.

Another study aimed at identifying GSHs through bioinformatic search of mCreI sites regions targeted by monomerized version of I-CreI homing endonuclease found and characterized in green algae as capable to make targeted staggered double-strand DNA breaks residing in loci that satisfy GSH criteria. Like previous work, several stably expressing sites were identified and proposed for synthetic biology applications in humans. However, local and global gene expression profiling following integration events in these sites have not been conducted.

All these potential GSH sites possess a shared limitation of being narrowed by lentiviral- or mCreI-based integration mechanisms. Additionally, safety assessments of some of these identified sites, as well as previously established AAVS1, CCR5 and Rosa26, were carried out by evaluating the differential gene expression of genes located solely in the vicinity of these integration sites, without observing global transcriptomic changes following integration.

A more comprehensive bioinformatic-guided and genome-wide search of GSH sites based on established criteria, followed by experimental assessment of transgene expression durability in various cell types and safety assessment using global transcriptome profiling would, thus, lead to the identification of a more reliable and clinically useful genomic region.

MC: If GSHs do not encode proteins, or RNAs with functions in gene expression, or other cellular processes what is their function in the genome?

EA: In addition to protein coding, functional RNA coding, regulatory and structural regions of the human genome, other less well understood and inactive DNA regions exist.

A large proportion of the human genome seems to have evolved in the presence of a variety of integrating viruses which, as they inserted their DNA into the eukaryotic genome over the course of million years, lead to an establishment of vast non-coding elements that we continue to carry to this day. Furthermore, partial duplications of functional human genes have resulted in the formation of inactive pseudogenes, which occupy space in the genome yet are not known to bear cellular functions.

Finally, functional roles of some non-coding portions of the human genome are not well understood yet. Our search of safe harbors was conducted using existing annotation of the human genome, and as more components of it are deciphered the identification of genomic regions safe for gene insertion will become more informed.

MC: Are you able to discuss why some regions of the genome were previously regarded as GSHs but are now recognized as non-GSHs?

EA: In the absence of other alternatives, AAVS1, CCR5 and hRosa26 sites were historically called GSHs, as they supported the expression of genes of interest in a variety of cell types and were suitable for use in a research setting.

Their caveats (mainly, location within introns of functional genes, closely surrounded by other known protein coding genes as well as oncogenes) however prevent them from being used for clinical applications. Therefore, in our paper we dont call them GSHs, and refer to our newly discovered sites as GSHs.

MC: You thoroughly scanned the genome to identify candidate loci for further study as potential GSHs. Can you discuss some of the technological methods you adopted here, and why?

EA: We used several publicly available databases to identify genomic coordinates of structural, regulatory and coding components of the human genome according to the GSH criteria we outlined in the beginning of our study (outside genes, oncogenes, lncRNAs etc.,). We used these coordinates and bioinformatic tools such as command lines bedtools to exclude these genomic elements as well as areas adjacent to them. This left us with genomic regions putative GSHs from which we could then experimentally validate by inserting reporter and therapeutic genes into them followed by transcriptomic analysis of GSH-integrated vs non-integrated cells.

MC: You narrowed down your search to test five, and then two GSHs. Can you expand on your choice of reporter gene when assessing two GSHs in cell lines?

EA: Oftentimes in research you go with what is available or what is of the most interest to the lab you are currently working in.

Our case was not an exception, and we initially (up until the T cell work) used the mRuby reporter gene as it was widely available and extensively utilized and validated in our lab at ETH Zurich back then.

When I moved to the Wyss Institute at Harvard, I began collaborating with Dr. Denitsa Milanova, who was interested in testing these sites in the context of skin gene therapy particularly the treatment of junctional epidermolysis bullosa caused by mutations in various anchor proteins connecting different layers of skin, among which is the LAMB3 gene. For this reason, we decided to express this gene in human dermal fibroblasts, together with green fluorescent protein to have a visualizable confirmation of expression. We hope we would be able to translate this study into clinics.

MC: Can you describe examples of how GSHs can be utilized in potential therapeutics?

EA: Current cell therapy approaches rely on random insertion of genes of interest into the human genome. This can be associated with potential side effects including cancerous transformation of therapeutic cells as well as eventual silencing of the inserted gene.

We hope that current cell therapies will eventually transition to therapeutic gene insertions precisely into our GSHs, which will alleviate both described concerns. Specific areas of implementation may involve safer engineering of T cells for cancer treatment: insertion of genes encoding receptors targeting tumor cells or cytokines capable of enhancing anti-tumor response.

Additionally, these sites can be used for the engineering of skin cells for therapeutic (as discussed earlier with the LAMB3 example) as well as anti-aging applications, such as expression of genes that result in youthful skin phenotype.

Finally, given the robustness of gene expression from our identified sites, they can be used for industry-scale bio-manufacturing: high-yield production of proteins of interest in human cell lines for subsequent extraction and therapeutic applications (e.g., production of clotting factors for patients with hemophilias).

MC: Are there any limitations to the research at this stage?

EA: A primary limitation to this study is the low frequency of genomic integration events using CRISPR-based knock-in tools. This means that cells in which the gene of interest successfully integrated into the GSH must be pulled out of the vastly larger population of cells without this integration.

These isolated cells would then be expanded to generate homogenous population of gene-bearing cells. Such pipeline is not ideal for a clinical setting and improvements in gene integration efficiencies are needed to help this technology easier translate into clinics.

Our lab is currently working on developing genome engineering tools which would eventually allow to integrate large genes into GSHs with high precision and efficiency.

MC: What impact might this study have on the cell and gene therapy development space?

EA: This study will hopefully lead to many researchers in the field testing our sites, validating them in other therapeutically relevant cell types and eventually using them in research as well as in clinics as more reliable, durable and safe alternatives to current viral based random gene insertion methods.

Additionally, since in our work we shared all putative GSHs identified by our computational pipeline, we hope researchers will attempt to test sites we havent validated yet by implementing the GSH evaluation pipeline that we outlined in the paper. This will lead to identification of more GSHs with perhaps even better properties for clinical translation or bio-manufacturing.

MC: What are your next steps in advancing this work?

We hope to one day translate our successful in vitro skin results and start using these GSHs in an in vivo context.

Additionally, we are looking forward to improving integration efficiencies into our GSHs, which would further support clinical transition of our sites.

Finally, we will evaluate the usability of our GSHs for large-scale production of therapeutically relevant proteins, thus ameliorating the pipeline of manufacturing of biologics.

Dr. Erik Aznauryan was speaking to Molly Campbell, Senior Science Writer for Technology Networks.

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Sailing the Genome in Search of Safe Harbors - Technology Networks

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Jasper Therapeutics Announces Management Changes to Strengthen Leadership Team – BioSpace

By daniellenierenberg

REDWOOD CITY, Calif., March 21, 2022 (GLOBE NEWSWIRE) --Jasper Therapeutics, Inc. (NASDAQ: JSPR), a biotechnology company focused on hematopoietic cell transplant therapies, today announced changes to its management team, including the promotions of Jeet Mahal to the newly created position of Chief Operating Officer, and of Wendy Pang, M.D., Ph.D., to Senior Vice President of Research and Translational Medicine. Both promotions are effective as of March 21, 2022. Jasper also announced that a new position of Chief Medical Officer has been created, for which an active search is underway. Judith Shizuru, M.D. PhD, co-founder, and Scientific Advisory Board Chairwoman will lead clinical development activities on an interim basis and Kevin Heller, M.D., EVP of Research and Development, will be transitioning to a consultant role.

Based on the recent progress with JSP191, our anti-CD117 monoclonal antibody, as a targeted non-toxic conditioning agent and our mRNA hematopoietic stem cell program we have decided to advance Jaspers organizational structure with the creation of the roles of Chief Operating Officer and Chief Medical Officer and by elevating our research and translational medicine team to report directly to the CEO, said Ronald Martell, CEO of Jasper Therapeutics. We also are pleased that Dr. Shizuru will lead clinical development activities on an interim basis, a role she served during the companys founding in 2019.

These changes will allow us to advance our upcoming pivotal trial of JSP191 in AML/ MDS and execute on our pipeline opportunities with a best-in-class organization, continued Mr. Martell. We also wish to thank Dr. Heller for his help advancing JSP191 through our initial AML/MDS transplant study.

In the two plus years since we founded Jasper and received our initial funding, the company has been able to advance JSP191 in two clinical studies, develop our mRNA stem cell graft platform and publicly list on NASDAQ, said Dr. Shizuru, co-founder and member of the Board of Directors of Jasper Therapeutics. These changes will strengthen the companys ability to advance the field of hematopoietic stem cell therapies and bring cures to patients with hematologic cancers, autoimmune diseases and debilitating genetic diseases."

Mr. Mahal joined Jasper in 2019 as Chief Finance and Business Officer and has led Finance, Business Development, Marketing and Facilities/ IT since the companys inception. Prior to joining Jasper, he was Vice President, Business Development and Vice President, Strategic Marketing at Portola Pharmaceuticals, where he led the successful execution of multiple business development partnerships for Andexxa, Bevyxxaand cerdulatinib. He also played a key role in the companys equity financings, including its initial public offering and multiple royalty transactions. Earlier in his career, Mr. Mahal was Director, Business and New Product Development, at Johnson & Johnson on the Xareltodevelopment and strategic marketing team. Mr. Mahal holds a BA in Molecular and Cell Biology from U.C. Berkeley, a Masters in Molecular and Cell Biology from the Illinois Institute of Technology, a Masters in Engineering from North Carolina State University and an MBA from Duke University.

Dr. Pang joined Jasper in 2020 and has led early research and development including leading creation of the companys mRNA stem cell graft platform and playing a pivotal role in advancing JSP191 across multiple clinical studies. Previously Dr. Pang was an Instructor in the Division of Blood and Marrow Transplantation at Stanford University and the lead scientist in the preclinical drug development of an anti-CD117 antibody program. She was the lead author on the proof-of-concept studies showing that an anti-CD117 antibody therapy targets disease-initiating human hematopoietic (blood cell-forming) stem cells in myelodysplastic syndrome (MDS). She has authored numerous publications on the characterization of hematopoietic stem and progenitor cell behavior in hematopoieticdiseases, as well as hematopoietic malignancies, including MDS and acute myeloid leukemia (AML), and in hematopoietic stem cell transplantation. Dr. Pang earned her AB and BM in Biology from Harvard University and her MD and PhD in cancer biology from Stanford University.

Dr. Shizuru is a Professor of Medicine (Blood and Marrow Transplantation) and Pediatrics (Stem Cell Transplantation) at StanfordUniversity.She is the clinician-scientist co-founder of Jasper Therapeutics. Dr. Shizuru is an internationally recognized expert on the basic biology of blood stem cell transplantation and the translation of this biology to clinical protocols.Dr Shizuruis a member of the Stanford Blood and Marrow Transplantation (BMT) faculty, the Stanford Immunology Program, and the Institute for Stem Cell Biology and Regenerative Medicine. Shehas been an attending clinicianattendedon the BMT clinical service since 1997.Currently, she oversees a research laboratory focused on understanding the cellular and molecular basis of resistance to engraftment of transplantedallogeneic bone marrow blood stemcells and the way in which bone marrow grafts modify immune responses.Dr. Shizuru earned her BA from Bennington College and her MD and PhD in immunology from Stanford University

About Jasper Therapeutics

Jasper Therapeutics is a biotechnology company focused on the development of novel curative therapies based on the biology of the hematopoietic stem cell. The company is advancing two potentially groundbreaking programs. JSP191, an anti-CD117 monoclonal antibody, is in clinical development as a conditioning agent that clears hematopoietic stem cells from bone marrow in patients undergoing a hematopoietic cell transplantation. It is designed to enable safer and more effective curative allogeneic hematopoietic cell transplants and gene therapies. Jasper is also advancing JSP191 as a potential therapeutic for patients with lower risk Myelodysplastic Syndrome (MDS). Jasper Therapeutics is also advancing its preclinical mRNA hematopoietic stem cell graft platform, which is designed to overcome key limitations of allogeneic and autologous gene-edited stem cell grafts. Both innovative programs have the potential to transform the field and expand hematopoietic stem cell therapy cures to a greater number of patients with life-threatening cancers, genetic diseases and autoimmune diseases than is possible today. For more information, please visit us at jaspertherapeutics.com.

Forward-Looking Statements

Certain statements included in this press release that are not historical facts are forward-looking statements for purposes of the safe harbor provisions under the United States Private Securities Litigation Reform Act of 1995. Forward-looking statements are sometimes accompanied by words such as believe, may, will, estimate, continue, anticipate, intend, expect, should, would,plan,predict,potential,seem,seek,future,outlookandsimilarexpressionsthat predict or indicate future events or trends or that are not statements of historical matters. These forward-looking statements include, but are not limited to, statements regarding the potentialof the Companys JSP191 and mRNA engineered stem cell graft programs. Thesestatementsarebasedonvariousassumptions,whetherornotidentifiedinthispressrelease, and on the current expectations of Jasper and are not predictions of actual performance. These forward-lookingstatementsareprovidedforillustrativepurposesonlyandarenotintendedtoserve as, and must not be relied on by an investor as, a guarantee, an assurance, a prediction or a definitivestatementoffactorprobability.Actualeventsandcircumstancesaredifficultorimpossible to predict and will differ from assumptions. Many actual events and circumstances are beyond the control of Jasper. These forward-looking statements are subject to a number of risks and uncertainties, including general economic, political and business conditions; the risk that the potential product candidates that Jasper develops may not progress through clinical development or receive required regulatory approvals within expected timelines or at all; risks relating to uncertainty regarding the regulatory pathway for Jaspers product candidates; the risk that prior study results may not be replicated; the risk that clinical trials may not confirm any safety, potency or other product characteristics described or assumed in this press release; the risk that Jasper will be unable to successfully market or gain market acceptance of its product candidates; the risk that Jaspers product candidates may not be beneficialtopatientsorsuccessfullycommercialized;patientswillingnesstotrynewtherapiesand the willingness of physicians to prescribe these therapies; the effects of competition on Jaspers business; the risk that third parties on which Jasper depends for laboratory, clinical development, manufacturing and other critical services will fail to perform satisfactorily; the risk thatJaspers business, operations, clinical development plans and timelines, and supply chain could be adversely affected by the effects of health epidemics, including the ongoing COVID-19 pandemic; the risk that Jasper will be unable to obtain and maintain sufficient intellectual property protection foritsinvestigationalproductsorwillinfringetheintellectualpropertyprotectionofothers;andother risks and uncertainties indicated from time to time in Jaspers filings with the SEC. If any of these risksmaterializeorJaspersassumptionsproveincorrect,actualresultscoulddiffermateriallyfrom the results implied by these forward-looking statements. While Jasper may elect to update these forward-lookingstatementsatsomepointinthefuture,Jasperspecificallydisclaimsanyobligation to do so. These forward-looking statements should not be relied upon as representing Jaspers assessmentsofanydatesubsequenttothedateofthispressrelease.Accordingly,unduereliance should not be placed upon the forward-lookingstatements.

Contacts:

John Mullaly (investors)LifeSci Advisors617-429-3548jmullaly@lifesciadvisors.com

Jeet Mahal (investors)Jasper Therapeutics650-549-1403jmahal@jaspertherapeutics.com

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Jasper Therapeutics Announces Management Changes to Strengthen Leadership Team - BioSpace

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The Incredible Story of Emily Whitehead & CAR T-Cell Therapy : Oncology Times – LWW Journals

By daniellenierenberg

Emily Whitehead:

Emily Whitehead

Warriors come in all shapes and sizes. Take for example Emily Whitehead, as fresh-faced a 16-year-old as has ever graced the planet. Her eyes nearly sparkle with intellectual curiosity and dreams for a fulfilling future. But Emily is not a typical teen. She is the first pediatric patient in the world to receive CAR T-cell therapy for relapsed/refractory acute lymphoblastic leukemia (ALL). She is a singular figure in the annals of medicine. She is a soldier on the front lines of the war on cancer. And like the shot heard round the world, her personal medical assault sparked a revolution in cancer care that continues to power forward.

It has been 10 years since the only child of Thomas and Kari Whitehead of Philipsburg, PA, received an infusion of CAR T cells at the hands of a collaborative medical team from the Children's Hospital of Philadelphia (CHOP) and the Hospital of the University of Pennsylvania. That team included, among others, luminary CAR T-cell therapy pioneer, Carl June, MD, the Richard W. Vague Professor in Immunotherapy in the Department of Pathology and Laboratory Medicine and Director of the Center for Cellular Immunotherapies at Penn's Perelman School of Medicine; as well as Stephan Grupp, MD, PhD, Professor of Pediatrics at the Perelman School of Medicine (at that time, Director of the Cancer Immunotherapy Program at CHOP) and now Section Chief for Cell Therapy and Transplant at the hospital. He had been working with June on cell therapies since 2000.

Tremendous progress has flowedgushedfrom the effort to save Emily Whitehead; many more lives have been saved around the globe since that fatefulyet nearly fatalundertaking. While all the progress that has come from this story must be our ultimate theme, it cannot be fully appreciated without knowing how it came to be.

In 2010, Emily, then 5 years old, went from a being a healthy youngster one day, to a child diagnosed with ALL. Chemotherapy typically works well in pediatric ALL patients; Emily was one of the exceptions. After 2 years of intermittent chemotherapy, she continued to relapse. And when a bone marrow transplant seemed the only hope left, her disease was out of control and the treatment just wasn't possible. The Whiteheads were told by her medical team in Hershey, PA, nothing more could be done. They were instructed to take Emily home where she could die peacefully, surrounded by family.

But peaceful surrender didn't interest the Whiteheads; they rejected any version of giving up. It ran contrary to Tom Whitehead's vision of her recovery, something he said was revealed to him in the whispers. He saw, in a prophetic whispering dream, that Emily would be treated in Philadelphia. More importantly, he saw she would survive. It is as if it happened yesterday, said Tom, remembering how unrelentingly he called doctors at CHOP and said, We're coming there, no matter what you can or cannot do. We're not letting it end like this.

Since we treated Emily, we have treated more than 420 patients with CAR T cells at CHOP. She launched a whole group to be treated with this therapy; thousands have been treated around the world.Stephan Grupp, MD, PhD

A combination of persistence and perfect timing provided the magic bullet. It was just the day before that CHOP received approval to treat their first pediatric relapsed/refractory ALL patient with CAR T cells in a trial. And standing right there, on the threshold of history, was that deathly sick little girl named Emily.

At that time, only a scant few terminal adult patients had ever received the treatment, which is now FDA-approved as tisagenlecleucel and developed in cooperation with CHOP and the University of Pennsylvania. When three adults were treated, two experienced quick and complete remission of their cancers. Could CAR T-cell therapy perform a miracle for Emily? A lot would ride on the answer.

On March 1, 2012, Emily was transferred to CHOP and a few days later an apheresis catheter was placed in her neck; her T cells were extracted and sent to a lab. Emily received more chemotherapy, which knocked out her existing immune system, and she was kept in isolation for 6 weeks. Waiting.

Finally, over 3 days in April, Emily's re-engineered T cells, weaponized with chimeric antigen receptors, were infused back into her weakening body. But Emily did not rise like a Phoenix from the ashes of ALL. Instead, she sunk into the feverish fire of cytokine release syndrome (CRS), and experienced a worse-than-anticipated reaction. The hope for a swift victory seemed to be disappearing.

I can still see Emily's blood pressure dropping down to 53/29, her fever going up to 105F, her body swelling beyond recognition, her struggle to breathe, said Tom, of the most nightmarish period of his life. Doctors induced a coma, and Emily was put on a ventilator. For 14 days, her death seemed imminent. Doctors told us Emily had a one in a thousand chance of surviving, said Tom. They said she could die at any moment. But she didn't.

Medical team members who fought alongside the young patient are unwavering heroes in Emily's story. But at the time of her massive struggle, they too were exhausted and battle-scarred, descending into the quicksand of what could have been a failing trial, grasping for some life-saving branch of stability. They knew if CRS could be overcome, the CAR T cells might work a miracle as they had done for those earlier adult patients. But the CRS was severe. There was no obvious antidote; time was running out.

I recall Dr. June saying he believed Emily was past the point where she could come back and recover, said her father. And he said if she didn't turn around, this whole immunotherapy revolution would be over.

The Whiteheads enjoy Penn State football games not far from their hometown. The family has often taken part in Penn State's THON, a 48-hour dance marathon that raises funds for childhood cancer.

June confirmed to Oncology Times that he and Grupp believed Emily would not survive the night. It was mentioned to the Whiteheads that perhaps they should just concentrate on comfort care measures and stop all the ICU interventions, he recalled. I believed she was going to die on the trial due to all the toxicity. I even drafted a letter to our provost to give a heads up.

When the first patient in a trial dies, that's called a Grade 5 toxicity, June noted. That closes the trial as well. It goes right into the trash bin and you have to start all over again. But fortunately, that letter never left my outbox. We decided to continue one more day, and an amazing event happened.

Grupp, offering context to the mysterious amazing event, said it was clear that Emily's extreme CRS was caused by the infusion of cells that he himself had placed in her fragile body. He said he felt an enormous sense of responsibility and incredible urgency as he watched the child struggle to live.

It was not until the CHOP/Penn team received results from a test profiling cytokines in Emily's body that a new flicker of hope sparked. Though Emily had many cytokine abnormalities, the one most strikingly abnormal, interleukin-6 (IL-6), caught the team's attention. It is not made by T cells, and should not have been part of the critical mix. Though there were very few cytokines that had drugs to target them individually, IL-6 was one that did. So the doctors decided to repurpose tocilizumab, an arthritis drug, as a last-ditch effort at saving their young patient.

We treated Emily with tocilizumab out of desperation, June admitted. Steve [Grupp] has told me that when he went to the ICU with tocilizumab as a rescue attempt for CRS, the ICU docs called him a cowboy. The ICU docs had given up hope for Emily. But she turned aroundunbelievably rapidly. Today, tocilizumab is the standard of care for CRS, and the only drug approved by the FDA for that complication. Emily's recovery was huge for the entire field.

Grupp reflected on the immensity of the moment. If things had gone differently, if Emily had experienced fatal toxicity, it would have been devastating to her family and to the medical team. And it might have ended the whole research endeavor. It would have set us back years and years. The impact that Emily and her family had on the field is nothing short of transformational, he declared.

Since we treated Emily, we have treated more than 420 patients with CAR T cells at CHOP. She launched a whole group to be treated with this therapy; thousands have been treated around the world, Grupp noted. And, if not for Emily, we wouldn't be in the position we are in todaywith five FDA-approved [CAR T-cell] products: four for adults and one for kids. And I think it also important to point out that the very first CAR-T approval, thanks to Emily, was in pediatric ALL.

June noted that between 2010 and the time of Emily's treatment in 2012, My work was running like a shoestring operation. I had to fire people because I couldn't get grants to support the infrastructure of the research. It was thought there was no way beyond an academic enterprise to actually make customized T cells, then mail and deliver them worldwide, he recalled.

But then everything changed. We experienced that initial success; it was totally exciting. It was a career-defining moment and the culmination of decades of research. It led to a lot of recognition, both for my contribution and for the team here at the University of Pennsylvania and at CHOP.

Today, hundreds of pharmaceutical and biotech companies are developing innovations. Hundreds of labs are making next-generation approaches to improve in this area, June noted. Today, I'm a kid in a candy shop because all kinds of things are happening. We have funding thanks to the amazing momentum from Emily. She literally changed the landscape of modern cancer therapy.

Grupp said the continuing CAR T-cell program at CHOP offers evidence of success in a broad perspective. There are two things to look at, he offered. The first is how well patients do with their therapy in terms of getting into remission. A month after getting their cells, are they in remission or not? A study with just CHOP patients showed that more than 90 percent met that bar (N Engl J Med 2014; doi: 10.1056/NEJMoa1407222). Worldwide, the numbers appear to be in the 80 percent range (N Engl J Med 2018; doi: 10.1056/NEJMoa1709866). So, I would say it is a highly successful therapy.

We now have trials using different cell types, like natural killer cells, monocytes, and stem cells, noted Carl June, MD, at Penn's Perelman School of Medicine. An entirely new field has opened because of our initial success. This is going to continue for a long time, making more potent cells that cover all kinds of cancer.

The other big question, Grupp noted: How long does remission last? We are probably looking at about 50 percent of patients remaining in remission long-term, which is to say years after the infusion. The farther out we go, the fewer patients there are to look at because it just started with Emily in 2012, reminded Grupp. We have Emily now 10 years out, and other patients who are at 5, 6, 7, 8 years out, but most were treated more recently than that. We need to follow them longer.

June said registries of patients treated with CAR T-cell therapy are being kept worldwide by various groups, including the FDA. CAR T-cell therapy happened fastest in the U.S., but it's gained traction in Japan, Europe, Australia, and they all have databases. The U.S. database for CAR T cells will probably be the best that exists, because the FDA requires people treated continue follow-up for at least 15 years, he explained.

This will provide important information about any long-term complications, and the relapse rate. If patients do get cancer again, will it be a new one or related to the first one we treated? We will follow the outcomes, he noted. Clinicians are teaching us a lot about how to use the informationat what stage of the disease the therapy is best used, and which patients are most likely to respond. This can move us forward.

June mentioned that Grupp is collaborating with the Children's Oncology Group ALL Committee led by Mignon Loh, MD, at the University of California in San Francisco.

They are conducting a national trial to explore using CAR T cells as a frontline therapy in newly diagnosed patients, he detailed. Emily was treated when she had pounds and pounds of leukemia in her body; ideally we don't want to wait so long. There are a lot of reasons to believe it would work as a frontline therapy and spare patients all the complications of previous chemotherapy and/or radiation. The good news is that the clinical trial is under way, and I suspect we may know the answer within 2 years.

The only true measure of success in Emily's case is the state of her health. When asked if she is considered cured, June said, All we can do is a lot of prognostication. We know with other therapies in leukemia, the most similar being bone marrow transplants, if you go 5 years without relapsing, basically you are considered cured. We don't know with CAR T cells because Emily is the first one. We have no other history. But she's at a decade now, and in lab data we cannot find any leukemia in her. So by all of the evidence we haveand by looking in the magic eight ballI believe Emily is cured.

One might think that going through such a battle for life would be enough for any one person, any one family. But for Emily and her parents, her survival was just the beginning of a larger assault. All of them saw the experience as a way to provide interest in continuing research, education for patients as well as physicians, and an extension of hope to other patients about to succumb to a cancerous enemy.

Tom thought back to one particular occasion, all those years ago, when Emily finally slept peacefully through the night in her hospital bed. I should have felt nothing but relief, but I heard a mother crying in the hallway. Her child, who has been in the room next door, had died that morning, he recalled. I am constantly reminded of how fortunate we are. There are so many parents fighting for their children who do not have a good outcome.

As soon as Emily regained her strength and resumed normal childhood activities, the family began travelling with members of the medical team, joining in presentations at meetings and conferences throughout the world. They wanted to give a human face to the potential of CAR T-cell therapy, and as such they willingly became a powerful tool to raise understanding and essential research dollars. In 2016, the Whiteheads founded the Emily Whitehead Foundation (www.emilywhiteheadfoundation.org) ...to help fund research for new, less toxic pediatric treatments, and to give other families hope.

We decided to hold what we called the Believe Ball in 2017. We asked lots of companies to sponsor a child who had received CAR T-cell treatment to come with their family to the ball at no cost to them. Each company's representative would be seated with the child and family they sponsored, and would meet the doctors and scientists involved in the research, as well as members of industry and pharma, to see exactly where research dollars are going. We implored these companies to move the cancer revolution forward with sponsorship. When it all shook out, we had around 35 CAR T-cell families together for the first time, said Tom.

He noted proudly that since the foundation's debut, donations have been consistent and now have totaled an impressive $1.5 million.

When the Emily Whitehead Foundation had a virtual gala recently, it awarded a $50,000 grantthe Nicole Gularte Fight for Cures Ambassador Awardto a young researcher working to get another trial started. The award is named for a woman who found her way to CAR T-cell trials at Penn through the Whitehead Foundation. The treatment extended her life by 5 years during which time Gularte became an advocate for other cancer patients, travelled with the Whiteheads, and made personal appearances whenever she thought she could be of help or inspiration. Eventually, she would relapse and succumb, but she assured Tom Whitehead, These were 5 of the best years of my life. I think my time here on Earth was meant to help cancer research move forward.'

While raising funds for progress is important, the Whiteheads' work is not just about bringing in money. It's also about education.

We want to send a message to all oncologists; they need to be more informed about these emerging treatments when their patients ask for help, Tom noted. In the beginning of CAR T-cell therapy, a lot of doctors were against it. It's hard to believe, but some still are, though not as much. We need more education so that oncologists give patients a chance to get to big research hospitals for cutting-edge treatments before everything else has failed.

June said he regularly interacts with patients Tom or the foundation refer to him. Such unawareness happens with all new therapies, he noted. The people most familiar with them are at academic medical centers. But only about 10 percent of patients actually go to academic centers, the rest are in community centers where newer therapies take much longer to roll out, he explained.

So much of Emily's life has been chronicled through the eyes of observers. But since her watershed medical intervention, she has grown into a well-travelled, articulate young woman who talks easily about her life. I used to let my father do all the talking, but I am finding my own voice now, she said, having granted an interview to Oncology Times.

I'm currently 16 years old and I'm a junior at high school. Just like when I was younger, cows are my favorite animals, she offered with a laugh. I still love playing with our chihuahua, Luna. In school, I love my young adult literature class because I really like reading. Besides that, I like art and film. And I'm in really good health today.

She mentioned her health casually, almost as an afterthought. I really don't have any memory of my treatment at this point, she revealed, but, the experiences that I've had since then have really shaped who I am. Traveling is a huge part of my life now and something I look forward to. We've been to conferences at a lot of distant places. I'm so grateful that I get to travel with my family and make these memories that I will have forever, while still being able to advocate for less toxic treatment options and raising money for cancer research. All of that is really important to me.

Reminded that she has already obtained fame as pediatric patient No. 1 for CAR T-cell therapy, Emily considered her status for a moment then commented, I don't really like to base the progress of the therapy on my story and what I went through. Instead, I like to take my experience and use it to advocate for all patients so that what happened to me does not have to be repeated and endured by another family. My hope is that CAR T-cell therapy will become a frontline treatment option and be readily available, so pediatric patients can get back to a normal life as soon as possible. I want to tell people if conventional treatments do not work, other options do exist. Overall, I am grateful that I can encourage others to keep fighting. That's the main thing; I am grateful.

After a brief pause, Emily continued, I always tell oncologists and scientists that the work they are doing is truly saving children's lives. It allows these kids to grow up, be with their friends and families, take vacations, play with their dogs, and someday go to college, just like me. They are not only saving patients' lives, they are saving families. The work they do does not go unnoticed or unappreciated. Again, I am really so grateful.

Appreciation is a two-way street, and June said he and his team appreciate and draw inspiration from Emily on a daily basis. Her picture hangs on the wall of our manufacturing center, June stated. Some of the technicians who were in high school when Emily was infused are now manufacturing CAR T cells. They learned so much from Emily's experience; she continues to be a big motivator. She's helped my team galvanize and see that the work can really benefit people.

Grupp said the success that is embodied in Emily Whitehead has spurred additional successes, and new inroads in CAR T-cell therapy. There are more applications now, especially in other blood cancerslymphoma and myeloma, in addition to leukemia. We've seen a lot of expansion there.

He noted a national trial is under way for an FDA-approved therapy called idecabtagene vicleucel, which can benefit multiple myeloma patients. All other CAR Ts target the same target, CD19. But this goes after an entirely different target, BCMA. The fact that we now have approval in something that isn't aimed at CD19 is very exciting. And there are others coming right behind it.

The field also has seen further expansion ...into adults being treated safely, because initially there was concern that these drug therapies were too powerful for safe treatment in older adults, detailed Grupp. Now we know that is clearly not the case, and that is great news, particularly because multiple myeloma most often occurs in people over 60.

The use of CAR T cells in solid tumors continues to be challenging, although Grupp noted, We have certainly seen hints of patients with solid tumors having major responses and going into remission with CAR T cells. It is still a small handful of patients, so we haven't perfected the recipe for solid tumors yet. But I am absolutely confident we will have the answers in a very short numberperhaps 2-4of years.

June said, since Emily's infusion, CAR T cells have matured and gotten better. There are many ways that has happened, he informed. We have different kinds of CAR designs to improve and increase the response rates, to decrease the CRS, or to target other kinds of bone marrow cancers. One that is not curable with a lot of therapies is acute myeloid leukemia (AML), so we have a huge group at Penn and CHOP working on AML specifically. And there is the whole field of solid cancer; we have teams working on pancreatic, prostate, breast, brain, and lung cancer now.

In addition to targeting different types of cancer, June said contemporary research is also exploring the use of different types of cells. Our initial CAR T trial used T cells, and that is what all the FDA-approved CARs are. But we now have trials using different cell types, like natural killer cells, monocytes, and stem cells. An entirely new field has opened because of our initial success. This is going to continue for a long time, making more potent cells that cover all kinds of cancer, not just leukemia and lymphoma.

Is this the beginning of the end of cancer? Is this that Holy Grail called a cure to cancer? It's a question June has pondered.

Some people do think that, he answered. They believe the immune system is the solution. And that's a huge statement. President Biden has made a big investment in this work, with the Cancer Moonshot. He's accelerated this research at the federal level. But we just don't know how long it is going to take. Fortunately, a lot of good minds are working hard to make an end to cancer a reality.

As the battle grinds on, June said he applies something he's learned over time, with reinforcement from Tom and Kari Whitehead. They were bulldogs. When it came to getting treatment for Emily, they just wouldn't take no for an answer. They demonstrated the importance of never giving up. That's what happened; they would not surrender. I think that is why Emily is alive today.

Valerie Neff Newitt is a contributing writer.

The Emily Whitehead Foundation and the Whitehead family take extraordinary advantage of a variety of media to reach patients and physicians and optimize educational opportunities.

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The Incredible Story of Emily Whitehead & CAR T-Cell Therapy : Oncology Times - LWW Journals

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A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy

By daniellenierenberg

Theranostics. 2020; 10(8): 36223635.

Institute of Molecular and Cell Biology, 61 Biopolis Drive, 138673, Singapore

Competing Interests: The authors have declared that no competing interest exists.

Received 2019 Oct 4; Accepted 2019 Dec 20.

The transcriptional co-regulators YAP and TAZ pair primarily with the TEAD family of transcription factors to elicit a gene expression signature that plays a prominent role in cancer development, progression and metastasis. YAP and TAZ endow cells with various oncogenic traits such that they sustain proliferation, inhibit apoptosis, maintain stemness, respond to mechanical stimuli, engineer metabolism, promote angiogenesis, suppress immune response and develop resistance to therapies. Therefore, inhibiting YAP/TAZ- TEAD is an attractive and viable option for novel cancer therapy. It is exciting to know that many drugs already in the clinic restrict YAP/TAZ activities and several novel YAP/TAZ inhibitors are currently under development. We have classified YAP/TAZ-inhibiting drugs into three groups. Group I drugs act on the upstream regulators that are stimulators of YAP/TAZ activities. Many of the Group I drugs have the potential to be repurposed as YAP/TAZ indirect inhibitors to treat various solid cancers. Group II modalities act directly on YAP/TAZ or TEADs and disrupt their interaction; targeting TEADs has emerged as a novel option to inhibit YAP/TAZ, as TEADs are major mediators of their oncogenic programs. TEADs can also be leveraged on using small molecules to activate YAP/TAZ-dependent gene expression for use in regenerative medicine. Group III drugs focus on targeting one of the oncogenic downstream YAP/TAZ transcriptional target genes. With the right strategy and impetus, it is not far-fetched to expect a repurposed group I drug or a novel group II drug to combat YAP and TAZ in cancers in the near future.

Keywords: TEAD, YAP, TAZ, Hippo, cancer therapy

The transcriptional co-regulators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are key players that mediate various oncogenic processes and targeting their activities has emerged as an attractive option for potential cancer therapy. YAP, as the name suggests, was initially identified as a protein that associates with Yes, a src family kinase (SFK) 1. The exact function of YAP remained elusive until it was demonstrated to be a potent transcriptional activator 2. YAP's paralog TAZ, identified from a screen for 14-3-3 interacting proteins, is also a transcriptional co-activator 3 (Figure ).

The oncogenic milestones of the transcriptional co-regulators YAP and TAZ. Discovery of YAP/TAZ and TEAD functions predate the discovery of the Hippo pathway. Role of YAP/TAZ in the Hippo pathway and the discovery of their oncogenic abilities in cell and animal models are considered significant. The initial studies from the groups that linked YAP/TAZ to oncogenic signaling pathway, stemness, actin cytoskeleton, fusion genes, drug resistance, metabolism, angiogenesis and immune suppression are also listed.

YAP and TAZ do not have a DNA-binding domain and they need to associate with a transcription factor in order to access DNA. It has now emerged that YAP/TAZ use predominantly the TEAD (TEA domain) family of transcription factors 4 to elicit most of their biologically relevant gene expression programs. ChIP-Seq data unraveled a significant overlap in YAP/TAZ and TEAD peaks throughout the genome, and also showed that some YAP/TAZ-responsive genes are also synergistically regulated by AP-1 transcription factors 5, 6. In addition to its interaction with TEADs, YAP/TAZ also communicates with the mediator complex and chromatin modeling enzymes like the methyltransferase and SWI/SNF complex to elicit changes in gene expression 7-9. YAP/TAZ also suppress gene expression and should be regarded as co-regulators rather than co-activators 10.

YAP/TAZ are now considered as effectors of a physiologically and pathologically important signaling pathway - popularly called the Hippo pathway 11. The Hippo pathway was initially identified in a genetic mosaic screen in Drosophila but the pathway components are evolutionarily conserved. It is now known that the primary function of the Hippo pathway is to suppress the activity of Yorkie - the Drosophila homolog of YAP 12. The Hippo pathway in mammals also inhibits YAP/TAZ through phosphorylation by the large tumor suppressor (LATS) family of Hippo core kinases 13, which leads to cytoplasmic sequestration via interaction with 14-3-3 proteins and/or degradation via ubiquitin proteasome pathway 14, 15.

YAP and TAZ were first shown to transform mammary epithelial cells 16, 17. The oncogenic role of YAP became apparent when it was shown to be a driver gene in a mouse model of liver cancer 18 (Figure ). In a conditional transgenic mouse model, YAP overexpression dramatically increases liver size and the mouse eventually develops hepatocellular carcinoma 19, 20. In addition to causing primary tumor growth, YAP also helps in the metastatic dissemination of tumor cells 21.

Over a decade of research has revealed that YAP/TAZ integrates the inputs of various oncogenic signaling pathways, such as EGFR, TGF, Wnt, PI3K, GPCR and KRAS. Through expression of the ligand AREG, YAP was first shown to communicate with the EGFR pathway 22 (Figure ). The genes regulated by YAP/TAZ collectively coordinate various oncogenic processes, such as stemness, mechanotransduction, drug resistance, metabolic reprogramming, angiogenesis and immune suppression (Figure ), many of which are considered to be cancer hallmarks 23.

YAP and TAZ regulate the expression of crucial transcription factors like Sox2, Nanog and Oct4 and are able to maintain pluripotency or stemness in human embryonic stem cells (ESCs) and in induced pluripotent stem (iPS) cells 24, 25 (Figure ). More specifically, TAZ has been shown to confer self- renewal and tumorigenic capabilities to cancer stem cells 26. Within the microenvironmental landscape of tissues, YAP/TAZ are increasingly recognized as mechanosensors that respond to extrinsic and cell-intrinsic mechanical cues. To this end, mechanical signals related to extracellular matrix (ECM) stiffness, cell morphology and cytoskeletal tension rely on YAP/TAZ for a mechano-activated transcriptional program 27-29. YAP/TAZ target genes, CTGF and CYR61, cause resistance to chemotherapy drugs like Taxol 30 and YAP/TAZ has emerged as a widely used alternate survival pathway that is adopted by drug-resistant cancer cells 31. YAP/TAZ activity is regulated by glucose metabolism and is connected to the activity of the central metabolic sensor AMP-activated protein kinase (AMPK) 32-35. YAP/TAZ reprograms glucose, nucleotide and amino acid metabolism in order to increase the supply of energy and nutrients to fuel cancer cells 36. Through expression of proangiogenic factors like VEGF and angiopoetin-2 37, 38, YAP is able to stimulate blood vessel growth to support tumor angiogenesis 39. YAP is also shown to recruit myeloid-derived suppressor cells in prostate cancers in order to maintain an immune suppressive environment 40. Active YAP also recruits M2 macrophages to evade immune clearance 41.

A TAZ fusion gene (TAZ-CAMTA1) alone, in the absence of any other chromosomal alteration or mutation, is sufficient to drive epithelioid hemangioendothelioma (EHE), a vascular sarcoma 42, 43. Furthermore, comprehensive analysis of human tumors across multiple cancer types from the TCGA database unraveled that YAP and TAZ are frequently amplified in squamous cell cancers in a mutually exclusive manner 44. In human cancers, there is also a good correlation between YAP/TAZ target gene signature and poor prognosis. To date, a proportion of every solid tumor type has been shown to possess aberrant YAP/TAZ activity. Further, many of the upstream Hippo components that negatively regulate YAP/TAZ are found inactivated across many cancer types 45. Thus, all of this paint a clear picture of the prominent role played by YAP and TAZ at the roots of cancers 46, 47.

There are more than fifty drugs that have been shown to inhibit YAP/TAZ activity 48, however, with the exception of verteporfin; none act directly on YAP/TAZ. The unstructured nature of YAP and TAZ renders them difficult to target using small molecules. Therefore, YAP/TAZ inhibition is achieved indirectly through targeting their stimulators or partners. In this review, we focus on small molecules, antibody and peptide-based drugs, as the majority of the drugs in the clinic belong to this class. Less attention is given to nucleotide-based molecules and to small molecule YAP/TAZ inhibitors whose targets are unknown. We classify the YAP/TAZ-inhibiting drugs into three groups with each group having its own combating strategy to counter YAP/TAZ activity (Figure ). Group I drugs target the upstream YAP/TAZ stimulators and enhance the LATS-dependent inhibitory phosphorylation of YAP/TAZ in order to restrain their transcriptional output. Group II drugs/candidates act directly on YAP/TAZ or TEAD and may either interfere with the formation of the YAP/TAZ-TEAD complex or inhibit TEADs directly and hence affect YAP/TAZ-TEAD transcriptional outcomes. Group III drugs' combat strategy is to target the oncogenic proteins that are transcriptionally upregulated by YAP and TAZ.

Classification ofYAP/TAZ-TEAD inhibiting drugs into three groups. Group I drugs (red font) act upstream and prevent the nuclear entry of YAP and TAZ, group I drug targets for potential pharmacological exploitation in order to generate repurposed YAP/TAZ-inhibiting drugs are circled. Group II drugs (green font) disrupt the formation of the YAP/TAZ-TEAD complex and they primarily bind to the TEAD family of transcription factors. Group Ill drugs (blue font) act on the downstream transcriptional targets in order to prevent YAP/TAZ-mediated oncogenicity.

Group I drugs target the upstream proteins (Figure ), inhibition of which culminates in the enhancement of the LATS-dependent inhibitory phosphorylation of YAP/TAZ 49, 50. However, some group I targets like SFKs 51-53, AMPK 33, 34 and phosphatases 54-56 act directly on YAP and TAZ and activate them. Majority of group I drugs are kinase inhibitors, in addition to restricting YAP/TAZ nuclear entry; they intriguingly promote TAZ, but not YAP degradation. A possible explanation for this is the presence of two phosphodegrons that render TAZ more prone to degradation 15. Some group I drugs, such as MEK/MAPK inhibitors 57, 58 and -secretase inhibitors (GSIs) 59 have the ability to actively reduce both YAP and TAZ levels. HDAC inhibitors however, reduce YAP, but not TAZ levels 60. Here, we have classified the group I drugs based on the nature of the drug target.

Drugs targeting the EGFR, GPCR, Integrin, VEGFR and adenylyl cyclase families as well as those targeting receptors like the -secretase complex and Agrin are shown to inhibit YAP/TAZ activity 51, 61-64.

YAP/TAZ exploits the transformative abilities of the ErbB receptors (EGFR family) to drive cell proliferation. By transcribing ErbB ligands, such as AREG 22, 65, TGF- 66, NRG1 67 as well as the ErbB receptors EGFR and ErbB3 67, YAP is able to activate ErbB signaling and promote tumorigenesis. Sustained EGFR signaling also disassembles the Hippo core complexes leading to an increased active pool of YAP/TAZ 68 that is ready to transcribe more ErbB ligands/receptors. Under these conditions, EGFR inhibitors like Erlotinib 22 and AG-1478 66 (Figure ) are able to act as YAP/TAZ inhibitors and may be used for EGFR-driven cancers requiring YAP/TAZ transcription.

Signaling from G-protein coupled receptors (GPCRs), transduced by the associated G subunit or by the G subunits, modulates YAP/TAZ activities 69. Inhibiting Gq/11 sub-type signaling, using losartan 70, or stimulating Gs sub-type, using dihydrexidine, has been shown to stimulate YAP inhibitory phosphorylation 69. Agonism of Gs has been recently exploited to facilitate YAP/TAZ inhibition that reverses fibrosis in mice 71. G inhibition using gallein has also been shown to restrict YAP/TAZ 72. Activating mutations in the Gq/11 types of GPCRs present in approximately 80% of the uveal melanoma patients generate an active pool of YAP 73, 74 but the signal transduction occurs via Trio-Rho/Rac signaling and not through the canonical Hippo pathway 74.

Integrin signaling negatively regulates the Hippo pathway complexes to drive YAP/TAZ activity 75, 76. Although blocking integrin activity using RGD peptides 63, cilengitide (cyclic RGD peptide) 77, function-blocking antibodies - BHA 2.1 76 and clone AIIB2 78 has been shown to increase YAP/ TAZ's inhibitory phosphorylation, disappointingly, the efficacy of integrin- blocking drugs against cancers has not been clinically proven 79. Interestingly, a function-blocking antibody against Agrin, an extrinsic stimulator of integrin signaling, abrogates YAP-dependent proliferation in mouse models 63, 80.

Among the kinase inhibitors tested in a biosensor screen for LATS activity, the VEGFR inhibitors are shown to potently activate LATS and thereby inhibit YAP and TAZ activity 81. Further, VEGFR2 signaling is also shown to induce actin cytoskeletal changes and promote YAP/TAZ activation 82. Therefore, VEGFR inhibitors like SU4312, Apatinib, Axitinib and pazopanib are able to inhibit the expression of YAP/TAZ-responsive genes in endothelial cells. But whether these drugs work as YAP/TAZ inhibitors in cancer cells remains to be seen.

Enhancing cyclic AMP (cAMP) levels using the adenylyl cyclase activator forskolin activates the LATS kinases through Protein kinase A (PKA) and Rho 69, therefore forskolin is also a YAP/TAZ inhibitor. cAMP is degraded by the cyclic nucleotide phosphodiesterases (PDE), the use of PDE inhibitors like theophylline, IBMX, ibudilast and rolipram also promotes YAP/TAZ-inhibitory phosphorylation 83, 84.

Notch and YAP/TAZ signaling are also closely linked, inhibiting notch activity by targeting the -secretase complex, either using DAPT or dibenzazepine has been shown to decrease YAP/TAZ expression levels in mouse livers and also reduce YAP activation and YAP-induced dysplasia in the intestine 20, 51, 59.

Integrin signaling activates focal adhesion kinase (FAK), SFK and integrin- linked kinase (ILK). Growth factor and GPCR signaling occurs through mitogen-activated protein kinase (MAPK) and phosphoinositide 3-OH kinase (PI3K) signaling. There is also significant crosstalk in the signaling from these membrane receptors. Given the availability of potent small molecule drugs targeting the downstream kinases, they are leveraged on to inhibit YAP or TAZ activities.

Members of downstream integrin signaling pathway including FAK, its counterpart PYK2, and ILK have emerged as negative regulators of the core Hippo pathway and thus activate YAP/TAZ. Membrane receptors, such as ErbB and GPCRs are unable to activate YAP upon genetic deletion of ILK. Therefore, pharmacological inhibition of ILK using a specific ILK inhibitor, QLT0267 potently inhibits YAP-dependent tumor growth in xenograft models 85. The FAK inhibitors PF-562271 and PF-573228 have also been shown to enhance the LATS-mediated inhibitory phosphorylation of YAP 63, 75. A multi-kinase inhibitor CT-707 that predominantly inhibits FAK, anaplastic lymphoma kinase (ALK) and PYK2 is able to render cancer cells vulnerable to hypoxia through YAP inhibition 86. Inhibiting PYK2 activity using the dual PYK2/FAK inhibitor PF431396 destabilizes TAZ and also inhibits YAP/TAZ activity in triple negative breast cancer cells 87.

The SFK member Src prevents the activation of LATS 75, 88, thereby relieves YAP/TAZ inhibition by LATS. Interestingly, SFKs, Src and YES are also shown to activate YAP through direct tyrosine phosphorylation 51-53. Treating cells with SFK inhibitors, such as Dasatinib, PP2, SU6656, AZD0530 and SKI-1 inactivates YAP 51-53, 75, 88. In -catenin-driven cancers, YES facilitates the formation of a tripartite complex comprising -catenin, YAP and TBX5 that drives cell survival and tumor growth 53, 89. The SFK inhibitor dasatinib also serves as YAP inhibitor in these cancers 53. Dasatinib, in addition to inhibiting SFKs may also potently inhibit PDGFR and Ephrin receptors, both of which are known to activate YAP/TAZ 90, 91. However, FAK and SFK inhibitors have shown very limited efficacy against solid tumors in clinical trials therefore their utility in YAP-driven cancers remains to be seen.

MEK (MAP kinase kinase) and YAP interact with each other and maintain transformed phenotypes in liver cancer cells 57. MEK inhibitors PD98059, U0126 and trametinib or MAPK inhibitors CAY10561 and {"type":"entrez-nucleotide","attrs":{"text":"FR180204","term_id":"258307209","term_text":"FR180204"}}FR180204 are able to trigger degradation of YAP in a Hippo-independent manner 57, 58. The finding that MEK inhibition causes YAP degradation is, however, difficult to reconcile if YAP and TAZ are shown to mediate resistance to the MEK inhibitor trametinib 92. The efficacy of trametinib is also being evaluated in EHE, a cancer that is caused by the TAZ-CAMTA1 fusion gene ({"type":"clinical-trial","attrs":{"text":"NCT03148275","term_id":"NCT03148275"}}NCT03148275).

PI3K inhibitors Wortmannin/LY294002 as well as the drug BX795, an inhibitor of its effector 3'-phosphoinositide-dependent kinase-1 (PDK1) prevents nuclear entry of YAP 68. PI3K is closely linked to the mammalian target of rapamycin (mTOR) pathway. mTOR inhibitors temsirolimus and MLN0128 have been shown to inhibit YAP activity in patients with idiopathic pulmonary fibrosis and in a mouse model of cholangiocarcinoma, respectively 93, 94. YAP levels in TSC1 mutant mouse could also be reduced by blocking mTOR using torin1 treatment that induces the autophagy-lysosomal pathway 95.

YAP/TAZ inhibition is an additional unexpected activity possessed by the few kinase inhibitors mentioned above. However, apart from YAP/TAZ inhibition, all other signaling events initiated by the target kinase are also shut down due to inhibitor treatment. If these signaling events are critical for cellular homeostasis, then, toxic side effects will outweigh clinical benefits and this cannot be uncoupled from YAP/TAZ inhibition. Therefore, kinase inhibitors that failed in the trials due to unacceptable toxcity or poor pharmacokinetics may not be repurposed as YAP/TAZ inhibitors in the clinic. Focus should be on the kinase inhibitors that are already in the clinic like EGFR, VEGFR, MEK, PI3K or mTOR inhibitors but efficacy needs to be proven in order to repurpose them as YAP/TAZ inhibitors. The kinase targeted by the inhibitor must activate YAP/TAZ in tumors, for the treatment to be efficacious and this restricts the use of kinase inhibitors to selective tumor types. Intriguingly, YAP/TAZ activation has emerged as a prominent survival strategy adapted by cancers that cause drug resistance to EGFR and its downstream MEK/MAPK inhibitors 31. In such scenarios, coupling a group II YAP/TAZ inhibitor with a EGFR pathway inhibitor might offer the intended treatment benefits.

The mevalonate pathway is essential for the biosynthesis of isoprenoids, cholesterol and steroid hormones. Statins as well as other mevalonate pathway inhibitors like zoledronic acid and GGTI-298 that target farnesyl pyrophosphate synthase and geranylgeranyltransferase, respectively are identified as drugs that restrict the nuclear entry of YAP and TAZ 96, 97. Studies have also shown that combining statins like simvastatin with the EGFR inhibitor gefitinib provides stronger anti-neoplastic effects 98. Atorvastatin and zoledronic acid have entered Phase II clinical trials in triple negative breast cancer to test if they improve the pathological complete response rates ({"type":"clinical-trial","attrs":{"text":"NCT03358017","term_id":"NCT03358017"}}NCT03358017).

Actin polymerization promotes YAP/TAZ nuclear localization and therefore, polymerization inhibitors like latrunculin A 27 and cytochalasin D 28, 29 inhibit YAP/TAZ. Myosin or myosin light-chain kinase inhibitors like blebbistatin and ML-7, respectively have a similar effect 27, 29. Interfering with the actomyosin cytoskeleton through other means, such as Rho inhibition (toxin C3 treatment), or by using Rho kinase inhibitors like Y27632 has also been shown to have an inhibitory effect on YAP/TAZ 27, 29. p21 activated kinase (PAK) family kinases are cytoskeletal regulators as well as Hippo inhibitors. The PAK allosteric inhibitor IPA3 prevents YAP's nuclear entry 63, 99, further, the PAK4 inhibitor PF-03758309 is also shown to reduce YAP levels 77.

YAP/TAZ inhibitory phosphorylation is dynamic and the protein phosphatases PP1 and PP2A are shown to associate with YAP/TAZ and aid in their dephosphorylation and activation. Inhibiting these phosphatases using okadaic acid or calyculin A increases YAP/TAZ phosphorylation and shifts YAP/TAZ to the cytoplasm 54-56. Some of the oncogenic functions of YAP/TAZ are also mediated by the protein-tyrosine phosphatase SHP2 100, therefore SHP2 inhibitors have also been shown to attenuate YAP/TAZ activity 101.

Cellular energy stress is closely linked with attenuation of YAP/TAZ activities 32. Drugs that enhance energy stress like the mitochondrial complex I inhibitors metformin and phenformin, enhance YAP/TAZ inhibitory phosphorylation, cytoplasmic localization and suppression of YAP/TAZ- mediated transcription 32. The energy stress induced by these drugs activates AMPK, which is shown to phosphorylate and stabilize AMOTL1 - a YAP/TAZ negative regulator 32. AMPK is also shown to directly phosphorylate and inactivate YAP by disrupting its interaction with TEADs 33, 34. Therefore, AMPK activators A769662 and AICAR (an AMP-mimetic) are YAP inhibitors 32-34.

Histone deacetylases (HDACs) are uniquely positioned to alter the transcription of target genes. Interestingly, HDAC inhibitors panobinostat, quisinostat, dacinostat, vorinostat and Trichostatin A transcriptionally repress the expression of YAP but not TAZ, and thereby reduce YAP-addicted tumorigenicity 60. Treatment of cholangiocarinoma cells with the HDAC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"CG200745","term_id":"34091806","term_text":"CG200745"}}CG200745 is also shown to decrease YAP levels 102. Although HDAC inhibitors are used to treat hematological malignancies their efficacy in solid cancers is questionable, however, combining HDAC inhibitor panobinostat with BET (bromodomain and extra-terminal) inhibitor I-BET151 achieves more effective YAP inhibition 103. There is also a clinical trial designed to evaluate the efficacy of HDAC/BET inhibitor combination in solid tumors and determination of YAP expression level after drug treatment is used as one of the objectives ({"type":"clinical-trial","attrs":{"text":"NCT03925428","term_id":"NCT03925428"}}NCT03925428). The BET family protein BRD4 is a part of the YAP/TAZ-TEAD transcriptional complex and inhibiting BRD4 using BET inhibitor JQ1 inhibits YAP upregulation and YAP-mediated transcription in KRAS mutant cells 104.

Many group I drugs can potentially be repurposed to treat YAP/TAZ- driven cancers 105. Among the group I drugs, only statins, trametinib and HDAC/BET inhibitors are being evaluated in clinical trials to test if they act against YAP/TAZ. Our prediction is that group I drugs that facilitate YAP/ TAZ inhibitory phosphorylation as well as degradation will have greater success in combating YAP/TAZ in cancers as YAP/TAZ degradation prevents their reactivation through other mechanisms. Importantly, the repurposing of group I drugs would also allow YAP/TAZ and its target gene(s) expression-based stratification amongst cancer patients.

Modalities that target either the TEAD family of transcription factors or YAP/TAZ are classified under this group (Figure ). The majority of the modalities, with the exception of verteporfin 106, target TEADs and are therefore predicted to act in the nucleus. By pairing with the TEAD family of transcription factors, YAP and TAZ upregulate the expression of many oncoproteins. The C-terminus of all TEADs possesses the YAP/TAZ-binding domain. The partnership between YAP/TAZ and TEAD is essential for the initiation of transcriptional program to drive oncogenesis. YAP is no longer oncogenic when sequestered by a dominant negative TEAD that lacks the DNA-binding domain 106. Similarly, a naturally occurring DNA-binding deficient TEAD isoform is also able to inhibit YAP/TAZ-mediated oncogenicity 107. Therefore, directly inhibiting TEAD or preventing YAP/TAZ-TEAD interaction is a promising and most direct strategy that warrants special attention 108.

Disruptors, stabilizers and destabilizers/degraders. A preformed YAP/TAZ-TEAD complex prevents access to drugs that occupy either the TEADs' surface or the palmitate-binding pocket (PBP), however, unassembled TEADs are accessible to drugs. Majority of the known YAP/TEAD-binding compounds are disruptors as they prevent the formation of the YAP/TAZ-TEAD complex. Two other classes of TEAD-binding compounds are stabilizers and destabilizers/degraders. Stabilizers either stabilize TEAD expression levels or enhance the formation of the YAP/TAZ-TEAD complex. Destabilizers bind to TEADs' surface or PBP and reduce TEAD expression levels through in situ denaturation, degraders on the other hand direct TEADs for proteasomal degradation.

Group I drugs target the upstream YAP/TAZ-activating proteins like the EGFR, GPCR, Src, or Integrins. As there are so many upstream YAP/TAZ activators, group I drugs are vulnerable to oncogenic bypass where inhibition of one group I YAP/TAZ activator leads to selection of cancer cells that activate YAP/TAZ via another group I activator. Strategically, Group II drugs may address this issue by directly targeting YAP/TAZ or TEAD, the converging points for various pathways and also the effectors for oncogenic transcription. However, Group II targeting modalities are still at the exploratory stage and it remains to be seen whether it is feasible to develop a Group II modality that works in clinic. We also need to be mindful of the possible associated toxicities due to YAP/TAZ-TEAD inhibition 109.

Most of the reported Group II modalities are disruptors; they target YAP/TAZ or TEAD and prevent their binary interaction. However, in addition to disruptors, in the future, we predict the emergence two other classes of group II compounds that would act as TEAD stabilizers and destabilizers/degraders (Figure ).

A small molecule benzoporphyrin drug named Verteporfin (VP) was shown to have the ability to bind to YAP and disrupt the YAP-TEAD interaction 106. VP is also able to inhibit YAP-induced excessive cell proliferation in YAP- inducible transgenic mice and in NF2 (upstream Hippo pathway component) liver-specific knockout mouse models 106. Although we do not understand the molecular details of VP binding to YAP, it is still undoubtedly the most popular YAP inhibitor within the scientific community. However, we need to be cautious as some of the tumor-inhibitory effects of VP are reported to be YAP- independent 110, 111. VP is photosensitive and proteotoxic and there is a need for better derivatives. A VP derivative, a symmetric divinyldipyrrine was shown to inhibit YAP/TAZ-dependent transcription but it is not clear if the compound specifically binds to YAP 112.

YAP and TAZ bind on the TEADs' surface; Inventiva Pharma has identified several compounds with benzisothiazole-dioxide scaffold that bind to the TEADs' surface and disrupt the YAP/TAZ-TEAD interaction. These compounds are currently in the lead optimization stage and have the potential to treat malignant pleural mesothelioma as well as lung and breast cancers that are driven by YAP/TAZ 113.

YAP cyclic peptide (peptide 17) and cystine-dense peptide (TB1G1) are also disruptors of YAP/TAZ-TEAD interaction in vitro but they have poor cell-penetrating abilities 114, 115. Interestingly, a peptide derived from the co-regulator Vgll4 appears to have remarkable cell-penetrating abilities and inhibits YAP-mediated tumorigenesis in animal models 116. Vgll proteins, named Vgll1-4 in mammals, belong to another class of co-regulators that pair with TEADs in a structurally similar, and therefore, in a mutually exclusive manner with YAP and TAZ 117, 118.

We identified a novel druggable pocket in the center of the TEADs' YAP/TAZ- binding domain 119 that could be occupied by fenamate drugs. Palmitate was subsequently shown to occupy this pocket, hereafter referred to as the palmitate-binding pocket (PBP). TEAD palmitoylation is shown to be important for stability and for the interaction with YAP 120, 121. Although the fenamate drug flufenamic acid competes with palmitate for binding to TEAD, higher concentrations are needed for it to be effective and it is not a disruptor of the interaction between YAP/TAZ with TEADs 122. However, covalently linking the fenamate to TEAD, using a chloromethyl ketone substitution, enables it to disrupt the YAP-TEAD interaction 123. The non-fused tricyclic compounds identified by Vivace Therapeutics could also be considered as fenamate analogs but it remains to be seen if they function as disruptors 124. Through structure-based virtual screen, vinylsulfonamide derivatives were identified as compounds that bind to PBP 125. Optimization of these derivatives yielded DC-TEADin02 a covalent TEAD autopalmitoylation inhibitor with an IC50 value of 200 nM. Interestingly, DC-TEADin02 is able to inhibit TEAD activity without disrupting the YAP-TEAD interaction.

Palmitate, by occupying the PBP, allosterically modulates YAP's interaction with TEAD 121, therefore it is conceivable that there might be small molecules that occupy the PBP and allosterically disrupt YAP/TAZ's interaction with TEADs. To this end, Xu Wu's group has identified and patented several potent compounds with alkylthio-triazole scaffold as PBP- occupying compounds that prevent YAP-TEAD interaction in cells 126. Another potent TEAD inhibitor that occupies the PBP is the small molecule K-975 127. K-975 also disrupts the YAP-TAZ-TEAD interaction and displayed anti-tumorigenic properties in malignant pleural mesothelioma cell lines much akin to the loss of YAP. Although palmitate is covalently attached to TEAD, it is a reversible modification and addition of PBP-occupying small molecules reduce the cellular palmitoylation status of TEADs 126. Moreover, the palmitoyl group is also removed from TEADs by depalmitoylases 128.

Being predominantly unstructured, YAP and TAZ are difficult to target directly. However, TEADs offer two attractive ways for targeting, one is to directly block the YAP/TAZ-binding pocket on the TEADs' surface with small molecules or peptides, whilst the other is to leverage on the PBP and allosterically disrupt YAP/TAZ interaction or inhibit TEADs (Figure ). However, the molecular determinants that confer YAP/TAZ disrupting ability to PBP-occupying small molecules are not clear. We do not know why flufenamate and DC-TEADin02 are unable to disrupt YAP/TAZ-TEAD interaction, like chloromethyl fenamate, K-975 and compounds with alkylthio-triazole scaffold.

The PBP could also be leveraged to allosterically enhance YAP/TAZ-TEAD stability or interaction. This prediction is subject to the identification of small molecules that functionally mimic the ligand palmitate (Figure ). Compounds with such an ability will enhance TEAD-dependent transcription and may have therapeutic value for regenerative medicine where enhancement of YAP/TAZ- TEAD activity is needed to repair damaged tissues 129. We recently identified that quinolinols occupy the PBP, stabilize YAP/TAZ levels and upregulate TEAD-dependent transcription 130. Enhanced YAP/TAZ levels increase the pool of assembled YAP/TAZ complex and therefore quinolinols could be considered as stabilizers (Figure ).

We identified a few chemical scaffolds that have the ability to occupy the PBP and destabilize TEAD (unpublished results). Addition of these compounds unfolds the TEADs' YAP/TAZ-binding domain and we call these compounds destabilizers (Figure ). Degraders could be generated when potent and selective TEAD surface or PBP-occupying compounds are coupled to proteolysis targeting chimera (PROTAC) 131 to direct TEAD proteasomal degradation. Therefore, destabilizers aim to reduce the cellular concentration of TEADs through in situ unfolding and degraders reduce TEAD levels through proteasomal degradation. Reducing the levels of their interacting partners deprives YAP/TAZ of their ability to activate transcription.

Any TEAD-binding compounds (disruptors, stabilizers or destabilizers/degraders) can only access unbound TEADs, as binding of YAP and TAZ blocks both the surface and the palmitate-binding pockets (Figure ). After accessing unbound TEADs, the disruptors and destabilizers/degraders reduce, whereas the stabilizers enhance, the formation of the YAP/TAZ-TEAD complex.

YAP/TAZ-mediated tumor development is due to the collective action of the repertoire of proteins that are expressed under their influence. However, some proteins are able to drive oncogenesis much better than others and they vary depending on the solid tumor and context. Therefore, drugs against these downstream YAP/TAZ targets including metabolic enzymes, kinases, ligands and proteins, such as BCL-xL, FOXM1 and TG2 are also used to combat YAP/TAZ-mediated oncogenicity (Figure ).

TAZ-dependent expression of ALDH1A1 (aldehyde dehydrogenase) is shown to impart stemness and tumorigenic ability; inhibition of ALDH1A1 using A37 reverses this transformation 132. GOT1 - the aspartate transaminase induced by YAP/TAZ, confers glutamine dependency to breast cancer cells and targeting this metabolic vulnerability using aminooxyacetate (AOA) represses breast cancer cell proliferation 133. Targeting the YAP/TAZ transcriptional target cyclooxygenase 2 (COX-2) using celecoxib inhibits cell proliferation and tumorigenesis in NF2 mutant cells 134. Interestingly, a positive feedback is seen in hepatocellular carcinoma cell lines where COX-2 is also shown to increase the expression of YAP 135. Inhibiting COX-2 using NS398 stimulates LATS-dependent phosphorylation of TAZ 136.

In hepatocellular carcinoma, Axl kinase has been shown to be crucial for mediating several YAP-driven oncogenic functions like cell proliferation and invasion 137. Similarly, YAP-driven Axl expression has been implicated in the development of resistance against EGFR inhibitors in lung cancer and sensitivity could be restored through Axl inhibition using TP-0903 138. YAP is shown to upregulate the expression of the kinase NUAK2 139 that, in turn activates YAP/TAZ by inhibiting LATS. Specific pharmacological inhibition of NUAK2 using WZ400 shifts YAP/TAZ to the cytoplasm and reduces cancer cell proliferation 140.

In a mouse model of prostate adenocarcinoma, the YAP-TEAD complex promotes the expression of the chemokine ligand CXCL5 that facilitates myeloid-derived suppressor cells (MDSC) infiltration and adenocarcinoma progression. Administering CXCL5 neutralizing antibody, or blocking CXCL5 receptor using the inhibitor SB255002, inhibits MDSC migration and tumor burden 40. The notch ligand Jagged-1 that is upregulated by YAP/TAZ is crucial for liver tumorigenesis 59, 141. Treating liver tumor cells with Jagged-1 neutralizing antibody greatly reduces oncogenic traits. The levels of integrin ligands CTGF and CYR61 that are also YAP target genes, could be reduced using the cyclopeptide RA-V (deoxybouvardin) leading to a reduction of YAP- mediated tumorigenesis in mst1/2 (Hippo homolog) knockout mouse model 142. Although neutralizing CTGF (FG-3019/pamrevlumab) and CYR61 (093G9) antibodies are available, they have not been effectively used against YAP/TAZ-driven cancers.

YAP mediates drug resistance to RAF- and MEK-targeted therapies in BRAF V600E cells, in part through the expression of the anti-apoptotic protein BCL- xL. BCL-xL inhibition using navitoclax sensitizes these cells to targeted therapies 92.

YAP-mediated proliferation through its target gene FOXM1 could be prevented in sarcoma cell lines and mouse models through the administration of thiostrepton that reduces FOXM1 levels 143.

Transglutaminase 2 (TG2) - the multifunctional transamidase is a YAP/TAZ target gene that is important for cancer stem cell survival and for maintaining integrin expression. TG2 inhibition using NC9 dramatically reduces tumorigenicity 144, 145.

We are aware that many of these target proteins also act upstream and stimulate YAP/TAZ by forming a positive feedback but we would nevertheless consider them in this group and not as group I as their expression is influenced by the TEAD-binding motif and YAP/TAZ.

Although attractive, toxicity issues and the identification of responsive patient population could be challenges in the successful implementation of the YAP/TAZ inhibitors in the clinic. YAP/TAZ inhibition might elicit toxicity 146; homozygous disruption of YAP in mice causes embryonic lethality, whereas TAZ knockouts are viable 147-150. Tissue-specific deletions of YAP in the heart 151, lung 152 or kidney 153 cause hypoplasia, whereas YAP/TAZ deletion in the liver cause hepatomegaly and liver injury 154. Surprisingly, YAP/TAZ knockouts in the intestine are well tolerated with no apparent tissue defects 155. All of these suggest that YAP and TAZ are crucial for development. However, they appear to be dispensable for adult tissue homeostasis. In most adult tissues, under normal homeostasis, YAP/TAZ are found restricted to the cytoplasm and are activated primarily in response to injury to initiate tissue regeneration. Therefore, it is predictable that administration of a YAP/TAZ inhibitor may not elicit severe toxicity. However, given the dynamic shuttling of YAP/TAZ/Yorkie between nucleus and cytoplasm 156-158, it is feasible that they still have a role in normal tissue homeostasis. Fittingly, YAP has been identified to be important for podocyte homeostasis and its functional inactivation compromises the glomerular filtration barrier and cause renal disease 109. Along similar lines, renal toxicity was observed in mice administered with K-975 - a YAP/TAZ-TEAD inhibitor 127. Renal toxicity in targeted therapy is very common and is seen in most of the kinase inhibitors used in oncology 159. Yet these kinase inhibitors are in the clinic as there is a therapeutic window, where the drug could be dosed to improve patient survival without causing much toxicity. The same could be envisaged for YAP/TAZ-inhibiting drugs.

Several drugs that act as YAP/TAZ inhibitors target multiple signaling pathways. Targeting multiple pathways could be a boon or a bane. Drug resistance is minimized in a multi-targeted approach as potential bypass mechanisms are also targeted. However, toxicity becomes an issue when the drug targets multiple important signaling pathways. For instance, raising cAMP through the use of PDE inhibitors activates a multitude of proteins like PKA, EPACs, ion channels and small GTPases. Similarly, GPCR modulators influence multiple pathways through signaling via G proteins, arrestins or GPCR kinases. To reduce toxic side effects, there are options available like selective targeting or biased signaling. Instead of hitting all the PDEs, the PDE enzyme that is the most potent activator of YAP/TAZ should be selectively targeted. Nonspecific PDE inhibitors cause more severe side effects than sub-type selective PDE inhibitors 160. Similarly, through stabilizing a particular GPCR conformation, certain small molecule GPCR modulators are able to effect signaling bias where one GPCR effector is preferentially activated over others, say G proteins over -arrestins, this way only a subset of signaling pathways get activated 161.

Another major challenge is the identification of patients responding to a YAP/TAZ inhibitor. YAP/TAZ expression is low in normal tissues and their levels are significantly elevated in cancers. Is YAP or TAZ positivity in tumors sufficient criteria to administer a YAP/TAZ inhibitor? YAP and TAZ might not be transcriptionally active or drivers in all tumors. Further, they could be expressing target genes that negatively regulate their activity 162, 163. There are also tumor types where YAP/TAZ or TEAD levels have no prognostic significance 46. These YAP/TAZ positive tumors are unlikely to respond to a YAP/TAZ inhibitor. Barring a few such scenarios, in many solid tumors, YAP or TAZ expression levels correlate well with higher-grade cancers or poor prognosis. Tumors with nuclear YAP or TAZ that are also positive for the downstream oncogenic YAP/TAZ target genes are likely to respond to a YAP/TAZ inhibitor and this should be used as criteria for patient stratification. As many of the YAP/TAZ-TEAD target genes are secreted proteins, the expression levels of these in the serum could also be estimated in addition to assessing their levels through immunohistochemistry.

As YAP and TAZ contribute to the acquisition of many hallmarks of cancer traits, targeting them is predicted to be more relevant for the management of several cancer types. It is still early to expect a newly developed drug against YAP/TAZ but it is nevertheless disconcerting to see that there are hardly any clinical trials that evaluate if known drugs could be repurposed as YAP/TAZ- inhibitors. Group I drugs are well suited to repurpose 105 but only statins ({"type":"clinical-trial","attrs":{"text":"NCT03358017","term_id":"NCT03358017"}}NCT03358017); trametinib ({"type":"clinical-trial","attrs":{"text":"NCT03148275","term_id":"NCT03148275"}}NCT03148275) and epigenetic modulators ({"type":"clinical-trial","attrs":{"text":"NCT03925428","term_id":"NCT03925428"}}NCT03925428) are being evaluated in clinical trials, assessment of the expression levels of YAP/TAZ after drug treatment is used as one of the clinical trial objectives. It is essential that we bolster our pharmacological arsenal so that we are prepared to combat YAP and TAZ. Group I drugs that failed in oncology trials are not expected to fare any better against YAP/TAZ. However, drugs that are already in the clinic like the kinase inhibitors targeting the EGFR or MEK, PDE inhibitors as well as GPCR modulators could be repurposed to combat YAP/TAZ. The cancer types need to be carefully stratified to ensure they are driven by YAP/TAZ through the upstream stimulator targeted by the drug. To overcome potential bypass mechanism or drug resistance, combinatory use of group I and II drugs could also serve as an avenue for cancer treatment. For the group III drugs, the situation may not be as promising, as they target only one of the many possible oncogenic proteins regulated by YAP/TAZ. Again, combinatory inhibition of few downstream target genes could be considered if they are collectively essential for oncogenic manifestation of YAP/TAZ-driven transcription. As they are new and untested, there is much excitement and progress in the development of novel group II compounds as drugs against YAP/TAZ. We are at an exciting juncture in the Hippo field where we could potentially see a novel group II drug or a repurposed group I drug to combat YAP/TAZ in the near future.

A.V. Pobbati and W. Hong are supported by the Agency for Science, Technology, and Research (A*STAR), Singapore. We thank Sayan Chakraborty, Gandhi T.K.B. and John Hellicar for critical reading of this review. We apologize to all authors whose work was not cited due to space constraints.

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A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy

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