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PM Modi Waives off Rs 6 Crore Tax on Imported Medicine for 6-month-old Baby Girl from Mumbai – News18

By daniellenierenberg

For baby Teera Kamat, who has been on the earth for a mere six months, every day has been a struggle for existence and a grim reminder to her parents about the fragile little being that needed a miracle to be saved. Mumbai-born Teera is suffering from Spinal Muscular Atrophy, a very rare medical condition that often does not let children live beyond 5 months of age and her condition requires a lot of money for the treatment.

On Wednesday, Prime Minister Narendra Modi, in a humanitarian move, decided to waive off Rs 6 crore as a GST amount against Rs 16 crore of imported medicines that are required to treat Teera. Baby Teera's parents Priyanka and Mihir Kamat have raised Rs 16 crore through crowdfunding for their daughter who needs a surgery to be cured. It also includes the cost of the medicine Zolgensma which has to be imported from the US. The tax exemption for baby's treatment amounts to at least Rs 6.5 crore and it includes 23 percent import duty and 12 percent Goods Services Tax.

The infant's parents had earlier appealed to PM Modi in October last year about Teeras medical condition and in January this year. The Leader of Opposition Devendra Fadnavis also wrote to the Prime Minister and Finance Minister Nirmala Sitaraman reiterating the request to exempt taxes on the medicine import.

It is a type of genetic disorder and a motor neuron disease that results in a person not having any control over movement of their muscles due because of the lack of nerve cells, in their spinal cord and/or brain stem.

Spinal muscular atrophy (SMA) results in muscle wasting and weakness. For someone suffering from SMA, it is very difficult to stand, walk and control their movements. Some intense forms of the SMA can also result in inability to breathe and swallow.

SMA can either occur at birth or even appear at stages of life and they can affect one's life expectancy depending upon the seriousness and the type of the SMA.

So far, there has been no cure of SMA, but certain medicines do help, such as nusinersen (Spinraza) and onasemnogene abeparvovec-xioi (Zolgensma), that help slow the disease's progress.

The types of SMA depend on when they start showing up in a patient and how the symptoms vary in them. There are basically four kinds of SMA, as National Institute of Neurological Disorders and Stroke list, which affects symptoms and life expectancy.

The first type of SMA, or Werdnig-Hoffmann disease appears before the infant is even 6 months of age. The child might be born with difficulty in breathing and the serious condition can turn fatal if there's no treatment.

Those with SMA type II will start showing symptoms of the disease usually when they are between 6 and 18 months of age. These children can sit but will not be able to walk or stand without helped and without treatment, they might just lose their power to sit as well.

Children with SMA type III or Kugelberg-Welander disease start showing symptoms after they are 18 months of age and can walk on their own. They however, experience difficultly in walking or running and other such physical exercises related to legs.

Those with SMA type IV usually develop the symptoms after they are over 21 years of age ad have minor muscle weakness and other issues. It doesn't affect one's life expectancy.

The USA Food and Drug Administration has approved the Zolgensma gene therapy for children who show the signs of the disease and are less than 2 years. Last year in August, the FDA also gave its nod to the orally-administered drug risdiplam (Evrysdi) for patients who are older than two months of age and are diagnosed with SMA.

Physical therapy, occupational therapy, and rehabilitation are some measures that can be taken to help improve posture, stop joint immobility and help in case of muscle weakness and atrophy.

You can find the link to the crowdfunding page for baby Teera here.

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Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice – Science

By daniellenierenberg

Endogenous activin A in ectopic bone formation

Heterotopic ossification (HO) is the formation of ectopic bone in soft tissues at sites of injury-induced inflammation. Similar to the development of normal endochondral bone, HO is initiated by a local mass of chondrocytes that progress through chondrogenesis, osteogenesis, and mineralization to form bone tissue. Using mouse models of both subcutaneous and intramuscular HO formation and single-cell RNA sequencing, Mundy et al. found that inflammatory cells and skeletal progenitor cells initially recruited to sites of HO formation expressed Inhba, which encodes the TGF- superfamily member activin A. Treating mice with an activin Aneutralizing antibody reduced the number of chondrogenic cells at HO sites and inhibited HO formation. These results demonstrate that this ligand plays an important role in the physiological progression in these mouse models of HO and suggest that interfering with activin A signaling may be effective in patients.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF- family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

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I got stung by a stingray, and all I got was this deeper understanding of venom medicine – NOVA Next

By daniellenierenberg

Three years ago, wading in the sun-warmed waters of the Florida Keys, I felt a sharp pinch and looked down at my feet in surprise. My friend Jen and I had driven down from Miami for a weekend full of strong Cuban coffee and Hemingways six-toed cats. Tempted by water so warm and aquamarine it was almost a cliche, we had stopped to swim at a roadside beach on Bahia Honda Key. I had eased in, careful to drag my feet slowly across the seafloor in a dance known to beachgoers as the stingray shuffle, hoping to alert any local sealife to my approach. But not careful enough.

My foot throbbing, I stumbled back to the beach toward Jen, who wondered if I might have stepped on some glass. But in the next half hour, as my ankle and foot ballooned and the pain ratcheted upfrom stinging to aching, from aching to bone breakingit became clear I must have been stung by something. Then my foot started to turn blue, and we drove to the hospital.

Bahia Honda? the nurse said. Youre the fourth person to come in with a stingray sting from there today.

The pain didnt subside until the next day, when my foot had returned to its normal color. That was the start of a six-week recovery, which also involved crutches, painkillers, heavy-duty antibiotics, and a horrible rash. I wouldnt wish the experiencewhich involves a level of discomfort that some have compared to a gunshot woundon anyone. But in retrospect, its an interesting one to consider. Because, it turns out, animal venoms like the one coursing through my veins on Bahia Honda Key are sought after for drug development, with seven FDA-approved drugs derived from venom toxins on the market so far. Harnessing their power to hurt opens up a world of possibilities for healing.

The Bahia Honda beach where the author had a run-in with a stingray. Image Credit: Giuseppe Milo, Flickr (CC BY 2.0)

Chemical biologist Mand Holford, who studies venom science at her lab at Hunter College, compares what was happening in my foot in the moment after the sting to a cluster bomb. The toxins in animal venom have been engineered by evolution over many millennia to incapacitate by affecting some component in the blood, brain, or cell membranes, she says. Youre getting invaded with 200 to 300 different toxins, all trying to figure out how to reach their target, moving through and rupturing cell membranes, doing all sorts of damage.

The nurse at the emergency room told me stingrays were migrating through the area, their path bringing them close to the cove where I went wading. Stingrays deliver their venom through one or more serrated barbs that lie along their tails. While at rest, a stingray keeps its barb tucked away, immunologist Carla Lima told me in an email. But when it feels threatenedsay, by the feet of a clueless human out for a swimit pushes its tail perpendicular to its body, puncturing that humans flesh with its venom-laden spine.

Lima studies toxins in venomous fish at the Butantan Institute in So Paolo, Brazil. Her research into stingray venom has shown that whats in that venom actually changes as a stingray matures. In the freshwater species she studieswhose venom properties are better explored than the marine stingray that got methe venom of young rays tends to contain toxins that cause pain to the target. Lima hypothesizes this may be to chase predators away. In contrast, the toxins in adult venom have a necrotizing effect, meaning they destroy tissue, which would be helpful for hunting.

Peptides, short chains of amino acids that play key roles in the biological functions of all kinds of organisms, make up a large part of most animal venomsand some are only found in those venoms. Lima and other researchers have identified the peptides porflan and orpotrin as two of the elements in the freshwater stingrays toxic cocktail, along with a number of different proteases, which are enzymes that break down peptides.

As I sat cradling my foot on the beach in Bahia Honda, similar proteases and related proteins worked to break down the structure of cells in my heel, helping the venom spread further, and to prompt an inflammatory reaction that led to the swelling I saw. The peptides, on the other hand, likely caused the arteries to constrict and blood to pool, creating more inflammation and blocking circulationperhaps the cause of my foot turning blue.

A southern stingray (Dasyatis americana) cruises the ocean floor off Grand Turk Island in the Caribbean. Image Credit: Nate Madden, Shutterstock

That a substance that causes so much pain and wreaks so much biological havoc can be used in medicine is what Holford calls the yin and yang of nature. And the fact that damage and healing are, at least in this case, two sides of the same coin forms the basis for the work she does in her lab, identifying new drug applications for various components of animal venom.

Venoms have great potential to contribute to drug development because they are both potent and highly targeted, Holford says, with peptides that fit physically into cell receptors and change how those cells function. Thanks to this dynamic, venom-based drugs can work almost instantaneously. And theyre not what people in the pharmaceutical business call leaky, meaning they tend to only act on the intended cell component and dont stop at other spots along the way causing side effects.

Most stingray venom research, like Limas, takes place in areas where stingrays pose a threat to people: tropical spots like Brazil and Australia. On a drug-development level, we still dont know much about it, Lima says. But we do know a lot about other venomsin particular those created by cone snails and snakes.

For one thing, not all venom toxins cause pain. Some peptides present in snake venom focus on manipulating proteins in the wound so blood flows freely, acting as natural anticoagulants. Other peptides in Gila monster venom promote insulin production, helpful for a hungry lizard that hasnt eaten for awhile. And yet other peptides in cone-snail venom do the opposite of what stingray venom does: paralyze and suppress pain, keeping the snails prey from going into fight-or-flight mode and slowing it down until the (also slow) snail can come nab it for a snack.

This last type of venom is one of the focuses at Holfords lab. Many cone-snail venom peptides are rich in cysteine amino acids, whose structure she compares to Velcro. That makes it relatively easy for them to stick in the hourglass-shaped pores on the surface of cells that let important minerals like sodium, calcium, and potassium flow in and out. The free movement of those minerals is part of how cells talk to each other.

With those channels shut down, neurons cant communicate with one another to indicate pain. Thats what makes Prialt, the commercial version of the cone snails ziconotide peptide, an effective pain medication. Holford and her colleagues are also exploring the potential of other related cone-snail peptides to help dampen signals firing too fast in someone having a heart attack or an epileptic seizure.

She even sees possible applications here for cancer treatment. Current chemotherapy regimens dont discriminate between normal cells and tumor, she says. But because venom peptides work on specific receptorsreceptors that some tumors grow too many of as part of their developmentthey could help create a cancer drug that specifically starves cancer cells of essential minerals, stopping their growth.

The saw-scaled viper (Echis carinatus) is one of the deadliest snakes in India, and its venom is the basis of the blood-thinning drug Tirofiban. Image Credit: Sagar Khunte, Wikimedia Commons (CC BY-SA 4.0)

The venom that nearly ruined my Florida Keys vacation (though I still got to enjoy some beautiful sunsets, and the seafood was fantastic) was incredibly sophisticated, honed by evolution to inflict pain and physiological damage with laser precision. It was almost comforting to learn this in the weeks after, as I hobbled around on my crutches and watched with fascinated disgust as the wound developed a stingray-shaped blister. (My boyfriend said it was a sign I was developing superpowers, but sad to say none appeared.)

We know from nature that these peptides work, Holford says. What we dont know is massive: where they work, how they work, how effective they are. And thats a huge game of Wheres Waldo. Holford and her colleagues have come up with a protocol for finding new venom components that have potential in drug applications, then figuring out how to get them there. The first step is a practical look at the natural world: identifying which animal species are creating venom, especially venom that can be extracted manually. Next, the team uses new technologies that Holford refers to as the omics genomics, transcriptomics, proteomicsto identify the toxins within those venoms, by examining the instructions the animals' DNA and RNA contain and the proteins built by following those instructions.

From there, the team is able to use that genetic code to manufacture more of a chosen peptide in the lab, which is especially useful when it comes to studying venoms that are produced in small quantities in nature. They then test the synthetic toxin on the animals natural prey to make sure its effective and further tweak it to ensure its as specifically targeted as it can be for humans. And finally, they start to think about drug delivery. Does this drug need to cross the blood-brain barrier? Would it work if administered orally? These are essential questions, since potential drugs that cant be delivered effectively cant really be drugs at all.

Much like the experience of the sting itself, the possibilities for new drugs here are dizzying. Most venom-based drugs on the market are derived from a single peptide. But my stingrays venom (just like other naturally occurring venoms) featured hundreds of peptides. And with the advent of the omics, drug development with venom has become more efficient. Time- and resource-intensive experiments can now be run much more quickly using computer modeling, making the whole process more viable and opening up a whole world of drug prospects.

Lima and her colleagues in Brazil, for example, are continuing to explore the realm of fish venom. One synthetic peptide derived from the venom of a species of toadfish shows particular promise. A 2017 study suggested that peptide, known as TnP, has powerful anti-inflammatory and therapeutic effects in mice. Effects that could potentially help stem the autoimmune reactions that lead to spinal cord damage in patients with multiple sclerosis.

As Holford and her team navigate the new technological landscape, theyre also looking for ways to simplify their process. One innovation Holford is excited about is organoids, in this case, venom glands grown independently in a laboratory. Growing organoids would make acquiring venom samples much easier, she says, and would not require sacrificing an animal for the initial sample.

Thats especially important with climate change and habitat loss fueling a looming biodiversity collapse that could take with it undiscovered venoms with the capacity to heal. In 10 years were heading toward this major shift thats coming if we dont change our attitudes and lifestyle, she says. We could lose a lot of things on the planet that are potentially lifesaving.

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Alberta Health to cover $2.8-million gene therapy treatment on case-by-case basis – Edmonton Journal

By daniellenierenberg

And for some families this wait is excruciating, he said at a Wednesday news conference.

Shandro said there is no specific budget or cap on how much the government will spend, but that it was working with drug manufacturer Novartis to provide access. Nearly 70 per cent of children with spinal muscular atrophy type 1 do not live past age two. The drug is typically only approved for children under two.

We just dont want kids to fall through the cracks, said Shandro.

Susi Vander Wyk, executive director of Cure SMA Canada, thanked the province for making a decision she said is saving lives.

Its a fairly new treatment, so we dont know the long-term future of it, but we sure know that it has an astounding impact on these babies, she said at the news conference.

The earlier they receive the treatment, the better their prognosis, Vander Wyk said.

Time is ticking for them.

For Lana Martin, whose two-year-old son Kaysen Martin received the Zolgensma treatment in December after fundraising and an anonymous $1.4 million donation, the news was a huge step towards more kids accessing the drug.

Its still early after Kaysens treatment, but hes already more confident in his movements and doesnt tire as easily, said Martin.

He can now officially completely roll from one side of the room to the other side of the room, and he was not able to do that before, she said.

Martin said it was difficult for her and her husband, normally private people, to advocate publicly for the drugs coverage, but shes glad they did.

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Transforming optimism: finding new ways to treat rare cancers – Cancer Research UK – Science Blog

By daniellenierenberg

Cancer is an extremely complex disease. There are over 200 different types, some of which are considered common and others which are classified as rare cancers. But what exactly does it mean if a cancer is rare?

Usually, it means it only affects a small handful of people, but doctors might also call a cancer rare if it starts in an uncommon place in the body, or if the cancer is an unusual type and requires special treatment.

For secondary central nervous system (CNS) lymphoma, its an incredibly rare cancer for a combination of these reasons.

Secondary CNS lymphoma is a type of lymphoma thats spread to the brain and spinal cord nervous system after originating elsewhere in the body. And as well as being a rare cancer, secondary CNS lymphoma is an aggressive cancer, which has relatively low survival rates.

However, the latest results from the Stand Up To Cancer-funded MARIETTA clinical trial, which details a new potentially transforming treatment, has shed a glimmer of hope for patients and doctors alike.

We spoke to Dr Kate Cwynarski, who led the study in the UK, about what the latest results could mean for patients with secondary CNS lymphoma.

With a rare cancer such as secondary CNS lymphoma, finding a large enough group of patients can be a real challenge. And in cases like this, researchers have to think on a global scale.

Its a rare disease. So the reality of it is that you would not get this information if we just performed a trial in the UK, says Cwynarski. International collaboration is the only way to do it.

The MARIETTA trial is the largest study focused on patients with secondary CNS lymphoma, involving 24 centres across 4 countries and recruiting a total of 79 patients. It involved the International Extranodal Lymphoma Group (IELSG) lead by Professor Andres Ferreri in Italy and it built on the success of prior research with this group. In the UK, the trial was managed by CRUK Southampton CTU.

In particular, findings from a previous clinical trial partly funded by us, which tested treatments for primary CNS lymphoma, a lymphoma thats only found in the brain, helped inform the design of this clinical trial.

The IELSG-32 trial tested the benefits of an intensive chemotherapy regimen known as MATRIX, followed by either whole brain radiotherapy or a stem cell transplant using the patients own cells.

Cwynarski describes the IELSG-32 trial as practice changing, and its from these impressive results that the MARIETTA trial was developed. So we adapted a strategy that was successful in treating primary CNS lymphoma in the IELSG-32 trial and added another chemotherapy regimen, called R-ICE, to help treat the systemic disease on top of the secondary brain disease.

Cwynarski specialises in lymphoma, so she has treated SCNSL patients both on and off the trial. One of the big benefits of this trial, she describes, is that the inclusion criteria for the cohort more accurately reflected the patients she sees in her clinic and referral practice.

This trial included patients up to 70 years of age. And it wasnt just focused on fit, young people. So I have to say I think it was meaningful, because it included the kind of patients that we actually see.

The trial also included people regardless of when their secondary CNS lymphoma was diagnosed, whether that was when someone was originally diagnosed with lymphoma, during treatment, or after their cancer had come back.

And the results look promising. A total of 49 patients (65%) responded to the treatment in some way, with 37 people going on to have a stem cell transplant. 100% of the patients who had the stem cell transplant had not seen their cancer recur a year after registering onto the trial. We are optimistic many will be cured of this aggressive lymphoma.

But the trial also picked up differences between groups. While the regime was effective to an extent in every sub-group, the most significant results were seen in patients whose CNS disease was discovered at initial lymphoma diagnosis. Within this group, 71% of patients had lived for 2 years without their cancer growing.

A result which has never been seen before.

The results of the trial have completely transformed the teams optimism when meeting new patients. We really have identified a regimen which is intensive, but its potentially curative, concludes Cwynarski, and the word cure is not something weve really used before when talking about this disease.

Recently, Cwynarski has been busy filling out a cohort of her patients DVLA forms, confirming they are fit to drive again after being 2 years treatment free. So thats an amazing success and it was very symbolic as a reminder that these people have been alive and off all treatment for 2 years.

Moments like this are a reflection of the huge impact the MARIETTA trial has had for real people, like Maureen Brewster.

Maureen was diagnosed with lymphatic cancer of the liver in 2011 and was under the watchful eyes of a consultant during her treatment. But in the summer of 2016, I started to have very extreme headaches, says Maureen.

After getting an emergency appointment, she was taken to A&E and admitted to hospital straight away. I was transferred to the National Neurology hospital in Russell Square for a biopsy. They thought I might have had a stroke. But it wasnt. Instead, Maureen was diagnosed with a secondary cancer in her brain.

When Maureen was transferred to UCLH, she was told about the MARIETTA trial. I could have chosen not to go on the trial, but being part of it meant that I would get more examinations and monitoring. So it was more reassuring to be on the trial, she says.

Maureen during treatment.

Maureen went through 8 tough months of chemotherapy before having a stem cell transplant in the summer of 2017. During one round of chemo in the hospital I became ill with an infection and really thought I was going to die. The last chemo prior to me having stem cell transplant was so strong it really had an impact on me and I couldnt eat I felt very poorly for a few weeks.

Maureens stem cell transplant went smoothly and prior to COVID-19, she was having regular check-ups and scans in hospital.

Prior to the first lockdown in March 2020, Maureen was able to do some volunteering and also go back to work, teaching a course on Project Management at a local adult college. In April 2019 I also secured a part-time job as a User Involvement Co-ordinator. It was great to get back to that level.

Dr Cwynarski emphasises that while the trial was a great success for some, it also exposed a group of patients who didnt do so well on the treatment.

The results threw up a real disparity and uncovered an unmet need in a particular group of patients. For the group of patients whose cancer had already failed to respond to a chemotherapy treatment, known as R-CHOP, at the follow up of 2 years, only 20% had not experienced their cancer progressing or getting worse.

We need to target this cohort of patients in a different way, says Cwynarski. So really the challenge is, can we identify experimental agents be it different biological agents or immunotherapies such as CAR T cell therapy in the patients who have relapsed, and maybe bringing these therapies into the frontline.


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Genetic Energy Boost Reverses Cellular Effects of Motor Neuron Disease – Technology Networks

By daniellenierenberg

A study examining the damage caused to nerve cells by motor neuron disease (MND) has shown that by targeting these cells energy centers, called mitochondria, neuronal function can be restored.

The research, conducted by a team at the University of Edinburgh, has been published in the journal Acta Neuropathologica.1MND is a broad term for a group of rare, progressive and sometimes fatal neurodegenerative conditions, including amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP) and progressive muscular atrophy (PMA).

The research team, led by Dr Arpan Mehta, alongside Dr Bhuvaneish Selvaraj and Professor Siddharthan Chandran, all based at the University of Edinburghs Euan MacDonald Centre for MND Research, focused their work on the axon of human motor neurons. This is the region of a motor neuron that conducts electrical signals released by the brain and carries them to the body part they are intended for. In some human motor neurons, the axon can be over a meter long. These processes are energy-guzzling, and that power is provided by mitochondria, known to generations of long-suffering biology students as the powerhouses of the cell.

The scientists noted that the axons of MND-affected neurons were shorter than normal, and their mitochondria were not as easily able to move around the cell as they were in healthy neurons.

Using stem cells taken from people who have a mutation in a gene called C9orf72 that is known to play a causal role in both the MND subtype ALS and frontotemporal dementia, Mehta and colleagues created a stem cell model of MND, and sought to repair these stricken neurons.

In their stem cell models of MND, the team showed that by increasing the levels of a protein named PGC1 that regulates mitochondrial energy metabolism, the motor neurons function could be returned to healthy levels.

Dr Arpan Mehta (right), alongside Euan MacDonald MBE, co-founder of the Euan MacDonald Centre.

Our data provides hope that by restoring the cells energy source we can protect the axons and their connection to muscle from degeneration. Work is already underway to identify existing licensed drugs that can boost the mitochondria and repair the motor neurons. This will then pave the way to test them in clinical trials.

The team focused solely on the most common genetic form of ALS in their study and acknowledge that MNDs such as ALS are caused by a range of genetic and environmental factors. Nevertheless, they hope that their findings can be applied to other forms of the disease.

Reference:Mehta AR, Gregory JM, Dando O, et al. Mitochondrial bioenergetic deficits in C9orf72 amyotrophic lateral sclerosis motor neurons cause dysfunctional axonal homeostasis. Acta Neuropathol. Published online January 4, 2021. doi:10.1007/s00401-020-02252-5

Correction: This article was updated on January 25, 2021 to amend a quote from Dr Mehta.

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Novel Treatment Leads to Dog’s Recovery – The Bark

By daniellenierenberg

Life for Miro, a 5-year-old German shepherd, has been what his owner describes as an emotional roller coaster over the past two years. Several peaks and valleys have dotted his metaphorical landscape as he has gone from premiere fitness to dealing with injuries and disease. But a clinical trial at the UC Davis veterinary hospital may have put him back on a positive track.

Working as a patrol dog with his handler/owner Martin Gilbertson, a ranger with California State Parks, Miro spent three years performing duties that required him to be at the top of his game. In early 2019, he was just that, having won the top dog award for his department.

By that summer, however, things started declining for Miro. He was diagnosed with lumbosacral intervertebral disc disease that caused spinal cord compression. UC Davis veterinary neurosurgeons performed a surgical decompression, and Miro eventually recovered after a lengthy recuperation period.

Miro with his handler Martin Gilbertson

Life was great, said Gilbertson. By early December 2019, Miro was cleared to return to work. I thought all the troubles were behind us.

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It only took a few weeks, though, until the roller coaster cleared a peak and started to descend.

In late December 2019, Miro collapsed for no apparent reason and started shaking in a way Gilbertson had never seen. So, the pair returned to UC Davis where Miro was diagnosed with myasthenia gravis, a disease in which there is a malfunction in the transmission of signals between the nerves and muscles. This causes muscle weakness, and an inability to walk or run properly, as well as potentially devastating neuromuscular disorders.

Gilbertson was devastated.

To go from the pinnacle of our profession to potentially being a couch potato at best for the rest of his life was a real gut check, he said.

But hope appeared a few weeks later when Neurology/Neurosurgery Service faculty members Drs. Pete Dickinson and Bev Sturges informed Gilbertson of a myasthenia gravis clinical trial they were beginning with the help of the schools Center for Companion Animal Health (CCAH) and the Veterinary Institute for Regenerative Cures.

I thought, What do we have to lose? stated Gilbertson. Dr. Dickinson told me that Miro would be the first dog to ever receive this new treatment. We were excited and grateful to be able to participate.

A computer program shows Miro's stride pattern on the Tekscan Strideway pressure walkway.

Over the next few months, Miro received three stem cell treatments, as well as traditional medications to treat myasthenia gravis. Additionally, part of Miros recovery involved examining his gait, which utilized a new piece of equipment aimed at better analyzing a dogs stride pattern. Thanks to CCAH funding, the school recently acquired a Tekscan Strideway pressure walkway that allows clinicians and researchers to better gauge a patients step pattern and make decisions about their optimal care and recovery. To fully understand a patients gait abnormalities associated with injuries or neuromuscular diseases, veterinarians and researchers rely on objective, quantitative ways to assess locomotor function. The Strideway system complements the force plates in the schools J.D. Wheat Veterinary Orthopedic Research Laboratory, which captures extensive information, but only for one gait step. The new pressure walkway expands the capabilities to quantify pressure, vertical force, and stride parameters (timing and spacing) on all limbs for several strides during walking, trotting or landing. Miros progress was able to be tracked with pinpoint accuracy throughout his recovery.

Before the trial, Miro could only walk about 10 steps before falling down. After the trial, he seemed fully recovered, and blood tests revealed no trace of antibodies to the disease. While the disease may not be completely gone from his system, the clinical trial seems to have repressed the disease to a point where it no longer inhibits Miro from his normal activities. Retired from his job, Miro now enjoys life as a family pet.

It is true that Miro is now in remission, but until more analysis of data is completed, it is still too early to determine if the stem cells were the driving force behind his recovery, since they were administered at the same time as standard-of-care medications. Miros results are being closely examined, along with the results of two other dogs that have completed the trial, to see if this stem cells treatment truly can be considered a cure for myasthenia gravis. Regardless of the final outcome of the study, Miros recovery, in one way or another, came from a novel combination of treatments pioneered at UC Davis.

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Scientists find uncommon hereditary disorder that influences the brain, heart and facial highlights – Microbioz India

By daniellenierenberg

Researchers at the National Institutes of Health have discovered a new genetic disorder characterized by developmental delays and malformations of the brain, heart and facial features. Named linkage-specific-deubiquitylation-deficiency-induced embryonic defects syndrome (LINKED), it is caused by a mutated version of the OTUD5 gene, which interferes with key molecular actions in embryo development.

The findings indicate that the newly identified pathway may be essential for human growth and may also underlie other disorders that are present at birth. The information will help scientists better understand such diseasesboth common and rareand enhance patient care. The results were reported Jan. 20, 2021 at Science Advances.

The project began when David B. Beck, M.D., Ph.D., a clinical fellow in the laboratory of Dan Kastner M.D., Ph.D., at the National Human Genome Research Institute (NHGRI) and co-first author, was asked to consult on a male baby who had been born with severe birth defects that included abnormalities of the brain, craniofacial skeleton, heart and urinary tract.

Our discovery of the dysregulated neurodevelopmental pathway that underlies LINKED syndrome was only possible through the teamwork of geneticists, developmental biologists and biochemists from NIH,. This collaboration provided the opportunity to pinpoint the likely genetic cause of disease, and then take it a step further to precisely define the sequence of cellular events that are disrupted to cause the disease.

Achim Werner, Ph.D., Investigator, National Institute of Dental and Craniofacial Research (NIDCR) and Lead Author

An in-depth examination of siblings and family members genomes, combined with hereditary bioinformatics analyses, revealed a mutation in the OTUD5 gene as the possible cause of the problem. Through outreach to other researchers working on similar problems, Beck found seven additional males ranging from 1 to 14 years of age who shared symptoms with the first patient and had varying mutations in the OTUD5 gene.

The gene comprises instructions for making the OTUD5 enzyme, which is involved in ubiquitylation, a process which molecularly alters a protein to change its purpose. Ubiquitylation plays a part in governing cell fate, where stem cells are taught to turn into specific cell types in the early stages of embryo development.

According to the genetic evidence, I was pretty sure OTUD5 mutations caused the disease, but I did not understand how this enzyme, when mutated, led to the symptoms seen in our patients, said Beck. For this reason we sought to work with Dr. Werners group, which specializes in using biochemistry to comprehend the functions of enzymes such as OTUD5.

To begin, the NIH team analyzed cells taken from patient samples, which were processed in the NIH Clinical Center. Usually, OTUD5 edits or eliminates molecular tags on particular proteins (substrates) to modulate their function. However, in cells from patients with OTUD5 mutations, this activity was diminished.

Using a method to reunite mature human cells into the stem cell-like state of embryo cells, the scientists discovered that OTUD5 mutations were linked to abnormalities in the development of neural crest cells, which give rise to tissues of the craniofacial skeleton, and of neural precursors, cells that eventually give rise to the brain and spinal cord.

In additional experiments, the team discovered that the OTUD5 enzyme acts on a few protein substrates called chromatin remodelers. This class of proteins alters the closely packed strands of DNA in a cells nucleus to make sure genes accessible for being turned on, or expressed.

With help from collaborators led by Pedro Rocha Ph.D., an investigator in the National Institute of Child Health and Human Development (NICHD), the group found that chromatin remodelers targeted by OTUD5 help enhance expression of genes that control the cell fate of neural precursors during embryo development.

Taken together, the investigators reasoned, OTUD5 normally keeps these chromatin remodelers from being tagged for destruction. However, while OTUD5 is mutated, its protective function is lost and the chromatin remodelers are destroyed, leading to abnormal development of neural precursors and neural crest cells. Ultimately, these changes can lead to some of the birth defects seen in LINKED patients.

This implies that the mechanism we discovered is a portion of a common developmental pathway that, when mutated at different points, will result in a spectrum of disease.

We were amazed to discover that OTUD5 elicits its effects via multiple, functionally related substrates, which shows a new principle of cellular signaling during early embryonic development, said Mohammed A. Basar, Ph.D., a postdoctoral fellow in Werners lab and co-first author of this study. These findings lead us to believe that OTUD5 may have far-reaching effects beyond those identified in LINKED patients.

In future work, Werners team plans to fully investigate the role which OTUD5 and similar enzymes play in development. The researchers hope the study can serve as a guiding framework for unraveling the causes of other undiagnosed diseases, ultimately helping clinicians better evaluate and care for patients.

Were finally able to provide families with a diagnosis, bringing an end to what is often a long and exhausting search for answers, said Beck.


Journal reference:

Beck, D.B.,et al.(2021) Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation.Science

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New Genetic Disorder Discovered That Affects Brain and Craniofacial Skeleton – Technology Networks

By daniellenierenberg

Researchers at the National Institutes of Health have discovered a new genetic disorder characterized by developmental delays and malformations of the brain, heart and facial features.

Named linkage-specific-deubiquitylation-deficiency-induced embryonic defects syndrome (LINKED), it is caused by a mutated version of theOTUD5gene, which interferes with key molecular steps in embryo development. The findings indicate that the newly identified pathway may be essential for human development and may also underlie other disorders that are present at birth. The information will help scientists better understand such diseases both common and rare and improve patient care. The results were reported Jan. 20, 2021 inScience Advances.

Our discovery of the dysregulated neurodevelopmental pathway that underlies LINKED syndrome was only possible through the teamwork of geneticists, developmental biologists and biochemists from NIH, said Achim Werner, Ph.D., an investigator at the National Institute of Dental and Craniofacial Research (NIDCR) and lead author. This collaboration provided the opportunity to pinpoint the likely genetic cause of disease, and then take it a step further to precisely define the sequence of cellular events that are disrupted to cause the disease.

The project began when David B. Beck, M.D., Ph.D., a clinical fellow in the laboratory of Dan Kastner M.D., Ph.D., at the National Human Genome Research Institute (NHGRI) and co-first author, was asked to consult on a male infant who had been born with severe birth defects that included abnormalities of the brain, craniofacial skeleton, heart and urinary tract. An in-depth examination of siblings and family members genomes, combined with genetic bioinformatics analyses, revealed a mutation in theOTUD5gene as the likely cause of the condition. Through outreach to other researchers working on similar problems, Beck found seven additional males ranging from 1 to 14 years of age who shared symptoms with the first patient and had varying mutations in theOTUD5gene.

The gene contains instructions for making the OTUD5 enzyme, which is involved in ubiquitylation, a process that molecularly alters a protein to change its function. Ubiquitylation plays a role in governing cell fate, where stem cells are instructed to become specific cell types in the early stages of embryo development.

Based on the genetic evidence, I was pretty sureOTUD5mutations caused the disease, but I didnt understand how this enzyme, when mutated, led to the symptoms seen in our patients, said Beck. For this reason we sought to work with Dr. Werners group, which specializes in using biochemistry to understand the functions of enzymes like OTUD5.

To start, the NIH team examined cells taken from patient samples, which were processed at the NIH Clinical Center. Normally, OTUD5 edits or removes molecular tags on certain proteins (substrates) to regulate their function. But in cells from patients withOTUD5mutations, this activity was impaired.

Using a method to return mature human cells to the stem cell-like state of embryo cells, the scientists found thatOTUD5mutations were linked to abnormalities in the development of neural crest cells, which give rise to tissues of the craniofacial skeleton, and of neural precursors, cells that eventually give rise to the brain and spinal cord.

In further experiments, the team discovered that the OTUD5 enzyme acts on a handful of protein substrates called chromatin remodelers. This class of proteins physically alters the tightly packed strands of DNA in a cells nucleus to make certain genes more accessible for being turned on, or expressed.

With help from collaborators led by Pedro Rocha Ph.D., an investigator at the National Institute of Child Health and Human Development (NICHD), the team found that chromatin remodelers targeted by OTUD5 help enhance expression of genes that control the cell fate of neural precursors during embryo development.

Taken together, the researchers concluded, OTUD5 normally keeps these chromatin remodelers from being tagged for destruction. But when OTUD5 is mutated, its protective function is lost and the chromatin remodelers are destroyed, leading to abnormal development of neural precursors and neural crest cells. Ultimately, these changes can lead to some of the birth defects seen in LINKED patients.

Several of the chromatin remodelers OTUD5 interacts with are mutated in Coffin Siris and Cornelia de Lange syndromes, which have clinically overlapping features with LINKED syndrome, said Werner. This suggests that the mechanism we discovered is part of a common developmental pathway that, when mutated at various points, will lead to a spectrum of disease.

We were surprised to find that OTUD5 elicits its effects through multiple, functionally related substrates, which reveals a new principle of cellular signaling during early embryonic development, said Mohammed A. Basar, Ph.D., a postdoctoral fellow in Werners lab and co-first author of the study. These findings lead us to believe that OTUD5 may have far-reaching effects beyond those identified in LINKED patients.

In future work, Werners team plans to more fully investigate the role that OTUD5 and similar enzymes play in development. The researchers hope the study can serve as a guiding framework for unraveling the causes of other undiagnosed diseases, ultimately helping clinicians better assess and care for patients.

Were finally able to provide families with a diagnosis, bringing an end to what is often a long and exhausting search for answers, said Beck.

Reference: Beck DB, Basar MA, Asmar AJ, et al. Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation. Sci Adv. 2021;7(4):eabe2116. doi:10.1126/sciadv.abe2116.

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Motor neurone disease: Researchers dare to hope existing drugs can reverse the deadly nerve damage – The New Daily

By daniellenierenberg

Have researchers in Scotland truly found a way to reverse the damage done to nerve cells by motor neurone disease (MND)?

A new study suggests that it all comes down to boosting the energy output from the mitochondria, the cells power supply.

How can that be done? Possibly with drugs that already exist.

The researchers, from the University of Edinburgh, are already looking for existing drugs that boost mitochondrial function and may be able to be repurposed to treat MND.

Overall, this is a startling development, albeit one that has to be tempered with wait-and-see caution. There are thousands of people around the world, sufferers and their families, desperate for even a glimmer of good news.

People with MNDprogressively lose the use of their limbs and ability to speak, swallow and breathe, whilst their mind and senses usually remain intact.

The average life expectancy is two and a half years.

Motor neuron cells are nerve cells that control movement. An axon is the long part of the motor neuron cell that connects to the muscle and it can be up to a metre long.

The starting point in the study was examining axonal dysfunction a common problem in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), a form of MND.

For axons to function properly, they need a lot of energy. The scientists wondered if the production of energy in the cells is put out of whack by MND. And if that was true, could it be fixed and if so, could the axon be restored to full function?

The researchers from the Euan MacDonald Centre for MND Research at the University of Edinburgh used stem cells taken from people with the C9orf72 gene mutation that causes both MND and frontotemporal dementia.

They discovered that, in these human stem cell models of MND, the axon was shorter than in healthy cells.

The study then found the back-and-forth movement along the axon was impaired.

And then, bingo! Once the mitochondrial performance was boosted, the axon think of a stunted tree suddenly getting water and nourishment regained its healthy function. Back to normal.

This then allowed the mitochondria to travel freely along the axon.

The study also examined human post-mortem spinal cord tissue from people with MND. The tissue was obtained from the Medical Research Council Edinburgh Brain and Tissue Bank.

These examinations supported the findings from the stem cells.

The first film shows mitochondria travelling along an axon in a healthy motor neuron:


This second film shows how the mitochondria becomes stalled as it attempts to travel along damaged motor neuron with the C9orf72 gene:


In this third film we see a damaged motor neuron with the C9orf72 gene restored to function after boosting the mitochondria:


Dr Arpan Mehta, the Lady Edith Wolfson Fellow and a PhD student at the University of Edinburgh, led the study. In a prepared statement he said:

The importance of the axon in motor nerve cells cannot be understated. Our data provides hope that by restoring the cells energy source we can protect the axons and their connection to muscle from degeneration.

Work is already underway to identify existing licensed drugs that can boost the mitochondria and repair the motor neurons. This will then pave the way to test them in clinical trials.

Although the research focused on the people with the commonest genetic cause of the ALS (amyotrophic lateral sclerosis) type of MND, researchers are hopeful that the results will also apply to other forms of the disease.

By the way: Frontotemporal dementia is a consequence of progressive damage to the frontal and/or temporal lobes of the brain.The right and left frontal lobes at the front of the brain are involved in mood, social behaviour, attention, judgement, planning and self-control.

Hence, damage can lead to reduced intellectual abilities and changes in personality, emotion and behaviour.

More than 2000 people have MND in Australia, of whom 60 per cent are male and 40 per cent female, according to figures from MND Australia.

The mean time from onset to confirmation of diagnosis is 10 to 18 months, and approximately 58 per cent of people with MND are under 65.

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Stem cells on the ballot – Science Magazine

By daniellenierenberg

California's ballot measures often reveal much about the broader U.S. policy environment. This is particularly true of the approval by the state's voters in November of Proposition 14, The California Stem Cell Research, Treatments, and Cures Initiative of 2020. Proposition 14 extends the 2004 ballot Proposition 71, which established the California Institute for Regenerative Medicine (CIRM) and authorized $3 billion in state-issued bonds for CIRM to fund stem cell and regenerative research and medicine (restricted to California). Proposition 14, which authorizes $5.5 billion over the next 10 years to continue CIRM's work, succeeded in part by informing voters of CIRM's successes and that its conflict-of-interest provisions are extremely strong. This state-level action is critical because, contrary to opponents' opinions, the overall policy environment for human stem cell research in the United States is in some ways worse now than when Proposition 71 passed.

Since 2004, CIRM has funded groundbreaking work on immune disorders, cancer, spinal cord injury, diabetes, and more. The result has been more than 90 stem cellrelated clinical trials (directly or indirectly supported by CIRM), almost 3000 scientific papers, and contributions to two cancer therapies approved by the U.S. Food and Drug Administration. The lives of many patients have improved because of CIRM. Notably, many CIRM-funded clinical trials rely on human embryonic or fetal stem cells, whereas the federal government currently does not fund any clinical trials using these types of cells.

Proposition 71 was motivated largely in response to restrictions on human embryonic stem cell research in the United States in 2004. However, although research was limited to a small number of human embryonic stem cell lines, there was no formal ban on federal funding of research on such stem cells. In addition, in 2004 there were no restrictions on federal funding of human fetal stem cell and tissue research; however, there is now near-complete blockage of federal funding for such research. And federal funding for human embryonic stem cell research is again at risk. On 4 September 2020, 22 Republican senators and 72 Republican House members wrote to President Trump requesting an end to all federal funding of human embryonic stem cell research. Could President Trump impose a ban that would be difficult to revoke? Or, could Republican senators manufacture a ban by legislative maneuvering on a budget reconciliation vote, which requires 60% support? Such maneuvering created the effectively permanent 1995 Dickey-Wicker amendment, which prohibits federal funding of any research in which human embryos are created or destroyed. Dickey-Wicker has limited research on in vitro fertilization methods and stalled progress on understanding early human development. It has not solved the problem of the many, perhaps 1 million frozen embryos in the United States that will not be used for in vitro fertilization and will be destroyed without benefit if not used for research. Vital long-term research is greatly harmed by the U.S. policy environment, with the likely outcome that many young scientists will avoid research using human embryonic stem cells and human fetal tissue.

Restrictions on valuable, ethical research appear particularly fool-hardy during a deadly pandemic. Research on viruses such as HIV and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) can benefit greatly from work using mice that utilize human fetal stem cells and tissues to generate a human-like immune system. These mice allow evaluation of a human immune system in the contexts of infection mechanisms, generation of immunity, and drug response. These studies can be supported with Proposition 14 funds in California, but not with federal funds. It is crucial for the incoming Biden administration to evaluate the need for federal funding in these important areas with high-quality scientific input and evidence.

California's vote on Proposition 14 should also help the rest of the country appreciate the need to increase investments in biomedical research at the U.S. National Institutes of Health and other federal agencies. Current biomedical research expenditures amount to only a tiny fraction of the costs of disease, so an objective evaluation of appropriately increased research funding relative to disease costs is warranted. Once again, California is showing the way.

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New gene therapy methods deliver promise – Spectrum

By daniellenierenberg

Special delivery: Stem cells can be modified to produce a therapeutic protein in the brain.

Laguna Design / Science Photo Library

Two unpublished studies detail improved methods for delivering gene therapies to the brain: One involves a type of stem cell that can produce gene-altering proteins on-site; the other taps an engineered virus to target neurons efficiently and noninvasively.

Researchers presented the work virtually on Monday and Tuesday at the 2021 Society for Neuroscience Global Connectome.

One of the biggest hurdles for targeted gene therapy is getting enough treatment to the right spot. In the first study, researchers overcame this obstacle by developing stem cells that produce a therapeutic protein inside the brain.

The team is using the approach to develop a treatment for Angelman syndrome, which is caused by mutation in or deletion of the maternal copy of the gene UBE3A. Because the paternal copy of the gene is typically silent, loss of the maternal copy results in an absence of UBE3A protein. People with Angelman syndrome usually have intellectual disability and motor impairments, and many are autistic.

The researchers had previously used modified stem cells to produce a protein that can activate the paternal copy of UBE3A. Transplanting the cells into the brains of Angelman syndrome model mice boosts levels of UBE3A protein, they found. However, the treatment required multiple direct injections into the animals brains.

In the new work, they instead tried injecting the cells into a pocket of cerebrospinal fluid at the base of the skull an approach that is less invasive and can be performed multiple times. They compared the results with direct injection into the animals hippocampus. In both cases, the mice had UBE3A expression in the brain for up to three weeks.

Mice that received direct injection of the stem cells had fewer Angelman syndrome traits than controls, as measured by their motor skills.

This suggests that though the new route is effective, it may not provide a high enough dosage, says Peter Deng, a postdoctoral researcher inKyle Finkslab at the University of California, Davis, who presented the work. And because the transplanted cells produce protein for only a limited period of time, the effects are temporary a limitation the team is addressing.

Deng and his colleagues also found that monkeys treated with the stem cells had the therapeutic protein throughout their brain and spinal cord three weeks after injection, which suggests the approach has potential for treating people.

The second approach presented at the conference improves the delivery of a more permanent form of gene therapy that uses adeno-associated viruses (AAVs).

Researchers typically inject these viruses directly into the brain, and the viruses usually only affect cells immediately surrounding the injection site.

Youre required to use a ton of the virus to penetrate the whole brain, says Jerzy Szablowski, assistant professor of neuroengineering at Rice University in Houston, Texas, who presented the work.

One potential workaround is to inject the AAV into the blood and use focused ultrasound to temporarily open up the blood-brain barrier, allowing the AAV to cross into the brain. Sometimes with this approach, however, the virus also inserts itself into other organs.

In their new work, the team developed AAVs that more easily cross the blood-brain barrier and more selectively target neurons than previous versions do. As a result, the new AAVs can be given in lower doses, reducing the amount of tissue affected outside the brain, Szablowski says.

To identify the most efficient AAV, Szablowski and his colleagues designed 2,100 new viruses, injected them all into the bloodstream of mice and applied focused ultrasound to the animals skulls. The mice had been engineered so that AAVs that successfully inserted themselves into a neuron got tagged with a marker. The team performed genomic sequencing on the mouse brains a few weeks later and read out the levels of viruses.

Compared with the previously most effective AAV, the top five newly identified AAVs targeted twice as many cells in the brain (including more neurons), and nearly half as many cells outside the brain, the researchers found.

The approach could be used to more efficiently deliver treatments for conditions such as Angelman syndrome or Parkinsons disease, the team says.

Read more reports from the 2021 Society for Neuroscience Global Connectome.

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Glioblastoma Tumors Triggered by the Healing Process of Brain Injury – Science Times

By daniellenierenberg

The growth ofglioblastoma tumors can be linked to the healing process that follows a brain injury, the researchers in Canada said. They believe that mutations derailed the new cells generated during the healing process in brain injuries, such as trauma and infection or stroke, that were supposed to replace the lost cells.

The researchers said that their data suggest that right mutations in particular brain cells could be modified by injury, which would create tumors. Dr. Peter Dirks, Head of the Division of Neurosurgery and a Senior Scientist in the Developmental and Stem Cell Biology program at SickKids, said that glioblastoma could be thought of as a wound that never heals.

They think that by studying the origins of glioblastoma, they can know how cancer originates and grows as it opens up new ideas about cancer treatment. Thestudywas headed by Dirks, molecular genetics professor Dr. Gary Bader, and senior scientist Dr. Trevor Pugh.

(Photo: Wikimedia Commons)Glioblastoma multiforme - MRT T1 axial mit Kontrastmittel. Histologie bioptisch gesichert.

According to Mayo Clinic, glioblastoma is an aggressive type of cancer that can either be found in the brain or the spinal cord that forms cells called astrocytes that support nerve cells. Although it can occur at any age, glioblastoma more common in older adults.

Unfortunately, options for treating glioblastoma are still limited, and patients have an average lifespan of 15 months after diagnosis. The researchers said that it is mostly because of the extensive heterogeneity observed within tumors as they harbor diverse cells, such as the glioblastoma stem cells.

Dirk's team believes that glioblastoma stem cells are responsible for tumor growth and recurrence after treatment. After a series of tests, they confirmed that each tumor contains multiple subpopulations of cancer stem cells, making its recurrence more likely that existing therapies cannot wipe away.

Moreover, they found that glioblastoma stem cells were comingled with the cancer stem cells within the tumors, which indicates that glioblastoma is starting to form when the healing process begins as new cells replace the lost cells due to injury. Dirks said that once the mutant cell becomes involved in the healing process, it can no longer stop multiplying, spurs tumor growth.

ALSO READ: Mobile Phones Caused Brain Tumour: Italian Court Rules

Further in their study, the researchers also classified two distinct molecular states that the tumors exhibited, Genetic Engineering & Biotechnology Newsreported. These are the "Developmental" and "Injury Response" states.

The Developmental state represents a hallmark of the glioblastoma stem cells, which is similar to the rapidly dividing stem cells in the growing brain of an infant after birth. On the other hand, the Injury Response state showed an increase in the number of immune pathways and inflammation makers that indicate a healing process.

Moreover, additional experiments established that the two states are at risk of various types of gene knockouts, which means that there are many therapeutic targets linked to inflammation that had not been previously linked to glioblastoma cells' growth.

The researchers found that the two states were patient-specific, which could lean toward the Developmental state of Injury Response state. Researchers are looking into these biases to make tailored therapies that are effective on different points of the two states.

READ MORE: Brain Tumor Vaccine: Combination Therapy Offers Promising Survival Results For Glioblastoma Brain Cancer Patients

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Hemostemix steps into the new year with capital and its critical clinical study data in hand – InvestorIntel

By daniellenierenberg

With a new management team spearheading Hemostemix Inc. (TSXV: HEM | OTC: HMTXF), the Company started 2021 with its critical clinical study data in hand. Raising over $4 million in 2020 and then in December adding an additional $4 million to the coffers ($2.75 million at a 50% premium), Hemostemix completed a 1-for-20 share consolidation as it charges into the New Year.

Receiving a copy of its entire clinical trial database relating to the clinical trial for Critical Limb Ischaemia (CLI) using its ACP-01 therapy (Angiogenic Cell Precursors) in November 2020 was a key event for Hemostemixs management team and it garnered real interest from the market.

Hemostemix Platform for Stem Cell Therapies

Based in Calgary and founded in 2006, Hemostemix is a clinical-stage biotechnology company specializing in blood-derived stem cell therapeutics with its lead product (ACP-01) in Stage 2 clinical trials for the treatment of CLI.

CLI is a disease caused by the narrowing of arteries in the limbs, particularly the legs, hands, and feet, causing chronic pain and soreness. Untreated CLI can sometimes require the amputation of the specific limb.

Stem cell treatments have been used for over 30 years to treat people with cancer conditions such as leukemia and lymphoma.

There are two main types of stem cell transplants: allogeneic and autologous. In an allogeneic stem cell transplant procedure, the patient receives stem cells from a donor. In an autologous stem cell transplant procedure, the patient provides themselves the stem cells for the procedure from various sources, including bone marrow or blood.

Hemostemixs autologous stem cell therapy platform uses the patients own blood to harvest the stem cells and the treatment helps to restore circulation in the damaged tissues.

Hemostemix has a strong intellectual property (IP) portfolio of 91 patents and has treated more than 500 patients with clinical results showing an improvement in 83% of the patients receiving its ACP-01 stem cell therapy.

Advantages with Hemostemixs process include the use of blood, which is safer and less invasive than extracting bone marrow, and since you are using the patients own blood, there is no immune rejection.

The clinical trials have shown that ACP-01 is safe and effective in the treatment of CLI. Now that Hemostemix has received the entire clinical trial database, it has entered into a contract with a new Clinical Research Organization (CRO) to complete the midpoint statistical analyses of the efficacy of ACP-01 and expects to publish the results this quarter.

Hemostemix Not a 1-Trick Pony Company

ACP-01 has the potential to treat other conditions such as Angina, Ischemic & Dilated Cardiomyopathy, and Peripheral Artery Disease (PAD). Currently, Hemostemix is preparing for Phase 2 trials for the treatment of Angina and is seeking joint-venture partners to fund the other Phase 2 trials.

Hemostemix has also developed NCP-01 (Neural Cellular Precursor) from blood with the potential, through building new neuronal lineage cells in a patient, to treat Alzheimers disease, Amyotrophic Lateral Sclerosis (ALS), Parkinsons disease, spinal cord injuries, and stroke-related issues. NCP-01 is currently in the R&D phase and is pre-clinical.

Market Size

According to the American Heart Association, Cardiovascular disease (CVD) accounted for approximately 1 of every 3 deaths in the United States in 2019.

Factors that increase the risk of CLI include diabetes, high cholesterol levels, high blood pressure, obesity, or smoking, all risk factors also associated with CVD.

Unfortunately, most of these factors are increasing at an alarming rate a study by the Centers for Disease Control and Prevention (CDC) in the United States, showed the prevalence of diagnosed diabetes has more than doubled from 3.3% in 1995 to 7.40% in 2015, affecting 23.4 million Americans.

According to a market research report released in 2019, the value of just the global CLI treatment market is projected to reach US$5.39 billion by 2025, up from US$3.13 billion in 2018, at an annual growth rate of 8%.

Competitive Landscape and Market Cap Comparisons

Even with Hemostemixs recent market surge, its market cap is only C$32.5 million. Similar-sized biotech companies focusing on CLI trade much higher.

Cynata Therapeutics Limited (ASX: CYP) is an Australian biotechnology company with a Phase 2 clinical-stage trial for its stem cell therapy for CLI using bone marrow and has a market cap of C$93.6 million.

Pluristem Therapeutics Inc. (NASDAQ: PSTI) is a Phase 3 bio-therapeutics company, based in Israel, that also has an allogeneic cell therapy for the treatment of CLI using the placenta and has a market cap of C$231.9 million.

In November 2020, Bristol-Myers Squibb Company (NYSE: BMY) bought MyoKardia, Inc. for US$13.1 billion. MyoKardia was a clinical-stage biopharmaceutical company that developed therapies for the treatment of cardiovascular diseases and its lead product was a Phase III clinical trial drug used in the treatment of hypertrophic cardiomyopathy (HCM).

As a company shifts from Phase 2 to Phase 3 clinical trials, the market cap often has a step-function shift higher, making it an ideal time to look at Hemostemix.


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Hemostemix steps into the new year with capital and its critical clinical study data in hand - InvestorIntel

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Stem Cell Assay Market Competitive Landscape Analysis with Forecast by 2025 – SoccerNurds

By daniellenierenberg

Stem Cell Assay Market: Snapshot

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues andtumors, wherein their toxicity, impurity, and other aspects are studied.

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With the growing number of successfulstem cell therapytreatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

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Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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TMR Research is a premier provider of customized market research and consulting services to business entities keen on succeeding in todays supercharged economic climate. Armed with an experienced, dedicated, and dynamic team of analysts, we are redefining the way our clients conduct business by providing them with authoritative and trusted research studies in tune with the latest methodologies and market trends.

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COVID-19 : Coronavirus I The hopeful treatment against COVID-19 with stem cells from the umbilical cord – Explica

By daniellenierenberg

It is known that mother cells are used as treatments for cancer, Parkinsons, spinal cord injury, type 1 diabetes, or Alzheimers diseases, among other. How could it be otherwise, nowadays also It has been revealed that these types of umbilical cord bodies could help people in the disease against COVID-19.

This information has been notified by the doctor Camilo Ricordi, director of Diabetes Research Institute (DRI) and from Cell Transplant Center at the University of Miami Miller School of Medicine, and his team of international collaborators (The Cure Alliance ), who have carried out an innovative test that has shown that Umbilical Cord Derived Mesenchymal Stem Cell (UC-MSC) Infusions Safely Reduce Risk of Death and Accelerate Recovery Time for the Most Severely Covid-19 Patients, as published in the magazine Stem Cells Translational Medicine (SCTM).

The study was cleared by the United States Food and Drug Administration (FDA) in April and started by The Cure Alliance, a organization non-profit, directed by Ricordi, of which research scientists are part who share knowledge with each other that serves to accelerate the cures of all kinds of diseases.

At the beginning of the pandemic, Ricordi created the Mini-Manhattan project, which has finally yielded important results on the possible treatment of stem cells from the umbilical cord to treat the disease of COVID-19 in people who have suffered serious consequences.

To conduct the study, the researchers analyzed the cases of 24 patients hospitalized at the University of Miami Tower and Jackson Memorial Hospital that they had developed severe acute respiratory distress because of coronavirus. As part of the trial, the scientists they tried giving two infusions of mesenchymal stem cells and placebos to hospitalized patients several days apart.

Its like the technology of a smart pump in the lungs to restore the normal immune response and reverse life-threatening complications, Ricordi notes in the study.

Our results confirm the powerful anti-inflammatory and immunomodulatory effect of UC-MSCs. These cells have clearly inhibited the cytokine storm, a hallmark of severe COVID-19. Add Giacomo Lanzoni, lead author of the research.

The results are critically important not only for COVID-19, but also for other diseases characterized by aberrant and hyper-inflammatory immune responses, such as autoimmune type 1 diabetes. We are eager to apply these cells in clinical trials to halt the progression of type 1 diabetes, he concludes. Lanzoni in Stem Cells Translational Medicine (SCTM)..

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January 2021: 2020 Papers of the Year – Environmental Factor Newsletter

By daniellenierenberg

Research funded by grantsPFAS linked with liver injury in children

Exposure to per- and polyfluoroalkyl substances (PFAS) in the womb may increase liver injury risk in children, according to NIEHS-funded researchers. This study is the first to examine the impact of early life exposures to a PFAS mixture on child liver injury. PFAS, a large group of synthetic chemicals found in a variety of consumer products, have been linked to immune dysfunction, altered metabolism, brain development, and certain cancers.

The study used data from 1,105 mothers and their children enrolled in the Human Early-Life Exposome, or HELIX, study in Europe. Using computational modeling, the scientists found that higher exposures to PFAS during pregnancy were associated with higher levels of liver enzymes in children. High liver enzyme levels may point to nonalcoholic fatty liver disease (NAFLD). The researchers also identified a profile for children at high risk for liver injury, characterized by high prenatal PFAS exposures.

Citation: Stratakis N, Conti DV, Jin R, Margetaki K, Valvi D, Siskos AP, Maitre L, Garcia E, Varo N, Zhao Y, Roumeliotaki T, Vafeiadi M, Urquiza J, Fernandez-Barres S, Heude B, Basagana X, Casas M, Fossati S, Grazuleviciene R, Andrusaityte S, Uppal K, McEachan RRC, Papadopoulou E, Robinson O, Haug LS, Wright J, Vos MB, Keun HC, Vrijheid M, Berhane KT, McConnell R, Chatzi L. 2020. Prenatal exposure to perfluoroalkyl substances associated with increased susceptibility to liver injury in children. Hepatology 72(5):17581770. (Synopsis(

In an NIEHS-funded study, researchers uncovered a previously unknown way that genes code for proteins. Rather than directions going one way from DNA through messenger RNA (mRNA) to proteins, the study showed that RNA can modify how DNA is transcribed into mRNA and translated to produce proteins.

Using mouse stem cells, the scientists found that mRNA modifies how DNA is transcribed using a reversible chemical reaction called methylation, which can change the activity of a DNA segment without changing the sequence. The researchers identified and characterized several proteins that recognized the methylated mRNA. They also discovered a group of RNAs called chromosome-associated regulatory RNAs (carRNAs) that used the same methylation process and controlled how DNA was stored and transcribed. The team found that a specific methylation modification, N6-methyladenosine, served as a switch to control carRNA levels, which regulated DNA transcription.

Citation: Liu J, Dou X, Chen C, Chen C, Liu C, Xu MM, Zhao S, Shen B, Gao Y, Han D, He C. 2020. N 6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367(6477):580586. (Synopsis(

Loss of the enzyme topoisomerase 1 (TOP1) leads to DNA damage in neurons and neurodegeneration, according to an NIEHS-funded study. TOP1 plays an important role in facilitating the expression of long genes that are important for neuronal function. The data suggest that TOP1 maintains proper gene function in the central nervous system.

The researchers deleted TOP1 in mouse neurons and examined behavior, development, and underlying indicators of neurodegeneration, such as inflammation. Mice lacking TOP1 showed signs of early neurodegeneration, with brains 3.5-times smaller at postnatal day 15 compared with controls. Although neurons developed normally, mice without TOP1 showed motor deficits, exhibited lower levels of nicotinamide adenine dinucleotide (NAD-plus) a compound critical in energy metabolism and died prematurely. However, when these mice received supplemental NAD-plus, they lived 30% longer, had less inflammation, and showed improved neuronal survival.

Citation: Fragola G, Mabb AM, Taylor-Blake B, Niehaus JK, Chronister WD, Mao H, Simon JM, Yuan H, Li Z, McConnell MJ, Zylka MJ. 2020. Deletion of topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration. Nat Commun 11(1):1962. (Synopsis(

NIEHS grantees found that a protein known as XPA bends DNA and pauses in response to DNA damage, revealing the location of damaged DNA and potentially promoting the recruitment of DNA repair proteins. Using single molecule experiments and imaging techniques, the researchers observed the biochemistry of a living cell.

The researchers used a new method to calculate the molecular weight of small proteins bound to DNA and tracked proteins involved in DNA repair in 3D using real-time single molecule imaging. XPA cycled through three distinct states on DNA: rapidly hopping over long distances of the DNA strand; slowly sliding over short ranges of DNA while bending local DNA regions; and pausing and forming complexes with bent DNA. XPA paused more frequently in the presence of more DNA damage. The work provided insight into a new damage sensor role for XPA.

Citation: Beckwitt EC, Jang S, Detweiler IC, Kuper J, Sauer F, Simon N, Bretzler J, Watkins SC, Carell T, Kisker C, Van Houten B. 2020. Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search. Nat Commun 11(1):1356. (Synopsis(

NIEHS grantees found that individual cells in a population respond differently to estrogen stimulation at both the level of single cells and alleles, which are other possible forms of a gene. These differences were not explained by estrogen receptor levels in the cells or receptor activation status.

The researchers treated human breast cancer cells with estrogen and examined two genes, GREB1 and MYC, whose activities are regulated by estrogen. Unexpectedly, individual cells exhibited large differences in the level of gene activation, even between alleles within the same cell. The scientists used automated high-throughput technologies to test small molecule inhibitors of the estrogen receptor regulators. One inhibitor, called MS049, markedly increased the response of individual alleles to estrogen. The researchers altered estrogenic response by inhibiting estrogen receptor regulators, establishing a previously unrecognized regulation path for estrogen to activate genes at the single cell level.

Citation: Stossi F, Dandekar RD, Mancini MG, Gu G, Fuqua SAW, Nardone A, De Angelis C, Fu X, Schiff R, Bedford MT, Xu W, Johansson HE, Stephan CC, Mancini MA. 2020. Estrogen-induced transcription at individual alleles is independent of receptor level and active conformation but can be modulated by coactivators activity. Nucleic Acids Res 48(4):18001810. (Synopsis(

NIEHS grantees showed that mice exposed to e-cigarette smoke (ECS) were more likely to develop lung adenocarcinomas, a type of lung cancer. They also found that exposed mice had higher levels of bladder urothelial hyperplasia, an abnormal increase in epithelial cells that can precede development of bladder tumors.

The researchers exposed one group of mice to ECS aerosols generated from e-juice containing nicotine and compared them to a second group of mice exposed to a control aerosol without ECS. A third group of mice was exposed only to filtered air. Of the ECS mice, 22.5% developed lung adenocarcinomas and 57.5% developed urothelial hyperplasia. Mice with ECS-induced lung adenocarcinomas were not more prone to developing urothelial hyperplasia, which suggested that the two outcomes were divergent events and might involve different mechanisms.

Citation: Tang MS, Wu XR, Lee HW, Xia Y, Deng FM, Moreira AL, Chen LC, Huang WC, Lepor H. 2019. 2019. Electronic-cigarette smoke induces lung adenocarcinoma and bladder urothelial hyperplasia in mice. Proc Natl Acad Sci U S A 116(43):2172721731. (Synopsis(

NIEHS grantees identified a novel pathway that controls the metabolic response of astrocytes, which are brain and spinal cord cells essential to maintaining central nervous system (CNS) health. Although astrocytes perform various functions, such as providing nerve cells with nutrients, they have been linked to CNS inflammation and multiple sclerosis (MS).

Using a mouse model of MS, researchers found that during the progressive phase of the disease, brain astrocytes switched on metabolic pathways that activated a protein called the mitochondrial antiviral signaling (MAVS) protein. It led to activation of several proinflammatory genes, triggering inflammation in the brain and spinal cord. If the scientists gave the mice the drug miglustat before the onset of MS, they were able to suppress MAVS activation and subsequent inflammation. The findings suggest a new role for MAVS in CNS inflammation and a potential therapeutic target for MS.

Citation: Chao CC, Gutierrez-Vazquez C, Rothhammer V, Mayo L, Wheeler MA, Tjon EC, Zandee SEJ, Blain M, de Lima KA, Takenaka MC, Avila-Pacheco J, Hewson P, Liu L, Sanmarco LM, Borucki DM, Lipof GZ, Trauger SA, Clish CB, Antel JP, Prat A, Quintana FJ. 2019. Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS. Cell 179(7):14831498.e22. (Synopsis(

NIEHS-funded researchers found that a mutation in the ultraviolet irradiation resistanceassociated gene (UVRAG), which is involved in cell regulation, can disrupt autophagy in mice. Autophagy is the process of removing damaged cells so the body can regenerate newer cells. The scientists say the UVRAG mutation causes increased inflammatory response and tumor development. The study provides the first genetic evidence connecting UVRAG suppression to autophagy regulation, inflammation, and cancer predisposition.

The researchers generated mice that expressed UVRAG with a frameshift mutation, which is a deletion or insertion in DNA that shifts the way the sequence is read. After inducing sepsis or intestinal colitis, they found that mice with the UVRAG mutation displayed increased inflammatory responses in both conditions and increased spontaneous tumor development compared with wild-type mice. The results indicate UVRAG could be one reason people are more susceptible to cancers as they age.

Citation: Quach C, Song Y, Guo H, Li S, Maazi H, Fung M, Sands N, O'Connell D, Restrepo-Vassalli S, Chai B, Nemecio D, Punj V, Akbari O, Idos GE, Mumenthaler SM, Wu N, Martin SE, Hagiya A, Hicks J, Cui H, Liang C. 2019. A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice. Nat Commun 10(1):5681. (Synopsis(

Exposure to polybrominated biphenyl (PBB) 153, a type of brominated flame retardant, alters DNA methylation in sperm, according to NIEHS grantees. DNA methylation refers to heritable changes in gene expression that occur with no alteration in the DNA sequence. Because PBB153 is toxic to living organisms following direct exposure, the study suggests it may also harm future generations.

The results of a Michigan PBB study showed that PBB153 was associated with gene methylation events in mens sperm. Based on this information, the research team conducted sperm studies and determined that exposure to PBB153 decreased methylation at regions of DNA that control imprinted genes, which are essential for fetal growth and play an important role in other aspects of development. These effects could explain some of the endocrine-related health effects that have been observed among children of PBB-exposed parents.

Citation: Greeson KW, Fowler KL, Estave PM, Thompson SK, Wagner C, Edenfield RC, Symosko KM, Steves AN, Marder EM, Terrell ML, Barton H, Koval M, Marcus M, Easley CA 4th. 2020. Detrimental effects of flame retardant, PBB153, exposure on sperm and future generations. Sci Rep 10(1):8567. (Synopsis(

NIEHS grantees determined that in mice, air pollution may play a role in the development of cardiometabolic diseases, such as diabetes, with effects comparable to eating a high-fat diet (HFD). They also established that effects were reversed when exposure to air pollution stopped.

The scientists divided male mice into three categories: those that received clean filtered air; those exposed to concentrated particulate matter 2.5 air pollution; and those that received clean filtered air and were fed an HFD. After 14 weeks, team members measured insulin resistance and glucose levels and assessed epigenetic changes, or chemical tags, that attach to DNA and affect gene expression.

Air pollution exposure was comparable to eating an HFD. Mice in the air pollution and HFD groups had impaired insulin resistance, high glucose, and reduced metabolism. After removing air pollution from the environment, health and epigenetic changes reversed within eight weeks.

Citation: Rajagopalan S, Park B, Palanivel R, Vinayachandran V, Deiuliis JA, Gangwar RS, Das LM, Yin J, Choi Y, Al-Kindi S, Jain MK, Hansen KD, Biswal S. 2020. Metabolic effects of air pollution exposure and reversibility. J Clin Invest 130(11):60346040. (Synopsis(

NIEHS researchers learned that mineralocorticoid receptors (MRs) control the gene profiles of neurons within the CA2 brain region, which is associated with learning and memory. MRs are a type of steroid receptor activated by corticosteroid hormones. The findings revealed the essential roles of MRs in the development and maintenance of CA2 neurons, as well as CA2-related behaviors.

In response to environmental stress, the body secretes corticosteroids that bind to MRs or glucocorticoid receptors and that induce gene expression changes in the brain. The CA2 region of the mouse and human hippocampus is enriched with MRs. Neuronal deletion of MRs at embryonic, early postnatal development, or adulthood stages in mice led to significantly reduced expression of CA2 molecular markers. Mice with CA2-targeted deletion of MRs showed disrupted social behavior and altered responses to novel objects. Therefore, MRs control both the identity and function of CA2 neurons.

Citation: McCann KE, Lustberg DJ, Shaughnessy EK, Carstens KE, Farris S, Alexander GM, Radzicki D, Zhao M, Dudek SM. 2019. Novel role for mineralocorticoid receptors in control of a neuronal phenotype. Mol Psychiatry; doi: 10.1038/s41380-019-0598-7 [Online 19 November 2019]. (Synopsis(

NIEHS researchers discovered a novel symbiotic interaction between mammalian cells and bacteria that boosts nicotinamide adenine dinucleotide biosynthesis in host cells. NAD is a cofactor that exists in all cell types and is necessary for life. Decreased levels of NAD are associated with aging, and elevated levels of its biosynthesis are important to sustain the higher metabolic needs of tumors.

The researchers showed that cancer cell lines infected with Mycoplasma hyorhinis were protected against toxicity by nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, which halt NAD biosynthesis. This same effect was observed in vivo, when infected versus noninfected cancer cells were injected in mice. Using a variety of screens and techniques, they showed that this resistance was the result of bacteria providing alternative NAD precursors to mammalian cells through the bacterial nicotinamidase PncA, bypassing the NAMPT-dependent pathway.

Citation: Shats I, Williams JG, Liu J, Makarov MV, Wu X, Lih FB, Deterding LJ, Lim C, Xu X, Randall TA, Lee E, Li W, Fan W, Li J-L, Sokolsky M, Kabanov AV, Li L, Migaud ME, Locasale JW, Li X. 2020. Bacteria boost mammalian host NAD metabolism by engaging the deamidated biosynthesis pathway. Cell Metab 31(3):564579.e7. (Synopsis( (Story)

New insights into how the liver adapts to an HFD may lead to novel treatments for obesity-related diseases such as NAFLD, according to a study by NIEHS researchers. They found that long-term consumption of a diet high in saturated fat led to dramatic reprogramming of gene regulation in the mouse liver.

NAFLD involves the buildup of excessive fat in the liver of an individual who is not a heavy user of alcohol, increasing the risk of liver damage. When the scientists fed mice an HFD, the mice became obese and showed other changes similar to metabolic syndrome in humans. Moreover, their livers became fatty and showed wide-ranging abnormalities at both molecular and cellular levels. The livers adaptation to the fat-rich diet was mediated by a protein called hepatocyte nuclear factor 4 alpha.

Citation: Qin Y, Grimm SA, Roberts JD, Chrysovergis K, Wade PA. 2020. Alterations in promoter interaction landscape and transcriptional network underlying metabolic adaptation to diet. Nat Commun 11(1):962. (Synopsis( (Story)

An NIEHS study reported a concerning rise in the prevalence of antinuclear antibodies (ANAs), which are commonly used biomarkers for autoimmunity. ANAs, which are produced by a persons own immune system, bind to and sometimes attack healthy cells. This study is the first to evaluate ANA changes over time in a representative sampling of the U.S. population. The findings may indicate an increase in autoimmune diseases.

Team members used the National Health and Nutrition Examination Survey to analyze serum ANAs in 14,211 participants aged 12 years and older from three time periods. ANA prevalence increased as follows.

The researchers found the largest ANA increases in adolescents, males, non-Hispanic whites, and adults older than 50 years compared with other subgroups.

Citation: Dinse GE, Parks CG, Weinberg CR, Co CA, Wilkerson J, Zeldin DC, Chan EKL, Miller FW. 2020. Increasing prevalence of antinuclear antibodies in the United States. Arthritis Rheumatol 72(6):10261035. (Synopsis( (Story)

Ubiquitin (Ub) stimulates the removal of topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) by tyrosyl-DNA phosphodiesterase 2 (TDP2) according to NIEHS researchers and their collaborators in Spain. The team also reported that TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ub interface. Because TDP2 works with a protein called ZATT to remove dangerous DNA-protein crosslinks, the work is important for understanding how cells handle this type of DNA damage.

Using X-ray crystallography and small angle X-ray scattering analysis, the scientists examined how Ub-dependent links and TDP2 function as they relate to DNA repair and other cellular pathways. Previous studies hypothesized that TDP2 interacts with K48-Ub chains to promote recruitment to TOP2-DPCs that are repaired using a proteasome-mediated TOP2 degradation pathway. However, the authors showed that TDP2 preferentially binds to K63-linked Ub3 and associates with K27 and K63 poly-Ub chains.

Citation: Schellenberg MJ, Appel CD, Riccio AA, Butler LR, Krahn JM, Liebermann JA, Cortes-Ledesma F, Williams RS. 2020. Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2. Nucleic Acids Res 48(11):63106325. (Synopsis(

NIEHS researchers and their collaborators concluded that a protein called tankyrase serves a critical role in mammalian embryonic genome activation (EGA). Using an in vitro culture system, the researchers identified and characterized tankyrase, a factor that allows EGA to occur. The characterization of tankyrase during the oocyte-to-embryo transition fills a gap in knowledge about how factors are activated in mammalian oocytes and early embryos and may lead to improved strategies for treating infertility.

Using a mouse model, the scientists depleted tankyrase from the embryos and observed that they could not perform EGA and stopped developing. They also found that tankyrase is necessary for gene transcription, protein translation, DNA damage repair, and modulation of beta-catenin in the early embryo. This study found a new role for tankyrase during normal development, revealing an essential function of this protein during the oocyte-to-embryo transition.

Citation: Gambini A, Stein P, Savy V, Grow EJ, Papas BN, Zhang Y, Kenan AC, Padilla-Banks E, Cairns BR, Williams CJ. 2020. Developmentally programmed tankyrase activity upregulates beta-catenin and licenses progression of embryonic genome activation. Dev Cell 53(5):545560.e7. (Synopsis( (Story)

NIEHS researchers showed that an enzyme called CLP1 plays an important role in transfer RNA (tRNA) processing by regulating the ligation of tRNAs. They also demonstrated that mature, functional tRNAs are generated from pre-tRNAs through a process called TSEN, or (tRNA splicing endonuclease)mediated splicing of introns. Mutations in CLP1 and the TSEN complex often lead to severe neurological disorders.

Using a technique that allowed Escherichia coli to produce several proteins at once, the scientists expressed and reconstituted the TSEN protein complex, which cleaved tRNA. TSEN complex alone was sufficient for removing tRNA introns, but CLP1, a binding partner for TSEN, was needed to correctly regulate the ligation step that generates mature tRNAs and tRNA intronic circular RNAs (tricRNAs). Genetic knockdown of CLP1 led to increases in mature tRNAs and tricRNAs, which suggested that CLP1 acts as a negative modulator of tRNA processing.

Citation: Hayne CK, Schmidt CA, Haque MI, Matera AG, Stanley RE. 2020. Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage. Nucleic Acids Res 48(14):76097622. (Synopsis(

Researchers at NIEHS and the National Toxicology Program developed the Tox21BodyMap to predict which organs in the human body may be affected by a chemical. The tool will help scientists generate novel hypotheses to test, prioritize chemicals for toxicity testing, and identify knowledge gaps.

To identify organs that could potentially be affected by a chemical, Tox21BodyMap used data from 971 high-throughput screening assays that evaluated approximately 10,000 unique chemicals. Specifically, it combined information about which gene an assay targets, how highly expressed that gene is in a human organ, and at what tested concentrations a chemical generated a positive assay result. The result was an overall picture of chemical bioactivity. The Tox21BodyMap provided multiple visualizations of the data, highlighting target organs on a map of the body, as well as showing a web of network connections and providing downloadable data.

Citation: Borrel A, Auerbach SS, Houck KA, Kleinstreuer NC. 2020. Tox21BodyMap: a webtool to map chemical effects on the human body. Nucleic Acids Res 48(W1):W472W476. (Synopsis(

In pregnant women, polyunsaturated fatty acids and their metabolic derivatives called eicosanoids are associated with infant size at delivery, according to NIEHS scientists and their collaborators. This work also provides novel longitudinal characterization of eicosanoids in blood plasma during different gestational ages of pregnancy. The results link inflammatory eicosanoids with adverse fetal growth outcomes.

The blood plasma concentration of polyunsaturated fatty acids, including omega-3 and omega-6, in study participants was found to be higher in cases of low birth weight and lower in cases of higher birth weight. Lower and higher birth weights were defined as equal to or less than the 10th percentile and equal to or greater than the 90th percentile for gestational age, respectively. In addition, certain eicosanoids, which are known to derive from inflammatory processes from these fatty acids, were found to be exclusively higher in pregnancy cases, which resulted in low birth weight.

Citation: Welch BM, Keil AP, van't Erve TJ, Deterding LJ, Williams JG, Lih FB, Cantonwine DE, McElrath TF, Ferguson KK. 2020. Longitudinal profiles of plasma eicosanoids during pregnancy and size for gestational age at delivery: a nested case-control study. PLoS Med 17(8):e1003271. (Synopsis(

Researchers at NIEHS and collaborators at the National Institute of Diabetes and Digestive and Kidney Diseases uncovered the neural basis behind the drive to select calorie-rich foods over nutritionally balanced diets. The findings partly explain the difficulty of dieting.

One group of mice received a standard diet (SD) consisting of regular chow, and another group ate an HFD. When the HFD mice were switched to a SD, they refused to eat. Even after fasting to stimulate their appetites, HFD mice preferred fatty food, rather than regular chow.

However, whenHFD mice were switched to a SD, regular chow no longer fully alleviated the response. The authors also saw that dopamine signaling, which is responsible for the pleasurable feelings from eating, were significantly diminished in the SD mice following HFD exposure.

Citation: Mazzone CM, Liang-Guallpa J, Li C, Wolcott NS, Boone MH, Southern M, Kobzar NP, Salgado IA, Reddy DM, Sun F, Zhang Y, Li Y, Cui G, Krashes MJ. 2020. High-fat food biases hypothalamic and mesolimbic expression of consummatory drives. Nat Neurosci 23(10):12531266. (Synopsis(

To uncover novel deletion patterns in mitochondrial DNA (mtDNA), NIEHS researchers and their collaborators developed LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. The team used the technique to reveal links between mitochondrial DNA replication, aging, and mitochondrial disease.

A mutation in POLG, a nuclear gene responsible for maintaining the mitochondrial genome, is known to be the most common cause of mitochondrial disease, a condition in which the mitochondria fail to produce enough energy for the body to function properly.

The scientists analyzed mtDNA from skeletal muscle biopsies of 41 patients with mitochondrial disease with wild-type and mutated POLG. They used LostArc to detect loss of mtDNA segments by mapping split-reads in the samples to a normal mtDNA reference. Thirty-five million deletion segments were detected in the biopsies. They spanned more than 470,000 unique segments, 99% of which were novel.

Citation: Lujan SA, Longley MJ, Humble MH, Lavender CA, Burkholder A, Blakely EL, Alston CL, Gorman GS, Turnbull DM, McFarland R, Taylor RW, Kunkel TA, Copeland WC. 2020. Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging. Genome Biol 21(1):248. (Synopsis(

Individual heterogeneity, or genetic variability among samples, can substantially affect reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), according to NIEHS scientists and their collaborators. iPSCs are stem cells that are derived from differentiated cells, such as fibroblasts, and they can both self-renew and are pluripotent, meaning they can be differentiated into other cell types. In a previous publication, the research team obtained fibroblasts from healthy diverse donors and observed that each persons fibroblasts had consistent differences in the ability to be reprogrammed to iPSCs. Ancestry was identified as a large contributing factor.

Using 72 dermal fibroblast-iPSCs from self-identified African Americans and White Americans, the researchers found ancestry-dependent and ancestry-independent genes associated with reprogramming efficiency. They also added 36 new genomic profiles of African American fibroblast-iPSCs pairs to publicly available databases, which will help address the underrepresentation of genomic data from non-European groups.

Citation: Bisogno LS, Yang J, Bennett BD, Ward JM, Mackey LC, Annab LA, Bushel PR, Singhal S, Schurman SH, Byun JS, Napoles AM, Perez-Stable EJ, Fargo DC, Gardner K, Archer TK. 2020. Ancestry-dependent gene expression correlates with reprogramming to pluripotency and multiple dynamic biological processes. Sci Adv 6(47):eabc3851. (Synopsis(

Researchers in the Division of the National Toxicology Program (DNTP) at NIEHS successfully compiled a rich resource to explore data on polycyclic aromatic compound (PACs) toxicity. This data-driven approach to contextualizing PAC hazard characterization allows researchers to predict eight different toxicity profiles of various PACs and other classes of compounds.

PACs are a structurally diverse class of human-made toxicants found widely in the environment. Unfortunately, information about human exposure and health effects of PACs is limited. To facilitate greater understanding of PAC toxicity in a cost-effective manner, DNTP researchers created an automated approach to identify PAC structures using computer workflows, algorithms, and clusters. Using existing data on similar compounds, the scientists categorized PACs based on structure and hazard characterization. The analysis results are available and searchable through an interactive web application.

Citation: Hsieh JH, Sedykh A, Mutlu E, Germolec DR, Auerbach SS, Rider CV. 2020. Harnessing in silico, in vitro, and in vivo data to understand the toxicity landscape of polycyclic aromatic compounds (PACs). Chem Res Toxicol; doi:10.1021/acs.chemrestox.0c00213 [Online 16 October 2020]. (Synopsis(

DNTP scientists and their collaborators used computational modeling to probe databases and to identify existing drugs that could be repurposed to fight SARS-CoV-2, the virus that causes COVID-19.

Proteases are enzymes that break down proteins. An essential step in the formation of infectious viral particles is the breakdown of precursor viral proteins by viral proteases. A class of antiviral drugs called protease inhibitors block the activity of viral proteases. The main protease (Mpro) of SARS-CoV-2 is a proposed target for COVID-19 drugs. The structure and activity of Mpro is highly conserved across the coronavirus family. In this study, previous data on drug interactions with SARS-CoV Mpro were used to develop quantitative structure-activity relationship models, which the team used to virtually screen all drugs in the DrugBank database. They identified 42 drugs that could be repurposed against SARS-CoV-2 Mpro.

Citation: Alves VM, Bobrowski T, Melo-Filho CC, Korn D, Auerbach S, Schmitt C, Muratov EN, Tropsha A. 2020. QSAR modeling of SARS-CoV Mpro inhibitors identifies sufugolix, cenicriviroc, proglumetacin, and other drugs as candidates for repurposing against SARS-CoV-2. Mol Inform; doi:10.1002/minf.202000113 [Online 28 July 2020]. (Synopsis(

DNTP scientists evaluated a high-throughput transcriptomics approach using liver and kidney tissue from 5-day assays in male rats to estimate the toxicological potency of chemicals.

Toxicity and carcinogenicity are typically assessed by the resource intensive two-year cancer bioassay. In the 5-day assays, the authors determined toxicological potency based on the most sensitive sets of genes active in the liver and kidney. For most chemicals, the results approximated the toxicological potency derived from the most sensitive histopathological effects independent of target tissue or organ observed in male rats in long-term assays. Notably, these approximations were similar in female rats, as well as in male and female mice. The findings suggest that estimates of transcriptomics-based potency from short-term in vivo assays can, in the absence of other data, provide a rapid and effective estimate of toxicological potency.

Citation: Gwinn WM, Auerbach SS, Parham F, Stout MD, Waidyanatha S, Mutlu E, Collins B, Paules RS, Merrick BA, Ferguson S, Ramaiahgari S, Bucher JR, Sparrow B, Toy H, Gorospe J, Machesky N, Shah RR, Balik-Meisner MR, Mav D, Phadke DP, Roberts G, DeVito MJ. 2020. Evaluation of 5-day in vivo rat liver and kidney with high-throughput transcriptomics for estimating benchmark doses of apical outcomes. Toxicol Sci 176(2):343354. (Synopsis(

Researchers from DNTP studied the effects of gestational and postnatal boron exposure on developing rat pups. The team was the first to show that pups exposed to boric acid, an oxidized form of boron commonly found in the environment, gained significantly less weight during postnatal development.

Pregnant rats were exposed to varying concentrations of boric acid once daily by oral gavage dosing, a technique that administered it directly to the stomach. Food intake, body weight, boron blood plasma levels, and any signs of morbidity were evaluated during gestation. After birth, the pups received boric acid at the same concentration as their mothers, and the scientists monitored the same parameters in the pups for the next 28 days. The team observed that the pups that received the highest dose of boric acid had a 23% reduction in weight gain.

Citation: Watson ATD, Sutherland VL, Cunny H, Miller-Pinsler L, Furr J, Hebert C, Collins B, Waidyanatha S, Smith L, Vinke T, Aillon K, Xie G, Shockley KR, McIntyre BS. 2020. Postnatal effects of gestational and lactational gavage exposure to boric acid in the developing Sprague Dawley rat. Toxicol Sci 176(1):6573. (Synopsis(

When scientists from DNTP analyzed the entire genetic code of tumors in rodent cancer studies, they determined that most rodent tumors whether arising spontaneously or induced by chemicals had DNA mutation signatures resembling those seen in human cancers.

Tumors can form as a result of DNA damage or they can arise spontaneously when physiological processes do not function properly. To understand the mechanism of cancer formation, members of the research team sequenced lung and liver tumor DNA from mice exposed to 20 carcinogens. They compared the sequences to those from tumors that formed spontaneously and from normal tissue. DNA signatures from exposure to 17 of the chemicals were similar to those from spontaneous tumors in mice. The finding suggests chemicals promote tumor formation through mechanisms that build on existing cancer processes.

Citation: Riva L, Pandiri AR, Li YR, Droop A, Hewinson J, Quail MA, Iyer V, Shepherd R, Herbert RA, Campbell PJ, Sills RC, Alexandrov LB, Balmain A, Adams DJ. 2020.The mutational signature profile of known and suspected human carcinogens in mice. Nat Genet 52(11):11891197. (Story)

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January 2021: 2020 Papers of the Year - Environmental Factor Newsletter

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Global Cell Therapy Market Report 2020: Market to Recover in 2023 – PRNewswire

By daniellenierenberg

DUBLIN, Dec. 31, 2020 /PRNewswire/ -- The "Cell Therapy Global Market Report 2020-30: COVID-19 Growth and Change" report has been added to's offering.

Cell Therapy Global Market Report 2020-30: COVID 19 Growth and Change provides the strategists, marketers and senior management with the critical information they need to assess the global cell therapy market.

Major players in the cell therapy market are Fibrocell Science Inc., JCR Pharmaceuticals Co. Ltd., PHARMICELL Co. Ltd., Osiris Therapeutics Inc., MEDIPOST, Vericel Corporation, Anterogen Co. Ltd., Kolon TissueGene Inc., Stemedica Cell Technologies Inc. and AlloCure.

The global cell therapy market is expected to decline from $7.31 billion in 2019 to $7.2 billion in 2020 at a compound annual growth rate (CAGR) of -1.54%. The decline is mainly due to the COVID-19 outbreak that has led to restrictive containment measures involving social distancing, remote working, and the closure of industries and other commercial activities resulting in operational challenges. The entire supply chain has been disrupted, impacting the market negatively. The market is then expected to recover and reach $10.0 billion in 2023 at a CAGR of 11.55%.

The cell therapy market consists of sales of cell therapy and related services. Cell therapy (CT) helps repair or replace damaged tissues and cells. A variety of cells are used for the treatment of diseases includes skeletal muscle stem cells, hematopoietic (blood-forming) stem cells (HSC), lymphocytes, mesenchymal stem cells, pancreatic islet cells, and dendritic cells.

North America was the largest region in the cell therapy market in 2019. Asia Pacific is expected to be the fastest-growing region in the forecast period.

The cell therapy market covered in this report is segmented by technique into stem cell therapy; cell vaccine; adoptive cell transfer (ACT); fibroblast cell therapy; chondrocyte cell therapy. It is also segmented by therapy type into allogeneic therapies; autologous therapies, by application into oncology; cardiovascular disease (CVD); orthopedic; wound healing; others.

In August 2019, Bayer AG, a Germany-based pharmaceutical and life sciences company, acquired BlueRock Therapeutics, an engineered cell therapy company, for $1 billion. Through this transaction, Bayer AG will acquire complete BlueRock Therapeutics' CELL+GENE platform, including a broad intellectual property portfolio and associated technology platform including proprietary iPSC technology, gene engineering, and cell differentiation capabilities. BlueRock Therapeutics is a US-based biotechnology company focused on developing engineered cell therapies in the fields of neurology, cardiology, and immunology, using a proprietary induced pluripotent stem cell (iPSC) platform.

The high cost of cell therapy hindered the growth of the cell therapy market. Cell therapies have become a common choice of treatment in recent years as people are looking for the newest treatment options. Although there is a huge increase in demand for cell therapies, they are still very costly to try. Basic joint injections can cost about $1,000 and, based on the condition, more specialized procedures can cost up to $ 100,000. In 2020, the average cost of stem cell therapy can range from $4000 - $8,000 in the USA. Therefore, the high cost of cell therapy restraints the growth of the cell therapy market.

Key players in the market are strategically partnering and collaborating to expand the product portfolio and geographical presence of the company. For instance, in April 2018, Eli Lilly, an American pharmaceutical company entered into a collaboration agreement with Sigilon Therapeutics, a biopharmaceutical company that focused on the discovery and development of living therapeutics to develop cell therapies for type 1 diabetes treatment by using the Afibromer technology platform. Similarly, in September 2018, CRISPR Therapeutics, a biotechnological company that develops transformative medicine using a gene-editing platform for serious diseases, and ViaCyte, a California-based regenerative medicine company, collaborated on the discovery, development, and commercialization of allogeneic stem cell therapy for diabetes treatment.

The rising prevalence of chronic diseases contributed to the growth of the cell therapy market. According to the US Centers for Disease Control and Prevention (CDC), chronic disease is a condition that lasts for one year or more and requires medical attention or limits daily activities or both and includes heart disease, cancer, diabetes, and Parkinson's disease. Stem cells can benefit the patients suffering from spinal cord injuries, type 1 diabetes, Parkinson's disease (PD), heart disease, cancer, and osteoarthritis.

According to Cancer Research UK, in 2018, 17 million cancer cases were added to the existing list, and according to the International Diabetes Federation, in 2019, 463 million were living with diabetes. According to the Parkinson's Foundation, every year, 60,000 Americans are diagnosed with PD, and more than 10 million people are living with PD worldwide. The growing prevalence of chronic diseases increased the demand for cell therapies and contributed to the growth of the market.

Key Topics Covered:

1. Executive Summary

2. Cell Therapy Market Characteristics

3. Cell Therapy Market Size And Growth 3.1. Global Cell Therapy Historic Market, 2015 - 2019, $ Billion 3.1.1. Drivers Of The Market 3.1.2. Restraints On The Market 3.2. Global Cell Therapy Forecast Market, 2019 - 2023F, 2025F, 2030F, $ Billion 3.2.1. Drivers Of The Market 3.2.2. Restraints On the Market

4. Cell Therapy Market Segmentation 4.1. Global Cell Therapy Market, Segmentation By Technique, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

4.2. Global Cell Therapy Market, Segmentation By Therapy Type, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

4.3. Global Cell Therapy Market, Segmentation By Application, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

5. Cell Therapy Market Regional And Country Analysis 5.1. Global Cell Therapy Market, Split By Region, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion 5.2. Global Cell Therapy Market, Split By Country, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

Companies Mentioned

For more information about this report visit

Research and Markets also offers Custom Research services providing focused, comprehensive and tailored research.

Media Contact:

Research and Markets Laura Wood, Senior Manager [emailprotected]

For E.S.T Office Hours Call +1-917-300-0470 For U.S./CAN Toll Free Call +1-800-526-8630 For GMT Office Hours Call +353-1-416-8900

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Regenerative Medicine: Market Trends and Legal Developments on the Horizon for 2021 – MedTech Intelligence

By daniellenierenberg

As the second wave of the pandemic engulfs us and the world works at warp speed to develop vaccines and therapies to respond, the importance of regenerative medicine has never been higher. Since 2017, Goldman Sachs has touted the sector as one of the most compelling areas for venture capital investment. With billions of dollars of global government spending being poured into the search for vaccines and therapies to respond to the novel coronavirus, and with the FDA having now granted approval to the first vaccines based on CRISPR mRNA gene-editing technologies, business models based on regenerative medicines are commanding record values. Despite the flood of cash into regenerative medicine, legal and ethical considerations will continue to cause much controversy.

Regenerative medicine ultimately accelerates the human bodys healing process. It is an area of biomedical sciences that involves medical treatments to repair or replace damaged cells, tissues, or organs. Instead of merely focusing on the symptoms, regenerative medicine uses cellular therapies, tissue engineering, medical devices, and artificial organs to improve peoples health. For example, stem cell therapies, tissue grafts, and organ transplants are all part of regenerative medicine.

Today, cellular and acellular regenerative medicines are often used in clinical procedures such as cell, immunomodulation, and tissue engineering therapies. They have the potential to effectively treat many chronic diseases, including Alzheimers, Parkinsons and cardiovascular disorders, osteoporosis, and spinal cord injuries.

A small number of unscrupulous actors, according to the FDA, however, have seized on the clinical promise of regenerative medicine to offer patients unproven treatments. The FDA and other regulators are challenged to provide assurances of safety for these therapies without stifling development, as well as to approve treatments based on manipulation of stem cells derived from human and animal embryos given the ethical issues involved.

In the future, stem cell research will play an increasingly outsized role in regenerative medicine techniques. In November 2020, voters in California narrowly passed Proposition 14, a referendum to approve $5.5 billion in new government funding for stem cell research. Other governments around the world are doing the same.

Today, the growing prevalence of chronic medical ailments and genetic disorders across the globe is a primary factor driving the regenerative medicine industrys growth, according to the Regenerative Medicine Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast 2020-2025. The increasing aging population, prone to various musculoskeletal, oncological, dermatological, and cardiological disorders, is a key growth driver. Widespread adoption of organ transplantation is another contributing factor to this growth in market share. The current pandemic that began in January 2020, however, has changed the paradigm for regenerative medicine.

Market applications are burgeoning. Regenerative medicine can prevent and cure disease through effective vaccines and efficacious therapies. It can minimize the risk of organ rejection post-transplant and speed recovery. Technological advancements in cell-based therapies, such as the development of 3-D bioprinting techniques and the adoption of artificial intelligence in the production of regenerative medicines, are also stimulating growth. These advancements also facilitate dermatological grafting procedures to treat burns, bone defects, and skin wounds. Other factors, including extensive research and development activities in medical sciences and improving healthcare infrastructure, are also predicted to drive the market even further.

According to the Alliance for Regenerative Medicine, there are approaching approximately 1,000 companies focusing on this evolving area worldwide. These new companies are focusing on gene therapy, cell therapy and tissue engineering therapeutic developers. More than half of these companies are in North America, followed by almost a quarter in Europe and Israel and approximately 20% in Asia. More than 50% of these companies are focusing on cell therapy and gene therapy.

From 2014 to 2019, the global regenerative medicine market experienced a nearly 16% CAGR. Companies involved in gene and cell therapies as well as other regenerative medicine areas raised $4.8 billion during the first half of 2019, including $2.6 billion in the second quarter. Meanwhile, companies in Europe and Israel saw an acceleration of fundraising, with $1.3 billion amassed in just the first half of 2019, representing a 17% increase over the same period in 2018. Project Warp Speed has attracted billions of dollars of U.S. government spending, and similar efforts are ongoing in China, Russia, the European Union and among other major powers. Consequently, regenerative medicine has never before benefited from such a combination of public and private investment.

Whenever the viability and quality of human life are at stake, ethical and legal considerations always arise.

The modern ethical controversy surrounding regenerative medicine began in 1998 when research scientists at the University of Wisconsin succeeded in deriving and growing stem cells from early-stage human embryos. Ethicists and right-to-life activists protested that scientists were taking away human life (embryos) to conduct scientific experiments. Left unchecked, so the argument went, doctors could usurp nature and play God by developing the power to create and terminate life. A society where human life could be fundamentally perverted by medicine conjured up comparisons to Nazi Germany and Frankenstein. In 2001, then-U.S. President George W. Bush cut off federal funding for any research involving newly created embryonic stem cell lines, but agreed to continue funding research on 60 existing stem cell lines, where the life and death decision ha[d] already been made. The State of California responded in 2004 and again in 2020 with voter-approved programs directing billions of funding into stem cell research, making the region the global hub of regenerative medicine.

The use of human-derived embryonic stem cells, or animal-derived stem cells, continues to cause much controversy among ethicists and society at large. Some fear the risks of enrolling humans in experimental stem cell studies. Others fear the use of organs from human-animal chimeras in transplantation.

While these techniques have the potential to cure disease and save lives, they also have the potential to forever alter the nature of life as we know it and fundamental aspects of our society.

In the United States, legal jurisdiction for regulating regenerative medicine on a federal level lies with the FDA and in a patchwork of state laws, R&D funding programs and non-binding, NGO-promulgated statements of policy. The main responsibility of the FDA is to protect the public from dangerous products and ensure its safety, including overseeing medications for humans and animals, vaccines, and more.

During the Trump Administration, the FDA has largely focused on enabling developers to gain product approvals through a less burdensome and costly process. In numerous policy statements, the FDA under President Trump has deferred questions about the efficacy of new regenerative health products to the free markets, so long as they posed no serious safety or toxicity concerns.

The U.S. federal government is now transitioning to an administration led by President-elect Biden. The president-elect has spent many years advocating for increased R&D funding and going for moonshots. With a new mandate from the U.S. electorate to address the coronavirus, more money will be earmarked for regenerative medicines and stem cell research. How this will affect the release of new products into the market remains to be seen.

Regenerative medicine is poised to change the way we live, work and interact like never before. The fourth industrial revolution is upon us. CRISPR gene-editing technologies, facilitated by quantum-computing capabilities at the edge of a computer network powered by 5G telecommunications bandwidths, artificial intelligence and machine learning, have changed the game for regenerative medicine. We can foresee a day when those suffering from paralysis regain movement, when a damaged heart reverses course through regeneration, and when a diagnosis of Alzheimers Disease no longer means neurodegeneration. What a wonderful day that will be.

Changing the traditional healthcare model and moving from cure to prevention will take time.

The rise in chronic disease and the effort to reduce healthcare costs presents a large opportunity for the field of regenerative medicine.

As the continent becomes a bigger player, western companies should explore the potential prospects.

Topics from regenerative medicine to artificial intelligence to cannabis will be discussed.

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Regenerative Medicine: Market Trends and Legal Developments on the Horizon for 2021 - MedTech Intelligence

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Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo – Science Advances

By daniellenierenberg


Remyelination failure in multiple sclerosis (MS) is associated with a migration/differentiation block of oligodendroglia. The reason for this block is highly debated. It could result from disease-related extrinsic or intrinsic regulators in oligodendroglial biology. To avoid confounding immune-mediated extrinsic effect, we used an immune-deficient mouse model to compare induced pluripotent stem cellderived oligodendroglia from MS and healthy donors following engraftment in the developing CNS. We show that the MS-progeny behaves and differentiates into oligodendrocytes to the same extent as controls. They generate equal amounts of myelin, with bona fide nodes of Ranvier, and promote equal restoration of their host slow conduction. MS-progeny expressed oligodendrocyte- and astrocyte-specific connexins and established functional connections with donor and host glia. Thus, MS oligodendroglia, regardless of major immune manipulators, are intrinsically capable of myelination and making functional axo-glia/glia-glia connections, reinforcing the view that the MS oligodendrocyte differentiation block is not from major intrinsic oligodendroglial deficits.

Remyelination occurs in multiple sclerosis (MS) lesions but its capacity decreases over time (13). Failed remyelination in MS leads to altered conduction followed by axon degeneration, which, in the long run, results in severe and permanent neurological deficits (4). MS lesions may or may not harbor immature oligodendroglia (oligodendrocyte progenitors and pre-oligodendrocytes), with these cells failing to differentiate into myelin-forming cells, suggesting that oligodendrocyte differentiation is blocked (57). So far, the mechanism underlying this block is poorly understood. It may result from adverse environmental conditions or the failed capacity of oligodendrocyte progenitors/pre-oligodendrocytes to migrate or mature efficiently into myelin-forming cells or even a combination of these conditions, all of which may worsen with aging. It has been shown that increasing remyelination either through manipulating the endogenous pool (8, 9) or by grafting competent myelin forming oligodendroglia (10, 11) or both (12) can restore the lost axonal functions, improve the clinical scores, and protect from subsequent axonal degeneration in experimental (13, 14) or clinical (3) settings.

There are multiple ways to investigate the oligodendroglial lineage in disease. Cells can be studied in postmortem tissue sections or purified from postmortem adult human brain for in vitro and transcriptomic/proteomic analysis. In this respect, in vitro experiments highlighted the heterogeneity of the adult human oligodendrocyte progenitor population in terms of antigen and microRNA expression, suggesting that remyelination in the adult human brain involves multiple progenitor populations (15). Moreover, single-cell transcriptomics characterized in detail the heterogeneity of human oligodendroglial cells, emphasizing changes in MS, with some subpopulations expressing disease-specific markers that could play a role in disease onset and/or aggravation (16, 17).

Yet, this MS signature could preexist or be acquired early at disease onset. Moreover, most of these MS postmortem analyses or experimental models cannot overlook the involvement of extrinsic factors such as immune factors that might add more complexity toward understanding the behavior of MS oligodenroglial cells.

Little is known about the biology of the MS oligodendroglial lineage, primarily due to the impossibility, for ethical reasons, to harvest oligodendroglial populations from patients and study the diseased cells and their matching controls in vitro or in vivo after cell transplantation. While cell-cell interactions and cell heterogeneity in diseased conditions generate more complexity when comparing control and pathological samples, the induced pluripotent stem cell (iPSC) technology provides a unique opportunity to study homogeneous populations of human oligodendroglial cells and gain further insights into monogenetic diseases and multifactorial diseases, such as MS. The iPSC technology has unraveled differences in oligodendroglia biology, in Huntingtons disease (18), and schizophrenia (19, 20), indicating that these cells can contribute autonomously to multifactorial diseases outcome. However, so far, little is known about the potential contribution of MS oligodendroglia to failed remyelination. While senescence affects iPSCneural precursor cells (NPCs) derived from patients with primary progressive MS (PPMS) (21), only few preliminary reports alluded to the fate of PPMS (22, 23) or relapsing-remitting (RRMS) (24) iPSC-derived oligodendroglia after experimental transplantation and did not study per se their capacity to differentiate into functional myelin-forming cells. We exploited a robust approach (25) to generate large quantities of iPSCs-derived O4+ oligodendroglial cells from skin fibroblasts (hiOLs) of three RRMS and three healthy subjects, including two monozygous twin pairs discordant for the disease. As a critical feature of the pluripotent-derived cells should be their ability to fully integrate and function in vivo, we compared the capacity of healthy and MS-hiOL derivatives to integrate and restore axo-glial and glial-glial functional interactions after engraftment in the developing dysmyelinated murine central nervous system (CNS). Our data show that in noninflammatory conditions, the intrinsic properties of iPSC-oligodendroglial cells to differentiate, myelinate, and establish functional cell-cell interactions in vivo are not altered in MS, making them candidates of interest for personalized drug/cell therapies as pluripotency maintains MS oligodendroglial cells in a genuine nonpathological state.

Fibroblasts were isolated from three control and three patients with MS and reprogrammed into iPSC. Pluripotent cells were differentiated into NPCs and further into O4+ hiOLs for 12 days in vitro under glial differentiation medium (GDM) conditions as previously described (25). hiOL cells were selected using flow cytometry for O4 before transplantation. Because our aim was to study the intrinsic properties of MS cells, we chose to engraft O4+ hiOLs in the purely dysmyelinating Shi/Shi:Rag2/ mouse model to avoid confounding immune-mediated extrinsic effects.

We first questioned whether MS-hiOLs differed from control-hiOLs wild type (WT) in their capacity to survive and proliferate in vivo. To this aim, we grafted MS- and control-hiOLs in the forebrain of neonatal Shi/Shi:Rag2/ mice. MS cells engrafted (one injection per hemisphere) in the rostral forebrain, spread primarily through white matter, including the corpus callosum and fimbria, as previously observed using control human fetal (11, 26, 27) and iPSC (25, 28) progenitors. With time, cells also spread rostrally to the olfactory bulb and caudally to the brain stem and cerebellum (fig. S1). Examining engrafted brains at 8, 12, and 16 weeks postgraft (wpg), we found that MS-hiOLs expressing the human nuclear marker STEM101 and the oligodendroglial-specific transcription factor OLIG2 maintained a slow proliferation rate at all times (5 to 19% of STEM+ cells), with no difference in Ki67+ MS-hiOLs compared to control (Fig. 1, A and C). Moreover, immunostaining for cleaved Caspase3 at 8 wpg indicated that MS cells survived as well as control-hiOLs (Fig. 1, B and D). Evaluation of the cell density of human cells based on STEM positivity at each stage revealed no significant difference between grafted MS-hiOLs and control cells (fig. S2).

(A and C) Immunodetection of the human nuclei marker STEM101 (red) combined with OLIG2 (green) and the proliferation marker Ki67 (white) shows that a moderate proportion of MS-hiOLs sustains proliferation (empty arrowheads in the insets) following transplantation in their host developing brain, with no significant difference in the rate of proliferation between MS- and control-hiOLs over time. (B and D) Immunodetection of the apoptotic marker Caspase3 (green) indicates that MS-hiOLs survive as well as control-hiOLs 8 wpg. Two-way analysis of variance (ANOVA) followed by Tukeys multiple comparison or Mann-Whitney t tests were used for the statistical analysis (n = 3 to 4 mice per group). Error bars represent SEMs. H, Hoechst dye. Scale bars, 100 m.

Because MS-hiOLs and control cells proliferated and survived to the same extent, we next questioned whether their differentiation potential into mature oligodendrocytes could be affected. We used the human nuclei marker STEM101 to detect all human cells in combination with SOX10, a general marker for the oligodendroglial lineage, and CC1 as a marker of differentiated oligodendrocytes. We found that the number of MS oligodendroglial cells (SOX10+) increased slightly but significantly with time, most likely resulting from sustained proliferation (Fig. 2, A and B). Moreover, they timely differentiated into mature CC1+ oligodendrocytes with a fourfold increase at 12 wpg and a fivefold increase at 16 wpg when compared to 8 wpg and with no difference with control-hiOLs (Fig. 2, B and C).

(A) Combined immunodetection of human nuclei marker STEM101 (red) with CC1 (green) and SOX10 (white) for control (top) and MS-hiOLs (bottom) at 8, 12, and 16 wpg. (B and C) Quantification of SOX10+/STEM+ cells (B) and CC1+ SOX10+ over STEM+ cells (C). While the percentage of human oligodendroglial cells increased only slightly with time, the percentage of mature oligodendrocytes was significantly time regulated for both MS- and control-hiOLs. Two-way ANOVA followed by Tukeys multiple comparison tests were used for the statistical analysis of these experiments (n = 3 to 4 mice per group). Error bars represent SEMs. *P < 0.05 and ****P < 0.0001. Scale bar, 100 m.

The absence of abnormal MS-hiOL differentiation did not exclude a potential defect in myelination potential. We further investigated the capacity of MS-hiOLs to differentiate into myelin-forming cells. We focused our analysis on the core of the corpus callosum and fimbria. MS-hiOLs, identified by the human nuclear and cytoplasmic markers (STEM101 and STEM121), evolved from a bipolar to multibranched phenotype (Fig. 3A and fig. S3: compare 4 wpg to 8 and 12 wpg) and differentiated progressively into myelin basic proteinpositive (MBP+) cells associated, or not, with T-shaped MBP+ myelin-like profiles of increasing complexity (Fig. 3A and figs. S3 and S4B). Myelin-like profiles clearly overlapped with NF200+ axons (fig. S4A) and formed functional nodes of Ranvier expressing ankyrin G and flanked by paranodes enriched for CASPR (fig. S4B) or neurofascin (fig. S4C), as previously observed with control-hiOLs (25).

(A) Combined detection of human nuclei (STEM101) and human cytoplasm (STEM 121) (red) with MBP (green) in the Shi/Shi Rag2/ corpus callosum at 8, 12, and 16 wpg. General views of horizontal sections at the level of the corpus callosum showing the progressive increase of donor-derived myelin for control- (top) and MS- (bottom) hiOLs. (B) Evaluation of the MBP+ area over STEM+ cells. (C and D) Quantification of the percentage of (C) MBP+ cells and (D) MBP+ ensheathed cells. (E) Evaluation of the average sheath length (m) per MBP+ cells. No obvious difference was observed between MS and control-hiOLs. Two-way ANOVA followed by Tukeys multiple comparison tests were used for the statistical analysis of these experiments (n = 6 to 14 mice per group). Error bars represent SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar, 200 m. See also figs. S3 and S5.

We further analyzed, in depth, the myelinating potential of MS-hiOLs, applying automated imaging and analysis, which provided multiparametric quantification of MBP as established in vitro (29) for each donor hiOL (three controls and three RRMS) at 4, 8, 12, 16, and 20 wpg in vivo (Fig. 3, B to D). We first examined the MBP+ surface area generated by the STEM+ cell population (Fig. 3B). While MS-hiOLs generated very low amount of myelin at 4 wpg, they generated significantly more myelin at 12, 16, and 20 wpg, with similar findings for control-hiOLs, highlighting the rapid progress in the percentage of myelin producing STEM+ cells in MS group over time. Detailed MBP+ surface area generated by the STEM+ cell population per donor is presented in fig. S5 and shows differences among hiOLs in the control and MS groups, respectively.

We also quantified the percentage of STEM+ cells expressing MBP and the percentage of MBP+ with processes associated with linear myelin-like features, which we called MBP+ ensheathed cells. Both parameters increased significantly with time for control-hiOLs, reaching a plateau at 16 wpg. The same tendency was achieved for MS-hiOLs with no significant differences between the control- and MS-hiOL groups (Fig. 3, C and D).

Myelin sheath length is considered to be an intrinsic property of oligodendrocytes (30). We analyzed this paradigm in our MS cohort at 12 and 16 wpg, time points at which sheaths were present at a density compatible with quantification. For those time points, we found that the average MS MBP+ sheath length was equivalent to that of control with 25.86 0.98 and 27.74 1.52 m for MS-hiOLs and 24.52 1.48 and 27.65 0.96 m for control-hiOLs at 12 and 16 wpg, respectively (Fig. 3F). In summary, our detailed analysis of immunohistochemically labeled sections indicates that MS-hiOLs did not generate abnormal amounts of myelin in vivo when compared to control-hiOLs.

Moreover, the myelinating potential of MS-hiOLs was further validated after engraftment in the developing spinal cord (4 weeks of age). Immunohistological analysis 12 wpg revealed that STEM+ cells not only populated the whole dorsal and ventral columns of the spinal cord with preferential colonization of white matter but also generated remarkable amounts of MBP+ myelin-like internodes that were found on multiple spinal cord coronal sections (fig. S6), thus indicating that their myelination potential was not restricted to only one CNS structure.

The presence of normal amounts of donor MBP+ myelin-like structures in the shiverer forebrain does not exclude potential structural anomalies. Therefore, we examined the quality of MS derived myelin at the ultrastructural level at 16 wpg in the Shi/Shi:Rag2/ forebrain. In the corpus callosum of both MS and control-hiOLs grafted mice, we detected numerous axons surrounded by electron dense myelin, which at higher magnification was fully compacted compared to the uncompacted shiverer myelin (Fig. 4, A to F) (25, 31). Moreover, MS myelin reached a mean g ratio of 0.76 1.15 comparable to that of control myelin (0.75 1.56) (Fig. 4G) and thus a similar myelin thickness. This argues in favor of (i) MS-hiOLs having the ability to produce normal compact myelin and thus its functional normality and (ii) a similar rate of myelination between the two groups and, consequently, an absence of delay in myelination for MS-hiOLs.

(A to F) Ultrastructure of myelin in sagittal sections of the core of the corpus callosum 16 wpg with control-hiOLs (A to C) and MS-hiOLs (D to F). (A and D) General views illustrating the presence of some electron dense myelin, which could be donor derived. (B, C, E, and F) Higher magnifications of control (B and C) and MS (E and F) grafted corpus callosum validate that host axons are surrounded by thick and compact donor derived myelin. Insets in (C) and (F) are enlargements of myelin and show the presence of the major dense line. No difference in compaction and structure is observed between the MS and control myelin. (G) Quantification of g-ratio revealed no significant difference between myelin thickness of axons myelinated by control- and MS-hiOLs. Mann-Whitney t tests were used for the statistical analysis of this experiment (n = 4 mice per group). Error bars represent SEMs. Scale bars, (A and D) 5 m , (B and E) 2 m, and (C and F) 500 nm [with 200 and 100 nm, respectively in (C) and (F) insets].

Myelin compaction has a direct impact on axonal conduction with slower conduction in shiverer mice compared to WT mice (10, 32). We therefore questioned whether newly formed MS-hiOLderived myelin has the ability to rescue the slow axon conduction velocity of shiverer mice in vivo (Fig. 5). As previously performed with fetal glial-restricted progenitors (11), transcallosal conduction was recorded in vivo at 16 wpg in mice grafted with MS- and control-hiOLs and compared with nongrafted shiverer and WT mice. As expected, conduction in nongrafted shiverer mice was significantly slower compared to WT mice. However, axon conduction velocity was rescued by MS-hiOLs and, to the same extent, by control-hiOLs.

(A) Scheme illustrating that intracallosal stimulation and recording are performed in the ipsi- and contralateral hemisphere, respectively. (B) N1 latency was measured following stimulation in different groups of Shi/Shi:Rag2/: intact or grafted with control or MS-hiOLs and WT mice at 16 wpg. MS-hiOLderived myelin significantly restored transcallosal conduction latency in Shi/Shi:Rag2/ mice to the same extent than control-derived myelin (P = 0.01) and close to that of WT levels. One-way ANOVA with Dunnetts multiple comparison test for each group against the group of intact Shi/Shi:Rag2/ was used. Error bars represent SEMs. *P < 0.05. (C) Representative response profiles for each group. Scales in Y axis is equal to 10 V and in the X axis is 0.4 ms.

Rodent oligodendrocyte progenitors and oligodendrocytes can be distinguished by cell stagespecific electrophysiological properties (33, 34). To assess the electrophysiological properties of oligodendroglial lineage cells derived from human grafted control- and MS-hiOLs, red fluorescent protein (RFP)hiOLs were engrafted in the Shi/Shi:Rag2/ forebrain and recorded with a K-gluconatebased intracellular solution in acute corpus callosum slices at 12 to 15 wpg (Fig. 6A). As previously described for rodent cells, hiOLs in both groups were identified by their characteristic voltage-dependent current profile recognized by the presence of inward Na+ currents and outwardly rectifying steady-state currents (Fig. 6B). We found that ~60 and ~44% of recorded cells were oligodendrocyte progenitors derived from MS and control progenies, respectively. No significant differences were observed in the amplitude of Na+ currents measured at 20 mV (Fig. 6D) or steady-state currents measured at +20 mV between MS- and control-derived oligodendrocyte progenitors (Isteady = 236.70 19.45 pA and 262.10 31.14 pA, respectively; P = 0.8148, Mann Whitney U test). We further confirmed the identity of these cells by the combined expression of SOX10 or OLIG2 with STEM101/121 and the absence of CC1 in biocytin-loaded cells (Fig. 6F, top). The remaining recorded cells (MS and control) did not show detectable Na+ currents after leak subtraction and were considered to be differentiated oligodendrocytes by their combined expression of SOX10, STEM101/121, and CC1 in biocytin-loaded cells (Fig. 6F, bottom). The I-V curve of these differentiated oligodendrocytes displayed a variable profile that gradually changed from voltage dependent to linear as described for young and mature oligodendroglial cells in the mouse (33). Figure 6C illustrates a typical linear I-V curve of fully mature MS-derived oligodendrocytes. No significant differences were observed in the amplitude of steady-state currents measured at +20 mV between MS- and control-derived oligodendrocytes (Fig. 6E). Overall, the electrophysiological profile of oligodendrocyte progenitors and oligodendrocytes derived from control and MS was equivalent and showed similar characteristics to murine cells (33, 34).

(A) Schematic representation of the concomitant Biocytin loading and recording of single RFP+ hiOL derivative in an acute coronal brain slice prepared from mice engrafted with hiOLs (control or MS) and analyzed at 12 to 14 wpg. (B and C) Currents elicited by voltage steps from 100 to +60 mV in a control-oligodendrocyte progenitor (B, left) and a MS-oligodendrocyte (C, left). Note that the presence of an inward Na+ current obtained after leak subtraction in the oligodendrocyte progenitor, but not in the oligodendrocyte (insets). The steady-state I-V curve of the oligodendrocyte progenitor displays an outward rectification (B, right) while the curve of the oligodendrocyte has a linear shape (C, right). (D) Mean amplitudes of Na+ currents measured at 20 mV in control and MS iPSCs-derived oligodendrocyte progenitors (n = 8 and n = 9, respectively, for four mice per condition; P = 0.743, Mann-Whitney U test). (E). Mean amplitudes of steady-state currents measured at +20 mV in control and patient differentiated iPSC-derived oligodendrocytes (n = 10 and n = 6 for 3 and four mice, respectively; P = 0.6058, Mann-Whitney U test). (F) A control iPSC-derived oligodendrocyte progenitor loaded with biocytin and expressing OLIG2, STEM101/121, and lacking CC1 (top) and an MS iPSCderived oligodendrocyte loaded with biocytin and expressing SOX10, CC1, and STEM101/121 (bottom). Scale bar, 20 m.

(A) Z-stack identifying a target and connected cell. One single grafted human RFP+ cell (per acute slice) was loaded with biocytin by a patch pipette and allowed to rest for 30 min. The white arrowheads and insets in (A) illustrate biocytin diffusion up to the donut-shaped tip of the human oligodendrocyte processes. Another biocytin-labeled cell (empty yellow arrowhead) was revealed at different morphological level indicating diffusion to a neighboring cell and communication between the two cells via gap junctions. (B and C) Split images of (A) showing the target (B) and connected (C) cell separately at different levels. Immunolabeling for the combined detection of the human markers STEM101/121 (red), OLIG2 (blue), and CC1 (white) indicated that the target cell is of human origin (STEM+) and strongly positive for OLIG2 and CC1, a mature oligodendrocyte, and that the connected cell is of murine origin (STEM-) and weakly positive for OLIG2 and CC1, most likely an immature oligodendrocyte. Scale bars, 30 m. See also fig. S7.

Studies with rodents have reported that oligodendrocytes exhibit extensive gap-junctional intercellular coupling between other oligodendrocytes and astrocytes (35). Whether oligodendrocytes derived from grafted human cells can be interconnected with cells in the adult host mouse brain was not known, and whether MS-hiOLs maintain this intrinsic property was also not addressed. Because biocytin can pass through gap junctions, we inspected biocytin-labeled cells for dye coupling (Figs. 6A and 7, A and B).

We found that two of seven MS-derived oligodendrocytes (~29%) and 5 of 21 control-derived oligodendrocytes (~24%) were connected with a single neighboring cell, which was either human or murine (Fig. 7), except in one case where three mouse cells were connected to the biocytin-loaded human cell. These findings reveal that gap junctional coupling can occur between cells from the same or different species, and MS-hiOLs can functionally connect to other glial cells to the same extent as their control counterparts.

To validate the presence of glial-glial interactions, we investigated whether the grafted hiOL-derived progeny had the machinery to be connected to one another via gap junctions. To this end, we focused on oligodendrocyte-specific Cx47 and astrocyte-specific Cx43 as Cx43/47 channels, which are important for astrocyte/oligodendrocyte cross talk during myelination and demyelination (36, 37). Combined immunolabeling for hNOGOA, CC1, OLIG2, and Cx47 revealed that MS-derived oligodendrocyte cell bodies and processes were decorated by Cx47+ gap junction plaques, which were often shared by exogenous MS-derived oligodendrocytes or by MS and endogenous murine oligodendrocytes (fig. S7A). In addition, colabeling exogenous myelin for MBP and Cx43 identified the presence of several astrocyte-specific Cx43 gap junction plaques between human myelin internodes, highlighting contact points between astrocyte processes and axons at the human-murine chimeric nodes of Ranvier (fig. S7B).

Last, colabeling of hNOGOA, with Cx47 and the astrocyte-specific Cx43, revealed coexpression of oligodendrocyte- and astrocyte-specific connexins at the surface of MS-derived oligodendrocyte cell bodies and at the level of T-shaped myelin-like structures (fig. S7C), thus implying connections between human oligodendrocytes and murine and/or human astrocytes, as a small proportion of the grafted hiOLs differentiated into astrocytes. Immunolabeling for human glial fibrillary acidic protein (GFAP), and Cx43 showed that these human astrocytes were decorated by Cx43+ aggregates, as observed in the host subventricular zone (fig. S8A).

Furthermore, immunolabeling for human GFAP, mouse GFAP, and Cx43 indicated that Cx43+ gap junctions were shared between human and mouse astrocytes as observed at the level of blood vessels (fig. S8B). These data validate interconnections between the grafted-derived human glia (MS and controls) with murine host glial cells and confirm their interconnection with the pan-glial network.

Two main hypotheses have been considered in understanding MS pathology and etiology: the outside-in hypothesis highlighting the role of immune regulators and environmental inhibitors as extrinsic key players in MS pathology and possibly its repair failure or the inside-out hypothesis pointing to the intrinsic characteristics of neuroglia including oligodendroglial cells as the main contributors in the MS scenario. Single-cell transcriptomic analysis revealed the presence of disease-specific oligodendroglia expressing susceptibility genes in MS brains (16) and altered oligodendroglia heterogeneity in MS (17). The question remains open as to whether these altered oligodendroglial phenotypes are acquired in response to the disease environment or whether they reflect intrinsic traits of the MS oligodendroglial population. On the other hand, the whole exome sequencing analysis in 132 patients from 34 multi-incident families identified 12 candidate genes of the innate immune system and provided the molecular and biological rational for the chronic inflammation, demyelination, and neurodegeneration observed in patients with MS (38) and revealed the presence of epigenetic variants in immune cells and in a subset of oligodendrocytes contributing to risk for MS (39).

While none of these hypotheses have been fully proven or rejected, research efforts for a better understanding of this multifactorial disease have continued. Impaired remyelination or oligodendrocyte differentiation block in MS is still considered a potentially disease-relevant phenotype (40, 41). Many histological and experimental studies suggest that impaired oligodendrocyte progenitor to oligodendrocyte differentiation may contribute to limited remyelination in MS, although some reports question the contribution of newly generated oligodendrocytes to remyelination (17, 42, 43). Understanding MS oligodendrocyte biology has been challenging mainly due to the following reasons: (i) oligodendroglial cells are not easily accessible to be studied in vivo; (ii) dynamic remyelination observed in patients with MS, which points to their individual remyelination potential, is inversely correlated with their clinical disability (3), highlighting even more complexity in oligodendrocyte heterogeneity between patients with MS; and (iii) exclusion of the role of immune system players in understanding MS oligodendrocyte biology being inevitable in most of clinical or experimental studies.

In such a complex multifactorial disease, one of the most accessible and applicable approaches to overcome these problems is the generation of large quantities of disease and control oligodendroglia using the iPSC technology, and to investigate their genuine behavior in vivo after engraftment in a B and T cellfree system. Using a very efficient reprogramming method (25), and the purely dysmyelinating Shi/Shi:Rag2/ mouse model to avoid confounding immune-mediated extrinsic effects, we show that MS-hiOLs derivatives survive, proliferate, migrate, and timely differentiate into bona fide myelinating oligodendrocytes in vivo as efficiently as their control counterparts. Nicaise and colleagues reported that iPSC-NPCs from PPMS cases did not provide neuroprotection against active CNS demyelination compared to control iPSC-NPCs (44) and failed to promote oligodendrocyte progenitor genesis due to senescence without affecting their endogenous capacity to generate myelin-forming oligodendrocytes (21, 22). However, their myelinating potential was not evaluated against control cells. Generation of iPSC-oligodendrocyte progenitors from patients with PPMS or RRMS has also been reported by other groups, yet with no evidence for their capacity to become functional oligodendrocytes in vivo (23, 24). Thus, so far, no conclusion could be made regarding the potential impact of disease severity (PPMS verses RRMS) on the functionality of the iPSC-derived progeny.

We compared side by side, and at different time points after engraftment, hiOLs from patients with RRMS and controls including two pairs of homozygous twins discordant for disease. We found no significant difference in their capacity to timely differentiate (according to the human tempo of differentiation) and efficiently myelinate axons in the shiverer mouse in terms of the percentage of MBP+ cells generated, amount of myelin produced, length of MBP+ sheaths, and the ultrastructure and thickness of myelin sheaths. MS-hiOLs also reconstructed nodes of Ranvier expressing nodal components key to their function. We not only verified that the grafted MS-hiOLs derivatives were anatomically competent but also established their functionality at the electrophysiological level using (i) in vivo recordings of transcallosal evoked potentials and (ii) ex vivo recordings of the elicited current-voltage curves of the grafted MS-hiOLs verses controls. Our data show that the grafted MS-hiOLs were able to rescue the established delayed latency of shiverer mice to the same extent as control cells, as previously reported for human fetal glial progenitors grafted in the same model (11). Moreover, at the single-cell level, MS-hiOLderived oligodendrocyte progenitors and oligodendrocytes did not harbor aberrant characteristics in membrane currents compared to control cells ex vivo. Thus, iPSC-derived human oligodendroglial cells shift their membrane properties with maturation as previously observed in vitro (45) and these properties are not impaired in MS.

The absence of differences among control and MS-derivatives might be due to different causes. One might consider that pluripotency induction could by in vitro manipulation, erase cell epigenetic traits and/or reverse cells to an embryonic state, and as a result, modulate their intrinsic characteristics. Yet, several reports have highlighted differences in the behavior of diseased iPSC-derived oligodendrocytes in comparison to those from healthy controls using the same technology in multifactorial diseases such as schizophrenia (19, 20), Huntingtons disease (18), and others (46). In this regard, direct reprogramming of somatic cells into the desired cell type, bypassing the pluripotent stage, could be an attractive alternative. However, so far only mouse fibroblasts have been successfully directly converted into oligodendroglial cells, and with relatively low efficiency (47, 48).

iPSCs were transduced with three transcription factors to generate hiOLs in a fast and efficient way (25). While we cannot rule out that the use of these three transcription factors may have obscured differences between MS and controls, results for controls are quite comparable to our previously published data based on human fetal oligodendrocyte progenitor engraftment in the Shi/Shi:Rag2/ developing forebrain (49) or fetal NPC engrafted in the Shi/Shi:Rag2/ demyelinated spinal cord (50), suggesting that transduction with the three transcription factors does not overly modify the behavior of the grafted human cells. It could also be argued that the absence of differences between control and MS monozygous twins is not surprising given their equal genetic background. Yet, comparing controls with nonsibling MS hiOLS (compare C1 with RRMS2 and RRMS3; C2 with RRMS1, RRMS2, and RRMS3; and C3 with RRMS1 and RRMS2) revealed no defect in myelination for MS cells as well.

Analysis of hiOLs from each donor showed differences within each group. This could result from phenotypic instability, heterogeneity among donors, or disease subtype. Yet, the clinical history of each patient suggests a certain homogeneity among the MS disease phenotype, all being RRMS. In addition, the equal survival and proliferation rates between both groups argue in favor of cell stability. These confounding observations sustain that differences in terms of myelination are most likely due to heterogeneity among individuals rather than phenotypic instability or disease subtype.

While most preclinical transplantation studies have focused on myelination potential as the successful outcome of axo-glia interactions, less is known about the capacity of the grafted cells to fulfill glial-glial interactions in the pan-glial syncytium, which could ensure maintenance of newly generated myelin (51) and cell homeostasis (52). Oligodendrocytes are extensively coupled to other oligodendrocytes and oligodendrocyte progenitors through the homologous gap junctions Cx47 (35). These intercellular interactions between competing oligodendroglial cells influence the number and length of myelin internodes and the initiation of differentiation (53, 54). Oligodendrocytes are also coupled to astrocytes through heterologous gap junctions such as Cx32/Cx30 and Cx47/Cx43 (55). Disruption of oligodendrocytes from each other and from astrocytes, i.e., deconstruction of pan-glial network, has been observed in experimental models of demyelination (unpublished data) and frequently reported in MS and neuromyelitis optica (37, 56, 57). Mutations in Cx47 and Cx32 result in developmental CNS and PNS abnormalities in leukodystrophies (58, 59). Moreover, experimental ablation of Cx47 results in aberrant myelination (60) and significantly abolished coupling of oligodendrocytes to astrocytes (35).

In view of the major role of Cx-mediated gap junctions among oligodendrocytes and between oligodendrocytes and astrocytes during myelin formation (55), we asked whether the MS-hiOL progeny was capable of making functional gap junctions with other glial cells, and integrating into the host panglial network. We show that grafted MS-hiOLs, in common with rodent oligodendrocytes, express Cx47 that was frequently shared not only between the human and murine oligodendrocytes (through Cx47-Cx47) but also in conjunction with the astrocyte Cx43 (via Cx47/Cx43). The dye-coupling study highlighted that MS-hiOLs, similar to control cells, were capable of forming functional gap junctions with neighbor murine or human glial cells, indicating that MS-hiOLs retained the intrinsic property, not only to myelinate host axons but also to functionally integrate into the host pan-glial network. While our study focused mainly on oligodendroglial cells, a small proportion of the grafted hiOLs differentiated into astrocytes expressing Cx43. These human astrocytes were detected associated with blood vessels or the subventricular zone, where they were structurally gap-junction coupled to mouse astrocytes as observed after engraftment of human fetal glial restricted progenitors (61).

Together, our data highlight that human skinderived glia retain characteristics of embryonic/fetal brainderived glia as observed for rodent cells (10). In particular, we show that MS-hiOLs timely differentiate into mature oligodendrocytes, functionally myelinate host axons and contribute to the human-mouse chimeric pan-glial network as efficiently as control-hiOLs. These observations favor a role for extrinsic rather than intrinsic oligodendroglial factors in the failed remyelination of MS. The International Multiple Sclerosis Genetics Consortium after analyzing the genomic map of more than 47,000 MS cases and 63,000 control subjects, implicated microglia, and multiple different peripheral immune cell populations in disease onset (62). Moreover, neuroinflammation appears to block oligodendrocyte differentiation and to alter their properties and thereby aggravate the autoimmune process (63). Furthermore, MS lymphocytes are reported to exhibit intrinsic capacities that drive myelin repair in a mouse model of demyelination (64). On the other hand, a recent study highlighted the presence of disease-specific oligodendroglia in MS (16, 17). However, it should be considered that most of the data in the later were collected using single nuclei RNA sequencing of postmortem tissues from MS or control subjects of different ages that were suffering from other disorders ranging from cancer to sepsis and undergoing various treatment, and so died for different reasons, that may have influenced the type or level of RNA expression by the cells. In addition, the presence of genetic variants that alter oligodendrocyte function in addition to that of immune cells was also found (39). While this oligodendrocyte dysfunction contributes to MS risk factor, whether it is involved in other aspects of MS such as severity, relapse rate, and rate of progression is not yet known.

Numerous factors may cause the failure of oligodendrocyte progenitor maturation comprising factors such as axonal damage and/or altered cellular and extracellular signaling within the lesion environment (65) without neglecting aged-related environmental and cellular changes (40). Although the cells generated in this study are more of an embryonic nature, and did not experienced the kind of inhibitory environment that is present in MS, our data provide valuable findings in the scenario of MS pathology highlighting that RRMS-hiOLs, regardless of major manipulators of the immune system, do not lose their intrinsic capacity to functionally myelinate and interact with other neuroglial cells in the CNS under nonpathological conditions. Whether RRMS-hiOLs or oligodendroglial cells directly reprogrammed from MS fibroblasts would behave similarly well, if challenged with neuropathological inflammatory conditions as opposed to conditions wherein the immune system is intact (presence of T and B cells), or whether they would reflect intrinsic aging properties will require further investigation.

In summary, our findings provide valuable insights not only into the biology of MS oligodendroglia but also their application for cell-based therapy and should contribute to the establishment of improved preclinical models for in vivo drug screening of pharmacological compounds targeting the oligodendrocyte progenitors, oligodendrocytes, and their interactions with the neuronal and pan-glial networks.

We examined side by side the molecular, cellular, and functional behavior of MS hiOLs with their control counterparts after their engraftment in a dysmyelinating animal model to avoid the effect of major immune modulators. We used three MS and three control hiOLs including two monozygous twin pairs discordant for the disease. We performed in vivo studies in mouse with sample size between three to six animals per donor/time point/assay required to achieve significant differences. Numbers of replicates are listed in each figure legend. Animals were monitored carefully during all the study time, and animal welfare criteria for experimentation were fully respected. All experiments were randomized with regard to animal enrollment into treatment groups. The same experimenter handled the animals and performed the engraftment experiments to avoid errors. The data were analyzed by a group of authors.

Shiverer mice were crossed to Rag2 null immunodeficient mice to generate a line of Shi/Shi:Rag2/ dysmyelinating-immunodeficient mice to (i) prevent rejection of the grafted human cells and allow detection of donor-derived WT myelin and (ii) investigate the original behavior of MS-derived oligodendrocytes in a B cell/T cellfree environment. Mice were housed under standard conditions of 12-hour light/12-hour dark cycles with ad libitum access to dry food and water at the ICM animal facility. Experiments were performed according to European Community regulations and INSERM ethical committee (authorization 75-348; 20/04/2005) and were approved by the local Darwin ethical committee.

Fibroblasts were obtained under informed consent from three control and three RRMS subjects including two monozygous twin pairs discordant for the disease. They were reprogrammed into iPSCs using the replication incompetent Senda virus kit (Invitrogen) according to manufacturers instructions. Table S1 summarizes information about the human cell lines used in this study. The study was approved by the local ethical committees of Mnster and Milan (AZ 2018-040-f-S, and Banca INSpe).

Human iPSCs were differentiated into NPC by treatment with small molecules as described (66, 67). Differentiation of NPCs into O4+ oligodendroglial cells used a poly-cistronic lentiviral vector containing the coding regions of the human transcription factors Sox10, Olig2, and Nkx6.2 (SON) followed by an IRES-pac cassette, allowing puromycin selection for 16 hours (25). For single-cell electrophysiological recordings, the IRES-pac cassette was replaced by a sequence encoding RFP. Briefly, human NPCs were seeded at 1.5 105 cells per well in 12-well plates, allowed to attach overnight and transduced with SON lentiviral particles and protamine sulfate (5 g/ml) in fresh NPC medium. After extensive washing, viral medium was replaced with glial induction medium (GIM). After 4 days, GIM was replaced by differentiation medium (DM). After 12 days of differentiation, cells were dissociated by accutase treatment for 10 min at 37C, washed with phosphate-buffered saline (PBS) and resuspended in PBS/0.5% bovine serum albumin (BSA) buffer, and singularized cells were filtered through a 70-m cell strainer (BD Falcon). Cells were incubated with mouse immunoglobulin M (IgM) antiO4-APC antibody (Miltenyi Biotech) following the manufacturers protocol, washed, resuspended in PBS/0.5% BSA buffer (5 106 cells/ml), and immediately sorted using a FACS Aria cell sorter (BD Biosciences). Subsequently, human O4+ hiOLs were frozen and stored in liquid nitrogen. Media details were provided in (25). hiOLS from each donor was assayed individually (no cell mix) and studied as follows for forebrain engraftment: immunohistochemistry (all donors, three to seven mice per time point), electron microscopy (C1 and RRMS1, four mice per donor at 16 wpg), in vivo electrophysiology (C1 and RRMS1, six mice per donor and eight mice per medium at 16 wpg), dye coupling, and ex-vivo electrophysiology (C1-RFP and RRMS3-RFP, six to seven mice per donor at 16 wpg). For spinal cord engraftment: immuno-histochemistry (C1 and RRMS3, 3 and 4 mice respectively at 12 wpg).

RRMS1: Disease duration at biopsy was 11 years. Started on Rebif 22 and switched to Rebif 44 because of relapses. Relapse was treated with bolus of cortisone 20 to 30 days before biopsy and then switched to natalizumab.

RRMS2: Disease duration at biopsy was 16 months. Relapse at disease onset. On Rebif 22 from disease onset until biopsy with no episodes. A new lesion was identified 3 months after biopsy. At the time of biopsy, the patient reported cognitive difficulties, no motor dysfunctions.

RRMS3: Disease duration at biopsy was 15 months. Relapse 6 months before biopsy with dysesthesias and hypoesthesia right thigh and buttock. Active lesion identified by magnetic resonance imaging at day 10. On Rebif smart 44 mcg, 50 days later, and skin biopsy 4 months later. A new gadolinium negative temporal lesion identified 2 months after biopsy and the patient switched to Tecfidera.

To assay hiOL contribution to forebrain developmental myelination, newborn Shi/Shi:Rag2/ pups (n = 148) were cryo-anesthetized, and control and RRMS hiOLs were transplanted bilaterally, rostral to the corpus callosum. Injections (1 l in each hemisphere and 105 cells/l) were performed 1 mm caudally, 1 mm laterally from the bregma, and to a depth of 1 mm as previously described (49, 68). Animals were sacrificed at 4, 8, 12, 16, and, when indicated, 20 wpg for immunohistological studies and at one time point for electron microscopy (16 wpg), ex vivo (12 to 15 wpg), and in vivo (16 wpg) electrophysiology.

To assay the fate of hiOLs in the developing spinal cord, 4-week-old mice (n = 4) were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg) (Alcyon) and xylazine (10 mg/kg) (Alcyon) and received a single injection at low speed (1 l/2 min) of hiOLs (1 l, 105 cells/l) at the spinal cord thoracic level using a stereotaxic frame equipped with a micromanipulator and a Hamilton syringe. Animals were sacrificed at 12 wpg for immunohistological studies.

Immunohistochemistry. Shi/Shi:Rag2/ mice grafted with control and RRMS hiOLs (n = 3 to 6 per group, donor and time point) were sacrificed by transcardiac perfusion-fixation with 4% paraformaldehyde in PBS. Tissues were postfixed in the same fixative for 1 hour and incubated in 20% sucrose in 1 PBS overnight before freezing at 80C. Serial horizontal brain and spinal cord cross sections of 12 m thickness were performed with a cryostat (CM3050S, Leica). Transplanted hiOLs were identified using anti-human cytoplasm [1:100; STEM121; Takara, Y40410, IgG1], anti-human nuclei (1:100; STEM101; Takara, Y40400, IgG1), and anti-human NOGOA (1:50; Santa Cruz Biotechnology, sc-11030, goat) antibodies. In vivo characterization was performed using a series of primary antibodies listed in tableS2. For MBP staining, sections were pretreated with ethanol (10 min, room temperature). For glial-glial interactions, oligodendrocyte-specific connexin was detected with anti-connexin 47 (1:200; Cx47; Invitrogen, 4A11A2, IgG1) and astrocyte-specific connexin, with anti-connexin 43 (1:50; Cx43; Sigma-Aldrich, C6219, rabbit), and sections were pretreated with methanol (10 min, 20C). Secondary antibodies conjugated with fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate (SouthernBiotech), or Alexa Fluor 647 (Life Technologies) were used, respectively, at 1:100 and 1:1000. Biotin-conjugated antibodies followed by AMCA AVIDIN D (1:20; Vector, A2006). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (1 g/ml; Sigma-Aldrich) (1:1000). Tissue scanning, cell visualization, and imaging were performed with a Carl Zeiss microscope equipped with ApoTome 2.

Electron microscopy. For electron microscopy, Shi/Shi:Rag2/ mice grafted with control and RRMS hiOLs (n = 4 per group) were perfused with 1% PBS followed by a mixture of 4% paraformaldehyde/5% glutaraldehyde (Electron Microscopy Sciences) in 1% PBS. After 2-hour postfixation in the same solution, 100-m-thick sagittal sections were cut and fixed in 2% osmium tetroxide (Sigma-Aldrich) overnight. After dehydration, samples were flat-embedded in Epon. Ultra-thin sections (80 nm) of the median corpus callosum were examined and imaged with a HITACHI 120 kV HT-7700 electron microscope.

Electrophysiological recordings were performed in mice grafted with MS- and control-hiOLs, and compared with nongrafted intact or medium injected Shi/Shi:Rag2/ mice and WT mice 16 weeks after injection (n = 4 to 6 per group) as described (11). Briefly mice were anesthetized with 2 to 4% isoflurane performed under analgesia (0.1 mg/kg buprecare) and placed in a stereotaxic frame (D. Kopf, Tujunga, CA, USA). Body temperature was maintained at 37C by a feedback-controlled heating blanket (CMA Microdialysis). Electrical stimulation (0.1 ms at 0 to 0.1 mA) was applied using a bipolar electrode (FHC- CBBSE75) inserted to a depth of 200 m into the left cortex at 2 mm posterior to bregma and 3 mm from the midline. At the same coordinates in the contralateral hemisphere, homemade electrodes were positioned for recording local field potentials (LFPs) generated by transcallosal electric stimulation. Electrical stimulation and evoked LFPs were performed by the data acquisition system apparatus (Neurosoft, Russia), and signals were filtered at 0.01 to 1 000 Hz. Each response latency (in ms) was measured as the time between the onset of stimulus artifact to the first peak for each animal. A ground electrode was placed subcutaneously over the neck.

Slice preparation and recordings. Acute coronal slices (300 m) containing corpus callosum were made from Shi/Shi:Rag2/ mice grafted with control (n = 7) and RRMS (n = 6) RFP+ hiOLs. They were prepared from grafted mice between 12 and 15 wpg as previously described (69). Briefly, slices were performed in a chilled cutting solution containing 93 mM N-methyl-d-glucamine, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM Hepes, 25 mM glucose, 2 mM urea, 5 mM Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2, and 10 mM MgCl2 (pH 7.3 to pH 7.4; 95% O2 and 5% CO2) and kept in the same solution for 8 min at 34C. Then, they were transferred for 20 min to solution at 34C containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 20 mM glucose, 5 mM Na-pyruvate, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.3 to pH 7.4; 95% O2 and 5% CO2). Transplanted RFP+ hiOLs were visualized with a 40 fluorescent water-immersion objective on an Olympus BX51 microscope coupled to a CMOS digital camera (TH4-200 OptiMOS) and an light-emitting diode light source (CoolLed p-E2, Scientifica, UK) and recorded in voltage-clamp mode with an intracellular solution containing 130 mM K-gluconate, 0.1 mM EGTA, 2 mM MgCl2, 10 mM Hepes, 10 mM -aminobutyric acid, 2 mM Na2-adenosine 5-triphosphate, 0.5 mM Na-guanosine 5-triphosphate, 10 mM Na2-phosphocreatine, and 5.4 mM biocytin (pH 7.23). Holding potentials were corrected by a junction potential of 10 mV. Electrophysiological recordings were performed with Multiclamp 700B and Pclamp10.6 software (Molecular Devices). Signals were filtered at 3 kHz, digitized at 10 kHz, and analyzed off-line.

Immunostainings and imaging of recorded slices. For analysis of recorded cells, one single RFP+ cell per hemisphere was recorded in a slice and loaded with biocytin for 25 min in whole-cell configuration. After gently removing the patch pipette, biocytin was allowed to diffuse for at least 10 min before the slice was fixed 2 hours in 4% paraformaldehyde at 4C. Then, the slice was rinsed three times in PBS for 10 min and incubated with 1% Triton X-100 and 10% normal goat serum (NGS) for 2 hours. After washing in PBS, slices were immunostained for SOX10, CC1, and STEM101/121. Tissues were incubated with primary antibodies for 3 days at 4C. Secondary antibodies were diluted in 2% NGS and 0.2% Triton X-100. Tissues were incubated with secondary antibodies for 2 hours at room temperature. Biocytin was revealed with secondary antibodies using DyLight-488 streptavidin (Vector Laboratories, Burlingame, USA, 1:200). Images of biocytin-loaded cells were acquired either with a Carl Zeiss microscope equipped with ApoTome 2 or a LEICA SP8 confocal microscope (63 oil objective; numerical aperture, 1.4; 0.75-m Z-step) and processed with National Institutes of Health ImageJ software (70).

We adapted the heuristic algorithm from (29) to identify STEM+ MBP+ OLs in tissue sections. The foundations of the quantitative method remained the same. A ridge-filter extracted sheath-like objects based on intensity and segments associated to cell bodies using watershed segmentation. Two additional features adapted the workflow beyond its original in vitro application. First, we added functionality to allow colocalization of multiple fluorescent stains, as we needed to quantify triple positive STEM+/MBP+/DAPI+ cell objects. Second, because oligodendrocyte sheaths are not parallel and aligned in situ as they are in dissociated nanofiber cell cultures, we adapted the algorithm to report additional metrics about MBP production locally and globally that do not rely on the dissociation of sheaths in dense regions.

Cell nuclei were identified using watershed segmentation of DAPI+ regions and then colocalized pixel-wise with STEM+ objects. The DAPI+ nuclei were then used as local minima to seed a watershed segmentation of the STEM+ stain to separate nearby cell bodies. Last, the identified STEM+ cell bodies were colocalized with overlapping MBP+ sheath-like ridges to define ensheathed cells. We reported the area of MBP overlapping with STEM fluorescence in colocalized regions associated with individual cells, as well as the number of single, double, and triple fluorescently labeled cells. In addition, different cellular phenotypes were noted in situ that were then captured with the adapted algorithm. Qualitatively, we observed cells with expansive MBP production without extended linear sheath-like segments that were not observed in previous applications of the algorithm. These cells were denoted as tuft cells, and were quantitatively defined as STEM+/MBP+/DAPI+ cells without fluorescent ridges that could be identified as extended sheath-like objects.

The myelination potential of three control and 3 MS hiOLs was evaluated at 4, 8, 12, 16, and 20 wpg (n = 2 to 7 per line and per time point; n = 6 to 14 per time point). For each animal, six serial sections at 180-m intervals were analyzed. The percentage of MBP+ cells (composed of ensheathed or tuft cells) was evaluated. Total MBP+ area per STEM+ cells and the average length of MBP+ sheaths per MBP+ cells were analyzed.

Cell survival, proliferation, and differentiation in vivo. The number of STEM101+ grafted cells expressing Caspase3, or Ki67, or SOX10 and CC1 was quantified in the core of the corpus callosum at 8, 12, and 16 wpg. For each animal (n = 3 per group), six serial sections at 180-m intervals were analyzed. Cell counts were expressed as the percentage of total STEM101+ cells.

Myelination by electron microscopy. G ratio (diameter of axon/diameter of axon and myelin sheath) of donor-derived compact myelin was measured as previously described (10). Briefly, the maximum and minimum diameters of a given axon and the maximum and minimum axon plus myelin sheath diameter were measured with the ImageJ software at a magnification of 62,000 for a minimum of 70 myelinated axons per animal. Data were expressed as the mean of the maximal and minimal values for each axon for mice from each group (n = 4 mice per group). Myelin compaction was confirmed at a magnification of 220,000.

Data are presented as means + SEM. Statistical significance was determined by two-tailed Mann Whitney U test when comparing two statistical groups, and with one-way or two-way analysis of variance (ANOVA) followed by Tukeys or Dunnetts (in vivo electrophysiology) multiple comparison tests for multiple groups. Because electrophysiological data in brain slices do not follow a normal distribution after a DAgostino-Pearson normality test, we also performed two-tailed Mann-Whitney U test for comparison between groups. Statistics were done in GraphPad Prism 5.00 and GraphPad Prism 8.2.1 (GraphPad Software Inc., USA). See the figure captions for the test used in each experiment.

Acknowledgments: Funding: This work was supported by the Progressive MS Alliance [PMSA; collaborative research network PA-1604-08492 (BRAVEinMS)] to G.M., J.P.A., A.B.-V.E., and T.K., the National MS Society (NMSS RG-1801-30020 to T.K. and A.B.-V.E.), INSERM and ICM grants to A.B.-V.E., the German Research Foundation (DFG CRC-TR-128B07 to T.K.), and the Italian Multiple Sclerosis Foundation (FISM) (project no. Neural Stem Cells in MS to G.M.). M.C.A. was supported by grants from Fondation pour laide la recherche sur la Sclrose en Plaques (ARSEP) and a sub-award agreement from the University of Connecticut with funds provided by grant no. RG-1612-26501 from National Multiple Sclerosis Society. During this work, S.M. was funded by European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS). B.G.-D. and M.J.F.L. were supported by the PMSA, PA-1604-08492 and the National MS Society (RG-1801-30020), respectively. B.M.-S. was supported by a Ph.D. fellowship from the French Ministry of Research (ED BioSPC). A.B. and M.C.A. thank respective imaging facilities, ICM Quant and IPNP NeurImag and their respective funding sources Institut des Neurosciences Translationnelles ANR-10-IAIHU-06 Fondation Leducq. Author contributions: Conceptualization: S.M. and A.B.-V.E. Methodology: S.M., L.S., B.M.-S., Y.K.T.X., B.G.-D., M.J.F.L., D.R., L.O., K.-P.K., H.R.S., J.P.A., T.K., G.M., T.E.K., M.C.A., and A.B.V.-E. Formal analysis: S.M., B.M-S., Y.K.T.X., M.C.A., and A.B.-V.E. Writing: S.M. and A.B.V.-E, with editing and discussion from all coauthors Funding acquisition: S.M. and A.B.V.-E. Supervision: A.B.V.-E. Competing interests: T.K. has a pending patent application for the generation of human oligodendrocytes. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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