In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.[3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase.

DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides. Nucleotides in DNA contain a deoxyribose sugar, a phosphate, and a nucleobase. The four types of nucleotide correspond to the four nucleobases adenine, cytosine, guanine, and thymine, commonly abbreviated as A,C, G and T. Adenine and guanine are purine bases, while cytosine and thymine are pyrimidines. These nucleotides form phosphodiester bonds, creating the phosphate-deoxyribose backbone of the DNA double helix with the nuclei bases pointing inward (i.e., toward the opposing strand). Nucleotides (bases) are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine (two hydrogen bonds), and guanine pairs with cytosine (stronger: three hydrogen bonds).

DNA strands have a directionality, and the different ends of a single strand are called the 3 (three-prime) end and the 5 (five-prime) end. By convention, if the base sequence of a single strand of DNA is given, the left end of the sequence is the 5 end, while the right end of the sequence is the 3 end. The strands of the double helix are anti-parallel with one being 5 to 3, and the opposite strand 3 to 5. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3 end of a DNA strand.

The pairing of complementary bases in DNA (through hydrogen bonding) means that the information contained within each strand is redundant. Phosphodiester (intra-strand) bonds are stronger than hydrogen (inter-strand) bonds. This allows the strands to be separated from one another. The nucleotides on a single strand can therefore be used to reconstruct nucleotides on a newly synthesized partner strand.[4]

DNA polymerases are a family of enzymes that carry out all forms of DNA replication.[6] DNA polymerases in general cannot initiate synthesis of new strands, but can only extend an existing DNA or RNA strand paired with a template strand. To begin synthesis, a short fragment of RNA, called a primer, must be created and paired with the template DNA strand.

DNA polymerase adds a new strand of DNA by extending the 3 end of an existing nucleotide chain, adding new nucleotides matched to the template strand one at a time via the creation of phosphodiester bonds. The energy for this process of DNA polymerization comes from hydrolysis of the high-energy phosphate (phosphoanhydride) bonds between the three phosphates attached to each unincorporated base. Free bases with their attached phosphate groups are called nucleotides; in particular, bases with three attached phosphate groups are called nucleoside triphosphates. When a nucleotide is being added to a growing DNA strand, the formation of a phosphodiester bond between the proximal phosphate of the nucleotide to the growing chain is accompanied by hydrolysis of a high-energy phosphate bond with release of the two distal phosphates as a pyrophosphate. Enzymatic hydrolysis of the resulting pyrophosphate into inorganic phosphate consumes a second high-energy phosphate bond and renders the reaction effectively irreversible.[Note 1]

In general, DNA polymerases are highly accurate, with an intrinsic error rate of less than one mistake for every 107 nucleotides added.[7] In addition, some DNA polymerases also have proofreading ability; they can remove nucleotides from the end of a growing strand in order to correct mismatched bases. Finally, post-replication mismatch repair mechanisms monitor the DNA for errors, being capable of distinguishing mismatches in the newly synthesized DNA strand from the original strand sequence. Together, these three discrimination steps enable replication fidelity of less than one mistake for every 109 nucleotides added.[7]

The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected E. coli.[8] During the period of exponential DNA increase at 37C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 108.[9]

DNA replication, like all biological polymerization processes, proceeds in three enzymatically catalyzed and coordinated steps: initiation, elongation and termination.

For a cell to divide, it must first replicate its DNA.[10] This process is initiated at particular points in the DNA, known as origins, which are targeted by initiator proteins.[3] In E. coli this protein is DnaA; in yeast, this is the origin recognition complex.[11] Sequences used by initiator proteins tend to be AT-rich (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair) and thus are easier to strand separate.[12] Once the origin has been located, these initiators recruit other proteins and form the pre-replication complex, which unzips the double-stranded DNA.

DNA polymerase has 5-3 activity. All known DNA replication systems require a free 3 hydroxyl group before synthesis can be initiated (note: the DNA template is read in 3 to 5 direction whereas a new strand is synthesized in the 5 to 3 directionthis is often confused). Four distinct mechanisms for DNA synthesis are recognized:

The first is the best known of these mechanisms and is used by the cellular organisms. In this mechanism, once the two strands are separated, primase adds RNA primers to the template strands. The leading strand receives one RNA primer while the lagging strand receives several. The leading strand is continuously extended from the primer by a DNA polymerase with high processivity, while the lagging strand is extended discontinuously from each primer forming Okazaki fragments. RNase removes the primer RNA fragments, and a low processivity DNA polymerase distinct from the replicative polymerase enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.

The primase used in this process differs significantly between bacteria and archaea/eukaryotes. Bacteria use a primase belonging to the DnaG protein superfamily which contains a catalytic domain of the TOPRIM fold type.[13] The TOPRIM fold contains an / core with four conserved strands in a Rossmann-like topology. This structure is also found in the catalytic domains of topoisomerase Ia, topoisomerase II, the OLD-family nucleases and DNA repair proteins related to the RecR protein.

The primase used by archaea and eukaryotes, in contrast, contains a highly derived version of the RNA recognition motif (RRM). This primase is structurally similar to many viral RNA-dependent RNA polymerases, reverse transcriptases, cyclic nucleotide generating cyclases and DNA polymerases of the A/B/Y families that are involved in DNA replication and repair. In eukaryotic replication, the primase forms a complex with Pol .[14]

Multiple DNA polymerases take on different roles in the DNA replication process. In E. coli, DNA Pol III is the polymerase enzyme primarily responsible for DNA replication. It assembles into a replication complex at the replication fork that exhibits extremely high processivity, remaining intact for the entire replication cycle. In contrast, DNA Pol I is the enzyme responsible for replacing RNA primers with DNA. DNA Pol I has a 5 to 3 exonuclease activity in addition to its polymerase activity, and uses its exonuclease activity to degrade the RNA primers ahead of it as it extends the DNA strand behind it, in a process called nick translation. Pol I is much less processive than Pol III because its primary function in DNA replication is to create many short DNA regions rather than a few very long regions.

In eukaryotes, the low-processivity enzyme, Pol , helps to initiate replication because it forms a complex with primase.[15] In eukaryotes, leading strand synthesis is thought to be conducted by Pol ; however, this view has recently been challenged, suggesting a role for Pol .[16] Primer removal is completed Pol [17] while repair of DNA during replication is completed by Pol .

As DNA synthesis continues, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs. In bacteria, which have a single origin of replication on their circular chromosome, this process creates a theta structure (resembling the Greek letter theta: ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.>[18]

The replication fork is a structure that forms within the nucleus during DNA replication. It is created by helicases, which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching prongs, each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; the templates may be properly referred to as the leading strand template and the lagging strand template.

DNA is always synthesized in the 5 to 3 direction. Since the leading and lagging strand templates are oriented in opposite directions at the replication fork, a major issue is how to achieve synthesis of nascent (new) lagging strand DNA, whose direction of synthesis is opposite to the direction of the growing replication fork.

The leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork. A polymerase reads the leading strand template and adds complementary nucleotides to the nascent leading strand on a continuous basis.

The lagging strand is the strand of nascent DNA whose direction of synthesis is opposite to the direction of the growing replication fork. Because of its orientation, replication of the lagging strand is more complicated as compared to that of the leading strand. As a consequence, the DNA polymerase on this strand is seen to lag behind the other strand.

The lagging strand is synthesized in short, separated segments. On the lagging strand template, a primase reads the template DNA and initiates synthesis of a short complementary RNA primer. A DNA polymerase extends the primed segments, forming Okazaki fragments. The RNA primers are then removed and replaced with DNA, and the fragments of DNA are joined together by DNA ligase.

As helicase unwinds DNA at the replication fork, the DNA ahead is forced to rotate. This process results in a build-up of twists in the DNA ahead.[19] This build-up forms a torsional resistance that would eventually halt the progress of the replication fork. Topoisomerases are enzymes that temporarily break the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix; topoisomerases (including DNA gyrase) achieve this by adding negative supercoils to the DNA helix.[20]

Bare single-stranded DNA tends to fold back on itself forming secondary structures; these structures can interfere with the movement of DNA polymerase. To prevent this, single-strand binding proteins bind to the DNA until a second strand is synthesized, preventing secondary structure formation.[21]

Clamp proteins form a sliding clamp around DNA, helping the DNA polymerase maintain contact with its template, thereby assisting with processivity. The inner face of the clamp enables DNA to be threaded through it. Once the polymerase reaches the end of the template or detects double-stranded DNA, the sliding clamp undergoes a conformational change that releases the DNA polymerase. Clamp-loading proteins are used to initially load the clamp, recognizing the junction between template and RNA primers.[2]:274-5

At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome:[22]

Replication machineries consist of factors involved in DNA replication and appearing on template ssDNAs. Replication machineries include primosotors are replication enzymes; DNA polymerase, DNA helicases, DNA clamps and DNA topoisomerases, and replication proteins; e.g. single-stranded DNA binding proteins (SSB). In the replication machineries these components coordinate. In most of the bacteria, all of the factors involved in DNA replication are located on replication forks and the complexes stay on the forks during DNA replication. These replication machineries are called replisomes or DNA replicase systems. These terms are generic terms for proteins located on replication forks. In eukaryotic and some bacterial cells the replisomes are not formed.

Since replication machineries do not move relatively to template DNAs such as factories, they are called a replication factory.[24] In an alternative figure, DNA factories are similar to projectors and DNAs are like as cinematic films passing constantly into the projectors. In the replication factory model, after both DNA helicases for leading stands and lagging strands are loaded on the template DNAs, the helicases run along the DNAs into each other. The helicases remain associated for the remainder of replication process. Peter Meister et al. observed directly replication sites in budding yeast by monitoring green fluorescent protein(GFP)-tagged DNA polymerases . They detected DNA replication of pairs of the tagged loci spaced apart symmetrically from a replication origin and found that the distance between the pairs decreased markedly by time.[25] This finding suggests that the mechanism of DNA replication goes with DNA factories. That is, couples of replication factories are loaded on replication origins and the factories associated with each other. Also, template DNAs move into the factories, which bring extrusion of the template ssDNAs and nascent DNAs. Meisters finding is the first direct evidence of replication factory model. Subsequent research has shown that DNA helicases form dimers in many eukaryotic cells and bacterial replication machineries stay in single intranuclear location during DNA synthesis.[24]

The replication factories perform disentanglement of sister chromatids. The disentanglement is essential for distributing the chromatids into daughter cells after DNA replication. Because sister chromatids after DNA replication hold each other by Cohesin rings, there is the only chance for the disentanglement in DNA replication. Fixing of replication machineries as replication factories can improve the success rate of DNA replication. If replication forks move freely in chromosomes, catenation of nuclei is aggravated and impedes mitotic segregation.[25]

Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular way. Because eukaryotes have linear chromosomes, DNA replication is unable to reach the very end of the chromosomes, but ends at the telomere region of repetitive DNA close to the ends. This shortens the telomere of the daughter DNA strand. Shortening of the telomeres is a normal process in somatic cells. As a result, cells can only divide a certain number of times before the DNA loss prevents further division. (This is known as the Hayflick limit.) Within the germ cell line, which passes DNA to the next generation, telomerase extends the repetitive sequences of the telomere region to prevent degradation. Telomerase can become mistakenly active in somatic cells, sometimes leading to cancer formation. Increased telomerase activity is one of the hallmarks of cancer.

Termination requires that the progress of the DNA replication fork must stop or be blocked. Termination at a specific locus, when it occurs, involves the interaction between two components: (1) a termination site sequence in the DNA, and (2) a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E. coli regulates this process through the use of termination sequences that, when bound by the Tus protein, enable only one direction of replication fork to pass through. As a result, the replication forks are constrained to always meet within the termination region of the chromosome.[26]

Within eukaryotes, DNA replication is controlled within the context of the cell cycle. As the cell grows and divides, it progresses through stages in the cell cycle; DNA replication takes place during the S phase (synthesis phase). The progress of the eukaryotic cell through the cycle is controlled by cell cycle checkpoints. Progression through checkpoints is controlled through complex interactions between various proteins, including cyclins and cyclin-dependent kinases.[27] Unlike bacteria, eukaryotic DNA replicates in the confines of the nucleus.[28]

The G1/S checkpoint (or restriction checkpoint) regulates whether eukaryotic cells enter the process of DNA replication and subsequent division. Cells that do not proceed through this checkpoint remain in the G0 stage and do not replicate their DNA.

Replication of chloroplast and mitochondrial genomes occurs independently of the cell cycle, through the process of D-loop replication.

In vertebrate cells, replication sites concentrate into positions called replication foci.[25] Replication sites can be detected by immunostaining daughter strands and replication enzymes and monitoring GFP-tagged replication factors. By these methods it is found that replication foci of varying size and positions appear in S phase of cell division and their number per nucleus is far smaller than the number of genomic replication forks.

P. Heun et al.(2001) tracked GFP-tagged replication foci in budding yeast cells and revealed that replication origins move constantly in G1 and S phase and the dynamics decreased significantly in S phase.[25] Traditionally, replication sites were fixed on spatial structure of chromosomes by nuclear matrix or lamins. The Heuns results denied the traditional concepts, budding yeasts dont have lamins, and support that replication origins self-assemble and form replication foci.

By firing of replication origins, controlled spatially and temporally, the formation of replication foci is regulated. D. A. Jackson et al.(1998) revealed that neighboring origins fire simultaneously in mammalian cells.[25] Spatial juxtaposition of replication sites brings clustering of replication forks. The clustering do rescue of stalled replication forks and favors normal progress of replication forks. Progress of replication forks is inhibited by many factors; collision with proteins or with complexes binding strongly on DNA, deficiency of dNTPs, nicks on template DNAs and so on. If replication forks stall and the remaining sequences from the stalled forks are not replicated, the daughter strands have nick obtained un-replicated sites. The un-replicated sites on one parents strand hold the other strand together but not daughter strands. Therefore, the resulting sister chromatids cannot separate from each other and cannot divide into 2 daughter cells. When neighboring origins fire and a fork from one origin is stalled, fork from other origin access on an opposite direction of the stalled fork and duplicate the un-replicated sites. As other mechanism of the rescue there is application of dormant replication origins that excess origins dont fire in normal DNA replication.

Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA; during rapid growth, this can result in the concurrent occurrence of multiple rounds of replication.[29] In E. coli, the best-characterized bacteria, DNA replication is regulated through several mechanisms, including: the hemimethylation and sequestering of the origin sequence, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), and the levels of protein DnaA. All these control the binding of initiator proteins to the origin sequences.

Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence; in addition, DnaA (required for initiation of replication) binds less well to hemimethylated DNA. As a result, newly replicated origins are prevented from immediately initiating another round of DNA replication.[30]

ATP builds up when the cell is in a rich medium, triggering DNA replication once the cell has reached a specific size. ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication. A certain number of DnaA proteins are also required for DNA replication each time the origin is copied, the number of binding sites for DnaA doubles, requiring the synthesis of more DnaA to enable another initiation of replication.

Researchers commonly replicate DNA in vitro using the polymerase chain reaction (PCR). PCR uses a pair of primers to span a target region in template DNA, and then polymerizes partner strands in each direction from these primers using a thermostable DNA polymerase. Repeating this process through multiple cycles amplifies the targeted DNA region. At the start of each cycle, the mixture of template and primers is heated, separating the newly synthesized molecule and template. Then, as the mixture cools, both of these become templates for annealing of new primers, and the polymerase extends from these. As a result, the number of copies of the target region doubles each round, increasing exponentially.[31]

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DNA replication Wikipedia IPS Cell Therapy IPS Cell ...

IPS Cell Therapy Comments Off on DNA replication Wikipedia IPS Cell Therapy IPS Cell … | December 17th, 2016

The Inaugural ISCT-ASBMT Cell Therapy Training Course One Scholars Experience

Beth Sage MBBS, PhD UCL Respiratory University College London, London, UK

Having been lucky enough to be selected as an international scholar for the inaugural Cell Therapy Training Course I was looking forward to leaving behind the rather disappointing British summer and heading towards the much warmer Houston fall. Having googled my destination and accommodation I boarded the plane with great excitement and hopes of enjoying the Texan heat whilst exploring the cosmopolitan offerings of Americas fourth largest city oh and learning something about cell therapy!

The course, chaired by Dave DiGiusto and John Barrett, was the first of its kind, a joint enterprise between the ISCT and the American Society of Blood and Marrow Transplantation. Following a competitive selection procedure 12 scholars from all over the world were invited to attend a 5 day intensive workshop with the primary objective of giving junior cell therapy researchers an insight into the process of taking their research project from bench to bedside, including processing, clinical trial design, regulatory requirements, commercialization and ethical research. Alongside didactic lecture based teaching there were tours of good manufacturing practice (GMP) facilities, both academic and commercial, and most anticipated by the scholars was the opportunity to participate in small group discussions, led by experts in the field, dissecting and improving the individual cell therapy projects.

To break the ice on the light first day each scholar gave a short presentation on their project. It was immediately clear that there is a great breadth of exciting, novel cell therapy projects under investigation throughout the world, from the use of modified T-cells in hematological malignancies to the development of a tissue-engineered oesophagus using amniotic fluid stem cells. Projects ranged from early pre-clinical to those embarking on a first in man clinical trial and every stage in between, making the session interesting and varied. Having fought the jet-lag, the session ended with an ice-breaker drinks and dinner before retiring to prepare for the days ahead.

Over the next few days we were exposed to a wealth of information with detailed talks on pre-clinical development of different cell therapies from CD34 cells to mesenchymal stromal cells, quality systems development and one of the most useful from my personal perspective, manufacturing and release testing of different products. We were able to visit different manufacturing facilities and to understand the processes involved in the production of a clinical grade therapy. It made us challenge the protocols we were developing in the lab as we gained an insight into how it would scale up into a commercially viable process a 26 day culture process of autologous cells requiring purification and multiple cytokine stimulations is significantly more challenging (and expensive) than allogeneic cells cultured for 14 days with no manipulation and simple media exchanges.

Once the process development sessions were complete, we switched gears to look at how to conduct cell therapy clinical trials, covering issues of producing products including normal donors that are used to treat multiple recipients, the challenges of pooling donor cells, how to run multicenter studies and most importantly (although I can say almost universally never thought about by the scholars) how to deal with a regulatory body audit. This really was a really informative session that opened our eyes to the challenges and complexities of working in the field of cell therapy trials.

Just when we were beginning to feel that our jobs over the next few years would be focused on clinical trial design, process validation and filling in an endless paper chain of regulatory documents we were brought back to where we all started the excitement of the translational science. This was, for me, a really interesting session on the importance of correlative studies not only to assess clinical trial performance but to provide mechanistic insights into the behavior of cells when delivered to patients with disease. As scientists we can design and perform many experiments to predict how manipulated cells will behave but the most important data of all comes from the patients themselves. For me, the importance of testing a novel therapy is not just to see if it works but how it works and, just as importantly, if it doesnt - why.

To end to course, our wise leaders Dave DiGiusto and John Barrett decided to test whether we had been listening, and each scholar had to deliver a detailed presentation on how their project had developed during the course. Each scholar had to address the potential pitfalls and specific challenges they faced in moving towards the clinic. Whilst many of us stayed up into the small hours worrying about aspects we were previously oblivious to, undoubtedly we found this one of the most rewarding moments. Despite the potential difficulties we were now aware of, we also felt better placed to solve them and could see a clearer path ahead.

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Cellular Therapy Training Course - ISCT

IPS Cell Therapy Comments Off on Cellular Therapy Training Course – ISCT | November 21st, 2016

Progress in basic research, starting with the work on neural stem cells in the middle 1990's to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author's group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient's own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment is now being investigated in preclinical research. Among the mixture of rod and cone photoreceptors in the donor cells, the percentage of cone photoreceptors is still low. Donor cells with more. cone photoreceptors will be needed. If that will work well, photoreceptor transplantation will be the first example of neural network reconstruction in the central nervous system. These efforts will reach to variety of retinal cell transplantations in the future.

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[Retinal Cell Therapy Using iPS Cells]. - ncbi.nlm.nih.gov

IPS Cell Therapy Comments Off on [Retinal Cell Therapy Using iPS Cells]. – ncbi.nlm.nih.gov | November 20th, 2016

Some of the promise of stem cell therapy has been realized. A prime example is bone marrow transplantation. Even here, however, manyproblems remain to be solved.

Challenges facing stem cell therapy include the following:

Adult stem cells Tissue-specific stem cells in adult individuals tend to be rare. Furthermore, while they can regenerate themselves in an animal or person they are generally very difficult to grow and to expand in the laboratory. Because of this, it is difficult to obtain sufficient numbers of many adult stem cell types for study and clinical use. Hematopoietic or blood-forming stem cells in the bone marrow, for example, only make up one in a hundred thousand cells of the bone marrow. They can be isolated, but can only be expanded a very limited amount in the laboratory. Fortunately, large numbers of whole bone marrow cells can be isolated and administered for the treatment for a variety of diseases of the blood. Skin stem cells can be expanded however, and are used to treat burns. For other types of stem cells, such as mesenchymal stem cells, some success has been achieved in expanding the cellsin vitro, but application in animals has been difficult. One major problem is the mode of administration. Bone marrow cells can be infused in the blood stream, and will find their way to the bone marrow. For other stem cells, such as muscle stem cells, mesenchymal stem cells and neural stem cells, the route of administration in humans is more problematic. It is believed, however, that once healthy stem cells find their niche, they will start repairing the tissue. In another approach, attempts are made to differentiate stem cells into functional tissue, which is then transplanted. A final problem is rejection. If stem cells from the patients are used, rejection by the immune system is not a problem. However, with donor stem cells, the immune system of the recipient will reject the cells, unless the immune system is suppressed by drugs. In the case of bone marrow transplantation, another problem arises. The bone marrow contains immune cells from the donor. These will attack the tissues of the recipient, causing the sometimes deadly graft-versus-host disease.

Pluripotent stem cells All embryonic stem cell lines are derived from very early stage embryos, and will therefore be genetically different from any patient. Hence, immune rejection will be major issue. For this reason, iPS cells, which are generated from the cells of the patient through a process of reprogramming, are a major breakthrough, since these will not be rejected. A problem however is that many iPS cell lines are generated by insertion of genes using viruses, carrying the risk of transformation into cancer cells. Furthermore, undifferentiated embryonic stem cells or iPS cells form tumors when transplanted into mice. Therefore, cells derived from embryonic stem cells or iPS cells have to be devoid of the original stem cells to avoid tumor formation. This is a major safety concern.

A second major challenge is differentiation of pluripotent cells into cells or tissues that are functional in an adult patient and that meet the standards that are required for 'transplantation grade' tissues and cells.

A major advantage of pluripotent cells is that they can be grown and expanded indefinitely in the laboratory. Therefore, in contrast to adult stem cells, cell number will be less of a limiting factor. Another advantage is that given their very broad potential, several cell types that are present in an organ might be generated. Sophisticated tissue engineering approaches are therefore being developed to reconstruct organs in the lab.

While results from animal models are promising, the research on stem cells and their applications to treat various human diseases is still at a preliminary stage. As with any medical treatment, a rigorous research and testing process must be followed to ensure long-term efficacy and safety.

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Stem Cell FAQ

IPS Cell Therapy Comments Off on Stem Cell FAQ | November 13th, 2016

1. Introduction

Hematopoietic stem cell transplantation (HSCT) has a half-century history. It is currently an indispensable treatment for not only incurable blood diseases such as aplastic anemia and severe hemolytic anemia, but also malignant hematological diseases such as leukemia and lymphoma. Although allergenic HSCT is also used to treat hereditary diseases, its indications are restricted because of critical complications including regimen-related toxicities involving conditioning, infection, and graft-versus-host disease.

Studies in recent decades have shown that HSCT can have a long-term effect in the treatment of hereditary diseases involving a responsible gene in hematogenous cells. Although the first successful gene therapy using lymphocytes or bone marrow cells for a patient with adenosine deaminase (ADA) deficiency inspired great hope in the future of gene therapy [1-3], subsequent gene therapy using HSCs for patients with X-linked severe combined immunodeficiency (SCID-X1) resulted in tumorigenesis [4]. In addition to the self-renewal and multilineage differentiation capacities of tissue stem cells, HSCs exhibit cell-cycle dormancy, which complicates their use in gene therapy.

However, as technological advances have increased the safety and efficiency of introducing genes into HSCs, gene therapy with HSCs is attracting attention again. In this chapter, advances in the technology of HSC gene therapy, e.g., vector design to avoid genotoxicity and increase transgenic efficiency by taking advantage of the special characteristics of HSCs, are reviewed. In addition, recent studies on HSC gene therapy for various hereditary diseases, such as thalassemia, Fanconi anemia, hemophilia, primary immunodeficiency, mucopolysaccharidosis, Gaucher disease, and X-linked adrenoleukodystrophy (X-ALD) are discussed.

The concept of the HSC was introduced by Till and McCulloch in 1961 [5]. Although a healthy adult produces approximately 1 trillion blood cells each day, they are considered to originate from a single HSC which can potentially be transplanted into a mouse [6, 7]. Generally stem cells are defined as cells capable of self-renewal and multilineage differentiation. In addition to these two characteristics, HSCs have the capability of cell-cycle dormancy, i.e. to enter a state of dormancy (G0 phase) in the cell cycle and can continue blood cell production over a lifetime while protecting themselves from various kinds of stress [8].

Fig. 1 shows HSC surface markers and the typical cytokines regulating HSCs. Stem cell factor (SCF) and thrombopoietin (TPO) are important direct cytokine regulators of HSCs. Although SCF promotes the proliferation and differentiation of hematopoietic progenitor cells, it is thought to not be essential for the initiation of hematopoiesis and HSC self-renewal [9]. TPO and its receptor, c-Mpl, are thought to play important roles in early hematopoiesis from HSCs. In contrast to the CD34+CD38-c-Mpl- population, CD34+CD38-c-Mpl+ cells show significantly better HSC engraftment [10]. Mice lacking either TPO or c-Mpl have deficiencies in progenitor cells of multiple hematopoietic lineages [11]. TPO-mediated signal transduction for the self-renewal of HSCs is negatively regulated by the intracellular scaffold protein Lnk [12, 13]. A signal from angiopoietin-1 via Tie2 regulates HSC dormancy by promoting the adhesion of HSCs to osteoblasts in the bone marrow niche and maintains long-term repopulating activity [14]. Although cytokine-induced lipid raft clustering of the HSC membrane is essential for HSC re-entry into the cell cycle, transforming growth factor- (TGF-) inhibits lipid raft clustering and induces p57Kip2 expression, leading to HSC dormancy [15, 16]. Recently, the hypoxic niche of HSCs has been demonstrated. It, along with the osteoblastic and vascular niches, are important for HSC dormancy [17-19]. They are targets in HSC gene therapy [20].

Hematopoietic stem cell (HSC) surface markers and typical cytokines that regulate HSCs. Stem cell factor (SCF) promotes the proliferation and differentiation of HSCs. Thrombopoietin (TPO) and its receptor, c-Mpl, play important roles in early hematopoiesis, especially self-renewal. Signals from angiotensin-1 via Tie2 and transforming growth factor - via its receptors regulate HSC dormancy. (This figure is based on the illustration by BioLegend, Inc. San Diego, CA, U.S.A. http://www.biolegend.com/cell_markers)

While making a HSC with few opportunities for cell division into a transgenic target, it is important to design a safe and efficient vector for inserting a gene into the host chromosome. Furthermore, since a hematogenous cell also has many cells which exhibit its function in the specialization process to a mature effector cell, it is also important to select differentiation-specific or non-specific promoters or enhancers during the vector design process.

Vectors derived from the Retroviridae family, RNA viruses with reverse transcriptase activity, are widely used for inserting genes in host chromosomes. Although adeno-associated virus (AAV) vectors can also insert genes into host chromosomes, this process is inefficient and partial. Gammaretroviruses and lentiviruses are members of the Retroviridae family that are commonly used as vectors in HSC gene therapy. Generally, the former is called simply a retroviral vector and the latter is called a lentiviral vector. When a gene is inserted in the chromosome of an HSC with a Retroviridae vector, genotoxicity can occur.

Retroviral vectors are commonly constructed from the Moloney murine leukemia virus (MoMLV) genome. Retroviral genomes have a gag/pol gene that codes for viral structure proteins, protease and reverse transcriptase, an env gene that codes for the envelope glycoprotein and the packaging signal. These genes are flanked by long terminal repeats (LTR) which contain enhancers and promoters. A retroviral vector consists of a packaging plasmid that does not have the packaging signal but does include the gag/pol gene, a transfer vector with the packaging signal, and the target gene cDNA. After transfection of these plasmids into producer cells (e.g., 297T cells, NIH3T3 cell, etc.), a target vector is obtained by collecting the culture solution.

Expression of a target gene can be inhibited by mechanisms such as methylation of CpG islands in the promoter region, insertion of a negative control region (NCR) into the LTR, and the presence of a repressor binding site (RBS) downstream of the 5 LTR. Other vectors, such as the murine stem cell virus (MSCV) vector [21], the myeloproliferative sarcoma virus vector, the negative control region deleted (MND) vector [22], and the MFG-S vector [23] were developed to improve the efficiency of transgene expression; they are widely used in clinical applications of gene therapy involving HSCs.

Since the retroviral viral genome cannot cross the nuclear membrane, it can be incorporated into a chromosome only during the phase of mitosis when the nuclear membrane has disassembled. Since many HSCs are thought to exist in a dormant phase, insertions into the HSC genome with a retroviral vector require a proliferation stimulus by cytokines. Although various combinations of cytokines to suppress the decrease in HSC self-renewal have been studied, stem cell factor (SCF), fms-related tyrosine kinase-3 (Flt-3) ligand, interleukin-3 (IL-3), TPO, among others, are commonly used [24, 25].

Human immunodeficiency virus type 1 (HIV-1), the representative lentivirus, differs from gammaretroviruses in that it can be incorporated during a non-mitotic phase. This is one advantage of lentiviral vectors in HSC gene therapy.

Both lentiviruses and gammaretroviruses have gag, pol, and env genes sandwiched between LTRs with promoter activity at both ends. In addition, lentiviruses have accessory genes (vif, vpr, vpu, nef) and regulatory genes (tat, rev). Double-stranded cDNA produced from the viral genome enters the cell, and a pre-integration complex is formed with a host protein. This complex can pass through the pores of the nuclear membrane during non-mitotic phases, allowing the viral genome to be inserted into the host cell chromosome.

HIV provirus (A) and the four plasmids of a third-generation lentiviral vector (B). The viral long terminal repeats (LTRs), reading frames of the viral genes, splice donor site (SD), splicing acceptor site (SA), packaging signal (), and rev-responsive element (RRE) are indicated. The packaging plasmid contains the gag and pol genes under the influence of the CMV promoter, intervening sequences, and the polyadenylation site (polyA) of the human -globin gene. As the transcripts of the gag and pol genes contain cis-repressive sequences, they are expressed only if rev promotes their nuclear export by binding to the RRE. All tat and rev exons have been deleted, and the viral sequences upstream of the gag gene have been replaced. The rev plasmid expresses rev cDNA. The SIN vector plasmid contains HIV-1 cis-acting sequences and an expression cassette for the transgene. It is the only portion transferred to the target cells and does not contain wild-type copies of the HIV LTR. The 5 LTR is chimeric, with the RSV enhancer and promoter replacing the U3 region to rescue transcriptional dependence on tat. The 3 LTR has an almost completely deleted U3 region, which includes the TATA box. As the latter is the template used to generate both copies of the LTR in the integrated provirus, transduction of this vector results in transcriptional inactivation of both LTRs; thus, it is a self-inactivating (SIN) vector. The envelope plasmid encodes a heterologous envelope to pseudotype the vector, here shown coding for vesicular stomatitis virus (VSV)-G. Only the relevant parts of the constructs are shown (Reproduced with modifications from [26]).

Although first-generation lentiviral vectors included modification genes, they were removed in the second generation because it was discovered that the modification genes are not required for infection during non-mitotic phases. In the third generation, further modifications included the deletion of tat, use of multiple vector plasmids, and introduction of self-inactivating (SIN) vectors. The structure of HIV-1 and a typical third-generation lentiviral vector system are shown in Fig. 2 [26]. Approximately one-third of the HIV-1 genome has been deleted, and the vector system has been divided into four plasmids, namely, the packaging plasmid, rev plasmid, SIN vector plasmid and envelope plasmid. To prevent production of wild type HIV-1, tat, a regulatory gene indispensable to viral reproduction was deleted, and the rev gene was moved to a separate plasmid. Moreover, since the HIV-1 LTR promoter is weak in the absence of tat, it was replaced with the cytomegalovirus (CMV) promoter in the packaging plasmid. Since an envelope plasmid can only infect CD4 positive cells with a HIV-1 envelope, the envelope gene was replaced with the vesicular stomatitis virus G glycoprotein (VSV-G) envelope. The SIN vector further improved safety by replacing the enhancer / promoter portion of the LTR, suppressing the activation of unnecessary genes with the integrated gene (Fig. 3) [27].

Mechanism of gene activation induced by vector insertion. The genomic integration site of an MLV-based retroviral vector is depicted. With this MLV vector design, the enhancer and promoter within the U3 region (blue rectangle) of the long terminal repeat (LTR) drive transcription of the transgene (indicated by the parallel arrow arising from the blue rectangle). Vector integration near Gene X is shown in the top panel. The enhancer elements located in the U3 region (blue rectangle) of the vector can interact with the regulatory elements upstream of Gene X to increase its basal transcription rate to inappropriately high levels, potentially altering the growth of the cell. Two alternatives for eliminating the use of the powerful enhancer in the U3 region include (1) middle panel: use of a self-inactivating (SIN) MLV-based vector in which the U3 region has been deleted. An internal cellular promoter is used to drive transgene expression and (2) bottom panel: use of a SIN lentiviral vector in which U3 (yellow rectangle) has been eliminated. This system also uses an internal cellular promoter to drive transgene expression (Reproduced with modification from [27]).

To improve the gene transfer into HSCs, Verhoeyen and colleagues designed lentiviral vectors displaying early-acting cytokines such as TPO and SCF. This vector can promote survival of CD34 positive HSCs and achieve selective transduction of long-term repopulating cells in a humanized mouse model (Fig. 4) [28, 29].

Lentiviral vector particles (HIV-1) display recombinant membrane envelope proteins such as stem cell factor (SCF), thrombopoietin (TPO), and vesicular stomatitis virus G glycoprotein (VSV-G). This vector can specifically target vector particles to hematopoietic stem cells (HSCs) expressing c-kit and c-mpl receptors for SCF and TPO, respectively. VSV-G envelope protein can bind to phospholipids in the HSC cell membrane. (Karlsson S, Gene therapy: efficient targeting of hematopoietic stem cells. Blood. 2005;106(10):3333)

The most serious problem with using viral vectors to incorporate a gene into a chromosome is the potential development of clonal proliferative diseases such as leukemia, which was observed in clinical trials involving gene therapy for SCID-X1 and chronic granulomatous disease (CGD). Although this problem of genotoxicity represents a great hurdle in the development of clinical applications for gene therapy, there is promising ongoing research on the mechanisms underlying genotoxicity and how to avoid it.

The mechanisms of retrovirus-induced oncogenesis are shown in Fig. 5 [30]. In oncogene capture, an acute transforming replication-competent retrovirus captures a cellular proto-oncogene and mediates transformation. This mechanism does not occur in replication-incompetent vectors. Second, the provirus 3 LTR can trigger increased transcription of a cellular proto-oncogene. Third, enhancers in the provirus LTRs can activate transcription from nearby cellular proto-oncogene promoters. Fourth, a novel isoform can be expressed when transcription from the provirus 5 LTR creates a novel truncated isoform of a cellular proto-oncogene via splicing. Fifth, an inserted provirus can disrupt transcription by causing premature polyadenylation. The same mechanisms can occur in cellular oncogenesis when a gene is inserted by a retroviral vector [30].

Retroviral mechanisms of oncogenesis. The detailed mechanisms are shown in the text. The integrated provirus is indicated by two LTRs. Cellular proto-oncogene promoter and exons are indicated by black and grey boxes respectively (Reproduced from [30]).

Even if a gene is inserted into a HSC similarly, it is also known that there are diseases which may develop a tumor, and diseases a tumor is not accepted to be. Each type of virus has a unique integration profile, and the following observations have been made [30]: (a) Different retroviral vectors have distinct integration profiles. (b) The route of entry does not appear to strongly affect distribution of integration sites. VSV-Gpseudotyped HIV vectors have an integration profile similar to HIV virions with the native HIV envelope despite differences in the route of entry. (c) The integration profile is largely independent of the target cell type, although the transcriptional program and epigenetic status of the target cell can influence integration site selection. (d) For lentiviruses, which can integrate independently of mitosis, the cell-cycle status of the target cell has only a modest effect on the distribution of integration sites.

In order to avoid genotoxicity, various SIN vectors have been developed and improved. In general, lentiviral vectors are considered to have a lower risk of oncogenesis than retroviral vectors [31]. However, when a HSC is the target cell, more attention should be required because tumorigenesis can occur when the cell with the inserted gene undergoes differentiation.

Diseases in which gene therapy using HSCs are being studied are shown in Table 1. They are roughly divided into hematological disorders, immunodeficiencies, and metabolic diseases. Most are congenital or hereditary diseases. The characteristic clinical features and recent basic science or clinical studies on HSC gene therapy for each disease are discussed below.

Clinical applications of hematopoietic stem cell gene therapy.

Hemoglobin A (HbA), comprising 98% of adult human hemoglobin, is a tetramer with two -globin and two -globin chains combined with a heme group. -thalassemia is an autosomal hemoglobin disorder caused by decreased -globin chain synthesis. Although individuals with -thalassemia minor (heterozygote) may be asymptomatic or have mild to moderate microcytic anemia, -thalassemia major (homozygote) progresses to serious anemia by one or two years of age, and hemosiderosis, iron overload caused by transfusion or increased iron absorption, develops. Since most patients develop life-threatening complications such as heart failure by adolescence, HSCT has been performed in patients with advanced disease [32]. In recent years, gene therapy using a lentiviral vector containing a functional -globin gene has been performed in an HbE/ -thalassemia (E/ 0) transfusion-dependent adult male, who subsequently did not require transfusions for over 21 months [33].

The human -globin locus is located in a large 70kb area which also contains some -like globulin genes (, G, A, , ). Gene switching takes place according to the development stage, and the -globin gene is transcribed and expressed specifically after birth. A powerful enhancer called the LCR (locus control region) exists on the 5 side of the promoter. The LCR contains five DNase I hypersensitive sites, referred to as HS5 to HS1 starting from the 5 side. Furthermore, HS5 contains CCCTC-binding factor (CTCF)-dependent insulator.

The structure of the lentiviral SIN vector used in gene therapy for -thalassemia is shown in Fig. 6. To improve safety, two stop codons were inserted into the packaging signal () of GAG, the HS5 portion with insulator activity was deleted, and two copies of the 250 base pair (bp) core of the cHS4 chromatin insulators (chicken -globin insulators) were inserted in the U3 region of the HIV 3 LTR. Furthermore, the amino acid at the 87th position of -globin was changed from threonine to glutamine. This altered -globin can be distinguished from normal adult -globin by high performance liquid chromatography (HPLC) analysis in individuals receiving red blood cell transfusion and +-thalassemia patients [33].

Diagram of the human -globin gene in a lentiviral vector. HIV LTR, human immunodeficiency type-1 virus long terminal repeat; +, packaging signal; cPPT/flap, central polypurine tract/DNA flap; RRE, rev-responsive element; p, human -globin promoter; ppt, polypurine tract; HS, DNase I Hypersensitive Sites (Reproduced with color modification from [33])

A clinical study using this vector was performed in two -thalassemia patients. As with autologous bone marrow transplantation, some of the patients marrow cells were cryopreserved as a backup. The lentiviral vector particles containing a functional -globin were introduced into the remaining cells. After the transfected cells were cultured for one week ex vivo, some were also cryopreserved. The patients were conditioned with intravenous busulfan (3.2 mg/kg/day for four days) without the addition of cyclophosphamide, before transplantation using the autologous gene-modified cryopreserved cells (Fig. 7) [34].

The first patient failed to engraft because the HSCs had been compromised by how they were handled, not because of any issues with the gene therapy vector, and ultimately used backup bone marrow. The second patient, as described previously, achieved long-term -globin production; one-third of the patients hemoglobin was produced by the genetically modified cells [33].

Furthermore, the detailed examination of the transgenic cells showed significantly increased expression of high mobility group AT-hook 2 (HMGA2), which interacts with transcription factors to regulate gene expression, in the clones where gene insertion occurred in the HMGA2 gene. The proportion of the HMGA2 overexpressing clones increased with time, to over 50% of transgenic cells at 20 months after gene therapy. In this patient, the HMGA2 overexpressing cells were only 5% of all circulating hematopoietic cells and there was no evidence of malignant transformation. However, researchers point out that there was expressive production of a truncated form of the HMGA2 protein. Since truncated or overexpressed HMGA2 is observed with some blood cancers and non-malignant expansions of blood cells, caution is recommended with this therapy [34].

Gene-therapy procedure for patient with b-thalassemia. a. Hematopoietic stem cells (HSCs) are collected from the bone marrow of a patient with -thalassemia and maintained them in culture. b, Lentiviral-vector particles containing a functional -globin gene were then introduced into the cells and allowed them to expand further in culture. c. To eradicate the patients remaining HSCs and make room for the geneticaaly modified cells, the patient underwent chemotherapy. d. The genetically modified HSCs were then transplanted into the patient (Reproduced from [34]).

Recently, researchers generated a LCR-free SIN lentiviral vector that combines two hereditary persistence of fetal hemoglobin (HPFH)-activating elements, resulting in therapeutic levels of A-globin protein produced by erythroid progenitors derived from thalassemic HSCs [35]. Both lentiviral-mediated -globin gene addition and genetic reactivation of endogenous -globin genes are considered potentially capable of providing therapeutic levels of hemoglobin F to patients with -globin deficiency [36]. In addition, a trial of -globin induction with -globin production using mithramycin, an inducer of -globin expression, to remove excess -globin proteins in -thalassemic erythroid progenitor cells was reported [37].

Fanconi anemia is a hereditary disease characterized by cellular hypersensitivity to DNA crosslinking agents. It leads to bone marrow failure, such as aplastic anemia, by approximately eight years of age. Since there is a high risk of developing malignancy, HSCT has been performed as a curative treatment for bone marrow insufficiency. Although the ten-year probability of survival after transplant from an Human leukocyte antigen (HLA) -identical donor is over 80%, results with other donors are not satisfactory. HSC gene therapy is considered an alternative in cases where there is no HLA-identical donor available [38-40].

There are currently 13 discovered Fanconi anemia complement groups and 13 distinct genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN) have been cloned. Mutations in FANCB are associated with an X-linked form of Fanconi anemia; mutations in the other genes are associated with autosomal recessive transmission. Although frequencies vary by geographical region, FANCA gene abnormalities are found in more than half of all Fanconi anemia patients [41]. Although one of the major hurdles in the development of gene therapy for Fanconi anemia is the increased sensitivity of Fanconi anemia stem cells to free radical-induced DNA damage during ex vivo culture and manipulation, retroviral and lentiviral vectors have been successfully employed to deliver complementing Fanconi anemia cDNA to HSCs with targeted disruptions of the FANCA and FANCC genes [20, 42-44]. In a phase I trial of FANCA gene therapy, gene transfer was performed with patient bone marrow-derived CD34+ cells and the MSCV retroviral vector [38]. Whether sufficient HSCs can be obtained is a potential problem in Fanconi anemia patients due to possible bone marrow insufficiency, but in this study, sufficient target CD34+ cells were obtained from most patients. Two patients had FANCA-transduced cells successfully infused. The procedure was safe, well tolerated, and resulted in transient improvements in hemoglobin and platelet counts [39]. However, transduced cell products were not obtained in one patient who required cryopreserved bone marrow. The first clinical study of FANCC gene therapy using a retroviral vector involved four patients. Although functional FANCC gene expression was observed in peripheral blood and bone marrow cells, the results were transient [43].

Engraftment efficiency of FANCA-modified cells using a lentiviral vector was studied in a mouse model. Rapid transduction with four hours of culture using only SCF and megakaryocyte growth and development factor and minimal differentiation of gene-induced cells is better than standard 96-hour culture using a variety of cytokines, including SCF, interleukin-11, Flt-3 ligand, and IL-3 [44]. Moreover, a recent trial demonstrated enhanced viability and engraftment of gene-corrected cells in patients with FANCA abnormalities with short transduction (overnight), low oxidative stress (5% oxygen), and the anti-oxidant N-acetyl-L-cysteine [20]. Lentiviral transduction of unselected Fanconi anemia bone marrow cells mediates efficient phenotypic correction of hematopoietic progenitor cells and CD34- mesenchymal stromal cells, with increased efficacy in hematopoietic engraftment [45]. In Fancg -/- mice, the wild-type mesenchymal stem and progenitor cells play important roles in the reconstitution of exogenous HSCs in vitro [46]. Recently, a new approach that directly injects lentiviral vector particles into the femur for FANCC gene transfer in mice was able to successfully introduce the FANCC gene to HSCs. This result provides evidence that targeting the HSCs directly in their native environment enables efficient and long-term correction of bone marrow defects in Fanconi anemia [47].

In recent years, the design of lentiviral vectors used for gene therapy in Fanconi anemia has improved. Although the vav and phosphoglycerate kinase (PGK) promoters are relatively weak, physiological levels of FANCA gene expression can be obtained in lymphoblastoid cells. CMV and spleen focus-forming virus (SFFV) promoters result in overexpression of FANCA. The PGK-FANCA lentiviral vectors with either a wild-type woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a mutated WPRE in the 3 region have higher levels of FANCA gene expression. In conclusion, lentiviral vectors with a mutated WPRE and a PGK promoter are considered the most suitable with respect to safety and efficiency for Fanconi anemia gene therapy [48].

There was a recent interesting report on the use of induced pluripotent stem cells (iPS cell). Instead of introducing a repaired gene into the HSCs of a patient with a FANCA gene abnormality, the modified gene was introduced into more stable somatic cells, e.g. fibroblasts, and iPS cells were derived from the genetically modified somatic cells. If HSCs can be produced from genetically modified iPS cells, hematological function can be efficiently reconstructed in patients with hematologic disorders [49].

Hemophilia is a common congenital coagulopathy caused by coagulation factor VIII (hemophilia A) or IX (hemophilia B) deficiency. Although the genes encoding both factor VIII (Xq28) and factor IX (Xq27) are located on the X chromosome and most cases are X-linked, many sporadic variations have been reported. Factor substitution therapies have been used to treat hemophilia for many years. However, there is great hope for gene therapy with hemophilia because coagulation factors have short half-lives (factor VIII, 8 to 12 hours; factor IX, 18 to 24 hours), and an inhibitor is produced in many cases. Furthermore, it is possible for gene therapy to suppress immunogenicity by introducing a mutant protein that lacks the domain with which the inhibitor interacts. Since both coagulation factors are usually produced in the liver, there are few studies involving HSCs. In addition to hepatocytes, trials introducing the modified gene directly into splenic cells, endothelial cells, myoblasts, fibroblasts, etc. have been reported [50-52]. Since the factor IX gene (34 kb) is smaller than the factor VIII gene (186 kb), hemophilia B gene therapy can be possible with an adenovirus vector or an AAV vector. Therefore, hemophilia B is progressing more as a field of gene therapy research even through there are five times more patients with hemophilia A [51-53].

Recently, human factor VIII variant genes were successfully introduced into the HSCs of a mouse with hemophilia A resulting in therapeutic levels of factor VIII variant protein expression. This variant factor VIII has changes in the B and A2 domain in addition to the A1 domain for improved secretion and reduced immunogenicity (wild-type factor VIII has six domains, A1, A2, B, A3, C1, and C2) [54]. To ameliorate the symptoms of hemophilia A, partial replacement of the mutated liver cells by healthy cells in hemophilia A mice was challenged with allogeneic bone marrow progenitor cell transplantation. In this study, the bone marrow progenitor cell-derived hepatocytes and sinusoidal endothelial cells synthesized factor VIII, showing that autologous gene-modified bone marrow progenitor cells have the potential to treat hemophilia [55].

Although HSCT has been widely performed as curative treatment for primary immunodeficiencies, gene therapy has been considered when there is no HLA-identical donor available. As previously shown, the first successful gene therapy was performed in a patient with ADA deficiency in the U.S. in 1990. Since the gene was introduced into T lymphocytes, frequent treatment was required. However, this treatment was associated with an unacceptable level of toxicity. Since transfected vector and normal ADA gene expression in T lymphocytes continued for two years after the cessation of treatment [1], gene therapy attracted attention. With advances in HSC gene-transfer technology, gene therapy for many primary immunodeficiencies can now be considered [56].

SCID-X1 is an X-linked disease caused by deficiency of the common (c) chain in the IL-2 receptor. Because the c chain is common to the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, in SCID-X1 patients, there are defects in T and natural killer (NK) cells, and B cell dysfunction are usually observed [57]. Patients begin suffering from various infections starting several weeks after birth. Without curative treatment, such as HSCT, patients die in infancy.

In SCID-X1, since T cells are lacking, engraftment of the gene-transduced cells can be achieved without pre-conditioning therapy. In the clinical studies of SCID-X1 patients in France and the U.K., the MFG retroviral vector was used with HSCs obtained from the patient. After gene therapy, many patients had improvements in immune function. However, since the genes regulating lymphocyte proliferation, such as LIM domain only 2 (LMO2), Bmi1, cyclin D2 (CCND2) are near the gene insertion region, there was a high frequency of T-cell leukemia after treatment. Furthermore, in the patients who developed leukemia, additional chromosomal changes, including activating mutations of Notch1, changes in the T cell receptor region, and deletion of tumor suppressor genes, e.g. cyclin-dependent kinase-2A (CDKN2A) were observed [58]. Almost gene integration sites by the retroviral vector were inside or near genes that are highly expressed in CD34 positive stem cells. Furthermore, the activity of protein kinases or transferases coded by these activated genes was stronger in CD3 positive T cells than CD34 positive cells [59]. Thus, gene integration mediated by a retrovirus influences the target cells dormant capacity for survival, engraftment, and proliferation.

Although continuous T cell production was founded in many cases, there was little reconstruction of myeloid cells and B cells, and some patients required continuous immunoglobulin substitution therapy. The use of conditioning therapy is also related to immunological reconstruction after c chain gene therapy. There is decreased NK cell reconstruction without conditioning therapy, so conditioning chemotherapy is required for the engraftment of undifferentiated stem cells [58]. A trial of SCID-X1 gene therapy in the U.S. involved three patients ranging from 10 to 14 years of age. They had poor immunological recovery after allergenic HSCT and T cell recovery was only observed in the youngest patient, suggesting there is a limit to the recovery of the function of the thymus in older children [60].

To study whether activation of genes near the region of gene insertion or inserted c chain gene expression itself induces oncogenicity during SCID-X1 gene therapy, a study of the human c chain gene being expressed under the control of the human CD2 promoter and LTR (CD2- c chain gene) was performed in mice. When the CD2- c chain gene was expressed in transgenic mice, a few abnormalities involving T cells were observed, but tumorigenesis was not observed and T and B cell functions were recovered in c chain-gene deficient mice. This study demonstrated that when the c c chain gene is expressed externally, SCID-X1 may be treated safely [61].

Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or c chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].

ADA is an enzyme that catalyzes the conversion of purine metabolism products adenosine and deoxyadenosine into inosine or deoxyinosine. ADA-SCID is an autosomal recessive disease that results in the accumulation of adenosine, deoxyadenosine, and deoxyadenosinetriphosphate (dATP). Accumulated phosphorylated purine metabolism products act on the thymus and cause the maturational or functional disorder of lymphocytes. Because ADA-SCID patients have both T and B cell production fail, patients have a severe combined immunodeficiency disease with a clinical presentation similar to SCID-X1 results, but unlike SCID-X1, many patients have a low level of T cells. Although enzyme replacement therapy with polyethylene glycolmodified bovine ADA (PEG-ADA) was developed to treat ADA-SCID, it is limited by the development of neutralizing antibodies and the cost of lifelong treatment.

In ADA-SCID, since T cell counts are increased by PEG-ADA, gene therapy to increase peripheral T cell counts was attempted during the early stages of gene therapy. Although adverse events were not observed and continuous expression of ADA was achieved in many patients, reconstruction of immune function was not obtained and substitution therapy with PEG-ADA remained necessary. Therefore, HSCs were no longer the target of gene therapy for ADA-SCID. Since ADA-SCID patients have T cells, nonmyeloablative conditioning was performed to achieve gene-transduced HSC engraftment [25, 65].

In a joint Italian-Israeli study started in 2000, ten ADA-SCID children were infused with CD34 positive cells transduced with a MoMLV retroviral vector containing the ADA gene after nonmyeloablative conditioning with busulfan (2mg/kg/day for two days). T cell counts or function were improved in nine out of the ten patients, and PEG-ADA was discontinued in eight. Many patients also had improvements in B or NK cell function, and immunoglobulin substitution therapy was discontinued in five patients. Although some patients had serious adverse events including prolonged neutropenia, hypertension, Epstein-Barr virus infection, and autoimmune hepatitis, there were no cases of treatment-induced leukemia [25].

As with SCID-X1, the retroviral vector gene insertion region is also near genes that control cell proliferation or self-duplication, such as LMO2, or proto-oncogenes [66]. In clinical studies performed in France, the U.S., and the U.K., none of the ADA-SCID patients had adverse events related to insertional mutagenesis, such as leukemia [67, 68]. Thus, HSC gene therapy for ADA-SCID using a lentiviral vector [69] is expected to become the alternative therapy in cases without a suitable donor for HSCT [70]. As an alternative to HSC-based gene therapy, a study using an AAV vector has reported ADA gene expression in various tissues, including heart, skeletal muscle, and kidney [71].

CGD is a disease caused by an abnormality in nicotinamide dinucleotide phosphate (NADPH) oxidase expressed in phagocytes, resulting in failure to produce reactive oxygen species and decreased ability to kill bacteria or fungi after phagocytosis. NADPH oxidase consists of gp91phox (Nox2) and p22 phox which together constitute the membrane-spanning component flavocytochrome b558 (CYBB), and the cytosolic components p47phox, p67phox, p40phox, and Rac. CGD is caused by a functional abnormality in any of these components. Mutations in gp91phox on the X chromosome account for approximately 70% of CGD cases. CGD patients are afflicted with recurrent opportunistic bacterial and fungal infections, leading to the formation of chronic granulomas. Although lifelong antibiotic prophylaxis reduces the incidence of infections, the overall annual mortality rate remains high (2%5%) and the success rate of HSCT is limited by graft-versus-host-disease and inflammatory flare-ups at infected sites [56].

In the initial trials of CGD gene therapy without any conditioning therapy, p47phox or gp91phox gene was inserted using a retroviral vector. The inserted gene was expressed in peripheral blood granulocytes three to six weeks after re-infusion and mobilization by granulocyte colony-stimulating factor (G-CSF), but there was no clinical effect within six months [72-74].

In a German study where gp91phox was inserted with busulfan conditioning (8mg/kg), there were fewer infections after gene therapy. Gene expression was observed in 20% of leukocytes in the first month, rising to 80% at one year. However, in the gene insertion region there are genes related to myeloid cell proliferation, such as myelodysplastic syndrome 1-ecotropic virus integration site 1 (MDS1/EVI1), PR domain containing protein 16 (PRDM16), SET binding protein 1 (SETBP1). Two patients developed myelodysplasia [75]. These two patients had monosomy 7, considered to be related to EVI1 activation. One died of severe sepsis 27 months after gene therapy. Although the gene-inserted cells remained expressed in this patient, methylation of the CpG site in the LTR of the viral vector was observed and the expression of the inserted gp91phox gene was decreased. Interestingly, methylation was restricted to the promoter region of the LTR; the enhancer region was not methylated. Therefore, although gp91phox gene expression was decreased, the activation of EVI1 near the inserted region occurred, leading to clonal proliferation [76]. Since there is a possibility that the transcription activity of genes related to myeloid cell proliferation near the gene insertion site will be increased, there remains a concern about tumorigenesis with peripheral stem cells mobilization by G-CSF in CGD patients, as with X-SCID [74].

Recently, next-generation gene therapy for CGD using lineage- and stage-restricted lentiviral vectors to avoid tumorigenesis [77] and novel approaches involving iPSs derived from CGD patients using zinc finger nuclease (ZFN)-mediated gene targeting were studied [78]. Specific gene targeting can be performed in human iPSs using ZFNs to induce sequence-specific double-strand DNA breaks that enhance site-specific homologous recombination. A single-copy of gp91phox was targeted into one allele of the "safe harbor" AAVS1 locus in iPSs [79].

WAS is a severe X-linked immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP), a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WAS gene result in a wide spectrum of clinical manifestations ranging from relatively mild X-linked thrombocytopenia to the classic WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, high susceptibility to developing tumors, and autoimmune manifestations [80]. Preclinical and clinical evidence suggest that WASP-expressing cells have a proliferative or survival advantage over WASP-deficient cells, supporting the development of gene therapy [56]. Furthermore, up to 11% of WAS patients have somatic mosaicism due to spontaneous in vivo reversion to the normal genotype, and in WAS patients, accumulation of normal T-cell precursors are sometimes seen [81].

In one preclinical study introducing the WAS gene into human T and B cells or mouse HSCs using a retroviral vector, recovery of T cell function and immune reactions to infection were observed [82, 83]. The first clinical study of WAS using HSCs involved two young boys in Germany. The WASP-expressing retroviral vector was transfected into CD34 positive cells obtained by apheresis of peripheral blood. Busulfan was used for conditioning therapy (4mg/kg/day for two days). Over two years, WASP gene expression by HSCs, lymphoid and myeloid cells, and platelets was sustained, and the number and function of monocytes, T, B, and NK cells normalized. Clinically, hemorrhagic diathesis, eczema, autoimmunity, and the predisposition to severe infections were diminished. Since comprehensive insertion-site analysis showed vector integration near multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis, careful monitoring for tumorigenesis is necessary, as with SCID-X1 and CGD [84, 85].

SIN lentiviral vectors using the minimal domain of the WAS promoter or other ubiquitous promoters, such as the PGK promoter, are currently being developed for WAS gene therapy. Preclinical studies using the HSCs obtained from mice or human patients have yield good results in terms of gene expression and genotoxicity [86-90].

Since a study using human embryonic stem cells (hESCs) and WAS-promoterdriven lentiviral vectors labeled by green fluorescent protein (GFP) showed highly specific gene expression in hESCs-derived HSCs, the WAS promoter will be used specifically in the generation of hESC-derived HSCs [91].

JAK3 deficiency is characterized by the absence of T and NK cells and impaired function of B cells, similar to SCID-X1. Treatment consists of HSCT with an HLA-identical or HLA-haplo-identical donor, often the parents of the patient, with T cell depletion. Engraftment is successful in most cases.

Although the recovery of T cell function is usually observed after HSCT, there are usually no improvements in B or NK cell function [92]. One case report involved introduction of JAK3 into the patients bone marrow CD34 positive cells using the MSCV retroviral vector. In this study, immunological recovery was not achieved although gene expression was observed for seven months [93]. Since JAK activation can cause T-cell lymphoma, tumorigenesis remains a concern with JAK gene therapy [92].

PNP metabolizes adenosine into adenine, inosine into hypoxanthine, and guanosine into guanine. PNP deficiency is an autosomal recessive metabolic disorder characterized by lethal T cell defects resulting from the accumulation of products from purine metabolism.

In PNP-deficient mice, transplantation of bone marrow cells transduced with a lentiviral vector containing human PNP resulted in human PNP expression, improved thymocyte maturation, increased weight gain, and extended survival. However, 12 weeks after transplant, the benefit of PNP-transduced cells and the percentage of engrafted cells decreased [94].

LAD-1 is a primary immunodeficiency disease caused by abnormalities in the leukocyte integrin CD11/CD18 heterodimer due to mutations in the CD18 gene. It is similar to canine leukocyte adhesion deficiency (CLAD). LAD-1 patients begin experiencing repeated serious bacterial infections immediately after birth.

In order to suppress gene activation near the gene insertion region in CLAD and to obtain the sufficient expression of the CD18 gene, researches have used various promoters with a lentiviral vector or foamy virus, a retroviral vector. In vivo animal experiments using a PGK or an elongation factor 1 promoter did not lead to symptom improvement [95-97], but improvement was seen with CD11b and CD18 promoters, respectively, with a SIN lentiviral vector in one animal study [98].

MPS is a general term for diseases characterized by glycosaminoglycan (GAG) accumulation into lysosomes as a result of deficiencies in lysosomal enzymes that degrade GAG. Although there are more than ten enzymes that are known to degrade GAG, MPS is divided into seven types: type I (-L-iduronidase deficiency, Hurler syndrome, Sheie syndrome, Hurler-Sheie syndrome), type II (iduronate sulfatase deficiency, Hunter syndrome), type III (heparan N-sulfatase deficiency, -N-acetylglucosaminidase deficiency, -glucosaminidase acetyltransferase deficiency, N-acetylglucosamine 6-sulfatase deficiency, Sanfilippo syndrome), type IV (galactose 6-sulfatase deficiency, Morquio syndrome), type VI (N-acetylgalactosamine 4-sulfatase deficiency, Maroteaux-Lamy syndrome), type VII (-glucuronidase deficiency, Sly syndrome), and type IX (hyaluronidase deficiency). Type II is X-linked; the other types are autosomal recessive. Although lysosomes are found in almost all cells, MPS mainly affects internal organs such as the brain, heart, bones, joints, eyes, liver, and spleen. The extent of disease, including mental retardation, varies with MPS type.

In types I, II, and VI, enzyme replacement therapy is performed. HSCT is performed in types I, II, IV, and VII. Gene therapy for types I, II, III, and VII type have been investigated. There are trials using an AAV or adenovirus vector to insert the modified gene into various cell types, including hepatocytes, muscle cells, myoblasts, and fibroblasts [99].

The first study of HSC gene therapy for MPS using a retroviral vector was performed on type VII mice in 1992, resulting in decreased accumulation of GAG in the liver and spleen but not in the brain and eyes [100]. Subsequent studies in type I and III animal models showed decreases in GAG accumulation in the kidneys and brain. Introductory efficiency and immunological reactions are considered challenges in HSC gene therapy for MPS [99].

Restoring or preserving central nervous system (CNS) function is one of the major challenges in the treatment of MPS. Since replaced enzymes easily cannot pass the blood-brain barrier (BBB), a high dose of enzyme is needed to improve CNS function. Gene therapy faces the same challenge. Even with high expression of enzyme by, for example, hepatocytes, the BBB prevents efficient delivery into the CNS. When a lentiviral vector is directly injected into the body, gene expression in brain tissue is observed, although the underlying mechanism is unknown. There are also trials where AAV vectors are directly injected into the CNS of mice or dogs and gene expression was observed in brain tissue [99].

Recently, a lentiviral vector using an ankyrin-1-based erythroid-specific hybrid promoter/enhancer (IHK) was used with HSCs to obtain gene expression only in erythroblasts for type I MPS. This approach resulted in decreased accumulation of GAG in the liver, spleen, heart, and CNS via enzyme expression in erythroblasts [101].

Gaucher disease is the most common lysosomal storage disorder. It is caused by deficiency of glucocerebroside-cleaving enzyme (-glucocerebrosidase), resulting in the accumulation of glucocerebroside in the reticuloendothelial system [102]. This autosomal recessive disease presents with hepatosplenomegaly, anemia, thrombocytopenia, and convulsions with or without mental retardation. It is classified into three types based on the clinical course or existence of neurological symptoms: type I (non-neuropathic, adult type), type II (acute neuropathic, infantile type), and type III (chronic neuropathic, juvenile type). Enzyme replacement therapy has been established in type I. As with MPS, since it is difficult to improve CNS symptoms with enzyme replacement therapy, HSCT is used, especially with type III. Gene therapy is considered in cases with little improvement with enzyme replacement therapy [103].

For Gaucher disease without CNS symptoms, a animal model using an AAV vector to produce enzyme in hepatocytes yielded good results [103]. HSC gene therapy using a retroviral vector was attempted in type I mice. The treated cells had higher -glucocerebrosidase activity than the HSCs from wild-type mice. Glucocerebroside levels normalized five to six months after treatment and no infiltration of Gaucher cells could be observed in the bone marrow, spleen, and liver [104]. In recent years, development of lentiviral vectors including the human glucocerebrosidase gene [105] and low-risk HSCT with nonmyeloablative doses of busulfan (25mg/kg) and no radiation therapy have been attempted in mice [106].

X-ALD is a peroxisomal disease in which a lipid metabolism abnormality causes demyelination of CNS tissues and dysfunction of the adrenal gland. It results from mutations in the ATP-binding cassette sub-family D (ABCD1) gene that codes for the adrenoleukodystrophy (ALD) protein. Behavioral disorders, mental retardation, or both occur by the age of five or six. Once symptoms appear, they progress to gait disorder and visual impairment within several months and the prognosis is poor. Increased levels of very long chain fatty acids (VLCFA), such as C25:0 or C26:0, are observed in the CNS, plasma, erythrocytes, leucocytes, etc. If the neurological defects are not severe, arrest of or improvement in symptoms can be obtained with HSCT [107].

One study has reported the introduction of wild-type ABCD1 using a lentiviral vector into peripheral blood CD34 positive cells of two patients with no HLA-identical donor. The patients received a transfusion of autologous gene-modified cells after myeloablative conditioning therapy. At three years of follow-up, ALD proteins were expressed in approximately 714% of neutrophils, monocytes, and T cells. Clinically, cerebral demyelination stopped 14 and 16 months after gene therapy, respectively, similar to results with allergenic HSCT [108, 109].

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Recent Advances in Hematopoietic Stem Cell Gene Therapy ...

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In 2006, Shinya Yamanaka of Kyoto University in Japan was the first to disprove the previous notion that reversible cell differentiation of mammals was impossible. He reprogrammed a fully differentiated mouse cell into a pluripotent stem cell by introducing four genes, Oct-4, SOX2, KLF4, and Myc, into the mouse fibroblast through gene-carrying viruses. With this method, he and his coworkers created induced pluripotent stem cells (iPS cells), the key component in this experiment.[1] Scientists have been able to conduct experiments that show the ability of iPS cells to treat and even cure diseases. In this experiment, tests were run on mice with inherited sickle-cell anemia. Skin cells were turned into cells containing genes that transformed the cells into iPS cells. These replaced the diseased sickled cells, curing the test mice. The reprogramming of the pluripotent stem cells in mice was successfully duplicated with human pluripotent stem cells within about a year of the experiment on the mice.[citation needed]

Sickle-cell anemia is a disease in which the body produces abnormally shaped red blood cells. Red blood cells are flexible and round, moving easily through the blood vessels. Infected cells are shaped like a crescent or sickle (the namesake of the disease). As a result of this disorder the hemoglobin protein in red blood cells is faulty. Normal hemoglobin bonds to oxygen, then releases it into cells that need it. The blood cell retains its original form and is cycled back to the lungs and re-oxygenated.

Sickle cell hemoglobin, however, after giving up oxygen, cling together and make the red blood cell stiff. The sickle shape also makes it difficult for the red blood cell to navigate arteries and causes blockages.[2] This can cause intense pain and organ damage. The sickled red blood cells are fragile and prone to rupture. When the number of red blood cells decreases from rupture (hemolysis), anemia is the result. Sickle cells die in 1020 days as opposed to the traditional 120-day lifespan of a normal red blood cell.

Sickle cell anemia is inherited as an autosomal (meaning that the gene is not linked to a sex chromosome) recessive condition.[2] This means that the gene can be passed on from a carrier to his or her children. In order for sickle cell anemia to affect a person, the gene must be inherited from both the mother and the father, so that the child has two recessive sickle cell genes (a homozygous inheritance). People who inherit one sickle cell gene from one parent and one normal gene from the other parent, i.e. heterozygous patients, have a condition called sickle cell trait. Their bodies make both sickle hemoglobin and normal hemoglobin. They may pass the trait on to their children.

The effects of sickle-cell anemia vary from person to person. People who have the disease suffer from varying degrees of chronic pain and fatigue. With proper care and treatment, the quality of health of most patients will improve. Doctors have learned a great deal about sickle cell anemia since its discovery in 1979. They know its causes, its effects on the body, and possible treatments for complications. Sickle cell anemia has no widely available cure. A bone marrow transplant is the only treatment method currently recognized to be able to cure the disease, though it does not work for every patient. Finding a donor is difficult and the procedure could potentially do more harm than good. Treatments for sickle cell anemia are generally aimed at avoiding crises, relieving symptoms, and preventing complications. Such treatments may include medications, blood transfusions, and supplemental oxygen.

During the first step of the experiment, skin cells (also known as fibroblasts) were collected from infected test mice and put in a culture. The fibroblasts were reprogrammed by infecting them with retroviruses that contained genes common to embryonic stem cells. These genes were the same four used by Yamanaka (Oct-4, SOX2, KLF4, and Myc) in his earlier study. The investigators were trying to produce cells with the potential to differentiate into any type of cell needed (i.e. pluripotent stem cells). As the experiment continued, the fibroblasts multiplied into identical copies of iPS cells. The cells were then treated to form the mutation needed to reverse the anemia in the mice. This was accomplished by restructuring the DNA containing the defective globin gene into DNA with the normal gene through the process of homologous recombination. The iPS cells then differentiated into blood stem cells, or hematopoietic stem cells. The hematopoietic cells were injected back into the infected mice, where they proliferate and differentiate into normal blood cells, curing the mice of the disease.[3][4][verification needed]

To determine whether the mice were cured from the disease, the scientists checked for the usual symptoms of sickle cell disease. They examined the blood for mean corpuscular volume (MCV) and red cell distribution width (RDW) and urine concentration defects. They also checked for sickled red blood cells. They examined the DNA through gel electrophoresis, checking for bands that display an allele that causes sickling. Compared to the untreated mice with the disease, which they used as a control, "the treated animals had marked increases in RBC counts, healthy hemoglobin, and packed cell volume levels".[5]

Researchers examined "the urine concentration defect, which results from RBC sickling in renal tubules and consequent reduction in renal medullary blood flow, and the general deteriorated systemic condition reflected by lower body weight and increased breathing."[5] They were able to see that these parts of the body of the mice had healed or improved. This indicated that "all hematological and systemic parameters of sickle cell anemia improved substantially and were comparable to those in control mice."[5] They cannot say if this will work in humans because a safe way to inject the genes for the induced pluripotent cells is still needed.[citation needed]

The reprogramming of the induced pluripotent stem cells in mice was successfully duplicated in humans within a year of the successful experiment on the mice. This reprogramming was done in several labs and it was shown that the iPS cells in humans were almost identical to original embryonic stem cells (ES cells) that are responsible for the creation of all structures in a fetus.[1] An important feature of iPS cells is that they can be generated with cells taken from an adult, which would circumvent many of the ethical problems associated with working with ES cells. These iPS cells also have potential in creating and examining new disease models and developing more efficient drug treatments.[6] Another feature of these cells is that they provide researchers with a human cell sample, as opposed to simply using an animal with similar DNA, for drug testing.

One major problem with iPS cells is the way in which the cells are reprogrammed. Using gene-carrying viruses has the potential to cause iPS cells to develop into cancerous cells.[1] Also, an implant made using undifferentiated iPS cells, could cause a teratoma to form. Any implant that is generated from using these iPS cells would only be viable for transplant into the original subject that the cells were taken from. In order for these iPS cells to become viable in therapeutic use, there are still many steps that must be taken.[5][7]

In the future, researchers hope that induced pluripotent cells may be used to treat other diseases. Pluripotency is a crucial part of disease treatment because iPS cells are capable of differentiation in a way that is very similar to embryonic stem cells which can grow into fully differentiated tissues. iPS cells also demonstrate high telomerase activity and express human telomerase reverse transcriptase, a necessary component in the telomerase protein complex. Also, iPS cells expressed cell surface antigenic markers expressed on ES cells. Also, doubling time and mitotic activity are cornerstones of ES cells, as stem cells must self-renew as part of their definition. As said, iPS cells are morphologically similar to embryonic stem cells. Each cell has a round shape, a large nucleolus and a small amount of cytoplasm. One day, the process may be used in practical settings to provide a fundamental way of regeneration.

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Induced pluripotent stem-cell therapy - Wikipedia

IPS Cell Therapy Comments Off on Induced pluripotent stem-cell therapy – Wikipedia | October 20th, 2016

Frontiers in Biotechnology

Biotechnology is an innovative science in which living systems and organisms are used to develop new and useful products, ranging from healthcare products to seeds. The field of Biotechnology is growing rapidly making tremendous impacts in Medical/Health Care, Food & Agriculture. The Global Biotechnology industry is in the growth phase of its economic life cycle. Over the five years to 2014, revenue and industry value added (IVA) growth have outpaced world GDP growth. The Frontiers in Biotechnology track will cover current technological aspects that aim at obtaining products with scientific, industrial, health and agricultural applications, from organisms with increasing levels of complexity from bacteria, yeast, plants, animal cells and virus. With the lectures and demonstrations on stem cell therapy, Embryo transfer technology, next generation sequencing, Drug discovery, biotechnology in food and dairy, etc The participants are expected to acquire knowledge in techniques and methodologies used in Biotechnology.

Pharmaceutical Biotechnology

Pharmaceutical Biotechnology is the science that covers all technologies required for producing, manufacturing and registration of biological drugs.Pharmaceutical Biotechnologyis an increasingly important area of science and technology. It contributes in design and delivery of new therapeutic drugs,diagnosticagents for medical tests, and in gene therapy for correcting the medical symptoms of hereditary diseases. The Pharmaceutical Biotechnology is widely spread, ranging from many ethical issues to changes inhealthcarepracticesand a significant contribution to the development of national economy.Biopharmaceuticalsconsists of large biological molecules which areproteins. They target the underlying mechanisms and pathways of a disease or ailment; it is a relatively young industry. They can deal with targets in humans that are not accessible withtraditional medicines.

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11th World Congress onBiotechnology and Biotech Industries Meet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 13thBiotechnology Congress, Nov 28-30, 2016, San Francisco, USA;Global Biotechnology Congress2016, May 11th 14th 2016, Boston, MA, USA;Biomarker Summit2016, March 21-23, 2016 San Diego, CA, USA; 14thVaccines Research & Development, July 7-8, Boston, USA;Pharmaceutical & BiotechPatent Litigation Forum, Mar 14 15, 2016, Amsterdam, Netherlands; 4thBiomarkers in Diagnostics, Oct 07-08, 2015 Berlin, Germany, DEU.

Medical Biotechnology

Medicine is by means of biotechnology techniques so much in diagnosing and treating dissimilar diseases. It also gives opportunity for the population to defend themselves from hazardous diseases. The pasture of biotechnology, genetic engineering, has introduced techniques like gene therapy, recombinant DNA technologyand polymerase chain retort which employ genes and DNA molecules to make adiagnosis diseasesand put in new and strong genes in the body which put back the injured cells. There are some applications of biotechnology which are live their part in the turf of medicine and giving good results.

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11th World Congress onBiotechnology and Biotech Industries Meet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 13thBiotechnology Congress, Nov 28-30, 2016, San Francisco, USA;Global Biotechnology Congress2016, May 11th 14th 2016, Boston, MA, USA;Biomarker Summit2016, March 21-23, 2016 San Diego, CA, USA; 14thVaccines Research & Development, July 7-8, Boston, USA;Pharmaceutical & Biotech Patent Litigation Forum, Mar 14 15, 2016, Amsterdam, Netherlands; 4thBiomarkers in Diagnostics, Oct 07-08, 2015 Berlin, Germany, DEU.

Molecular Biotechnology

Molecular biotechnology is the use of laboratory techniques to study and modify nucleic acids and proteins for applications in areas such as human and animal health, agriculture, and the environment.Molecular biotechnologyresults from the convergence of many areas of research, such as molecular biology, microbiology, biochemistry, immunology, genetics, and cell biology. It is an exciting field fueled by the ability to transfer genetic information between organisms with the goal of understanding important biological processes or creating a useful product.

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11th World Congress onBiotechnology and Biotech IndustriesMeet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 13thBiotechnology Congress, Nov 28-30, 2016, San Francisco, USA; GlobalBiotechnology Congress2016, May 11th-14th 2016, Boston, MA, USA;BIO Investor Forum, October 20-21, 2015, San Francisco, USA;BIO Latin America Conference, October 14-16, 2015, Rio de Janeiro, Brazil;Bio Pharm America 20158th Annual International Partnering Conference, September 15-17, 2015, Boston, MA, USA.

Environmental Biotechnology

The biotechnology is applied and used to study the natural environment. Environmental biotechnology could also imply that one try to harness biological process for commercial uses and exploitation. It is the development, use and regulation of biological systems for remediation of contaminated environment and forenvironment-friendly processes(green manufacturing technologies and sustainable development). Environmental biotechnology can simply be described as the optimal use of nature, in the form of plants, animals, bacteria, fungi and algae, to producerenewable energy, food and nutrients in a synergistic integrated cycle of profit making processes where the waste of each process becomes the feedstock for another process.

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11th World Congress onBiotechnology and Biotech IndustriesMeet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 13thBiotechnology Congress, Nov 28-30, 2016, San Francisco, USA; GlobalBiotechnology Congress2016, May 11th 14th 2016, Boston, MA, USA;Biomarker Summit2016, March 21-23, 2016 San Diego, CA, USA; 14thVaccines Research & Development, July 7-8, Boston, USA;Pharmaceutical & BiotechPatent Litigation Forum, Mar 14 15, 2016, Amsterdam, Netherlands

Animal Biotechnology

It improves the food we eat meat, milk and eggs. Biotechnology can improve an animals impact on the environment. Animalbiotechnologyis the use of science and engineering to modify living organisms. The goal is to make products, to improve animals and to developmicroorganismsfor specific agricultural uses. It enhances the ability to detect, treat and prevent diseases, include creating transgenic animals (animals with one or more genes introduced by human intervention), using gene knock out technology to make animals with a specific inactivated gene and producing nearly identical animals by somatic cell nuclear transfer (or cloning).

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11th World Congress onBiotechnology and Biotech Industries Meet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 13thBiotechnology Congress, Nov 28-30, 2016, San Francisco, USA;Global Biotechnology Congress2016, May 11th 14th 2016, Boston, MA, USA;Biomarker Summit2016, March 21-23, 2016 San Diego, CA, USA; 14thVaccines Research & Development, July 7-8, Boston, USA;Pharmaceutical & BiotechPatent Litigation Forum, Mar 14 15, 2016, Amsterdam, Netherlands; 4thBiomarkers in Diagnostics, Oct 07-08, 2015 Berlin, Germany, DEU.

Agricultural Biotechnology

Biotechnology is being used to address problems in all areas of agricultural production and processing. This includesplant breedingto raise and stabilize yields; to improve resistance to pests, diseases and abiotic stresses such as drought and cold; and to enhance the nutritional content of foods. Modern agricultural biotechnology improves crops in more targeted ways. The best known technique is genetic modification, but the term agricultural biotechnology (or green biotechnology) also covers such techniques asMarker Assisted Breeding, which increases the effectiveness of conventional breeding.

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Industrial Biotechnology

Industrial biotechnology is the application of biotechnology for industrial purposes, includingindustrial fermentation. The practice of using cells such as micro-organisms, or components of cells like enzymes, to generate industrially useful products in sectors such as chemicals, food and feed, detergents, paper and pulp, textiles andbiofuels. Industrial Biotechnology offers a premier forum bridging basic research and R&D with later-stage commercialization for sustainable bio based industrial and environmental applications.

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Microbial Biotechnology

Microorganisms have been exploited for their specific biochemical and physiological properties from the earliest times for baking, brewing, and food preservation and more recently for producingantibiotics, solvents, amino acids, feed supplements, and chemical feedstuffs. Over time, there has been continuous selection by scientists of special strains ofmicroorganisms, based on their efficiency to perform a desired function. Progress, however, has been slow, often difficult to explain, and hard to repeat. Recent developments inmolecular biologyand genetic engineering could provide novel solutions to long-standing problems. Over the past decade, scientists have developed the techniques to move a gene from one organism to another, based on discoveries of how cells store, duplicate, and transfer genetic information.

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Food Biotechnology

Food processing is a process by which non-palatable and easily perishable raw materials are converted to edible and potable foods and beverages, which have a longer shelf life. Biotechnology helps in improving the edibility, texture, and storage of the food; in preventing the attack of the food, mainly dairy, by the virus like bacteriophage producing antimicrobial effect to destroy the unwanted microorganisms in food that cause toxicity to prevent the formation and degradation of other toxins andanti-nutritionalelements present naturally in food.

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Genetic Engineering and Biotechnology

One kind of biotechnology is gene technology, sometimes called genetic engineering orgenetic modification, where the genetic material of living things is deliberately altered to enhance or remove a particular trait and allow the organism to perform new functions. Genes within a species can be modified, or genes can be moved from one species to another. Genetic engineering has applications inmedicine, research, agriculture and can be used on a wide range of plants, animals and microorganisms. It resulted in a series of medical products. The first two commercially prepared products from recombinant DNA technology were insulin andhuman growth hormone, both of which were cultured in the E. coli bacteria.

The field of molecular biology overlaps with biology and chemistry and in particular, genetics and biochemistry. A key area of molecular biology concerns understanding how various cellular systems interact in terms of the way DNA, RNA and protein synthesis function.

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Biotechnology Investor & partnering Forum

The Biotech Investor & Partnering Forum is one of the unique conclave focused on the management and economics of biotechnology which became so important as the field is growing on a fast paced. From agriculture and environment sectors to pharmaceutical and healthcare products and services, the industries and institutions emerging from the biotech revolution Bio-Based Economy represent one of the largest and most steadily growing building blocks of the Global economy. The social impact is overwhelming, generating tremendous progress in quality of life but also difficult issues that needs responsible management based on consumer & bio-industry perspective, solid ethical principles, growing intellectual property rights complexity, long drug development times, Bio security, unusual market structures and highly unpredictable outcomes are just some of the challenges facing biotechnology management today.

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Nano Biotechnology

Nano biotechnology, bio nanotechnology, and Nano biology are terms that refer to the intersection of nanotechnology and biology. Bio nanotechnology and Nano biotechnology serve as blanket terms for various related technologies. The most important objectives that are frequently found inNano biologyinvolve applying Nano tools to relevantmedical/biologicalproblems and refining these applications. Developing new tools, such as peptide Nano sheets, for medical and biological purposes is another primary objective in nanotechnology.

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8thWorldMedicalNanotechnologyCongress& Expo during June 9-11, Dallas, USA; 6thGlobal Experts Meeting and Expo onNanomaterialsand Nanotechnology, April 21-23, 2016 ,Dubai, UAE; 12thNanotechnologyProductsExpo, Nov 10-12, 2016 at Melbourne, Australia; 5thInternationalConference onNanotekand Expo, November 16-18, 2015 at San Antonio, USA; 11thInternational Conference and Expo onNano scienceandMolecular Nanotechnology, September 26-28 2016, London, UK; 18thInternational Conference onNanotechnologyand Biotechnology, February 4 5, 2016 in Melbourne, Australia; 16thInternational Conference onNanotechnology, August 22-25, 2016 in Sendai, Japan; International Conference onNano scienceand Nanotechnology, 7-11 Feb 2016 in Canberra, Australia; 18thInternational Conference onNano scienceand Nanotechnology, February 15 16, 2016 in Istanbul, Turkey; InternationalNanotechnologyConference& Expo, April 4-6, 2016 in Baltimore, USA.

Animal biotechnology

Animal biotechnology is a branch of biotechnology in which molecular biology techniques are used to genetically engineer animals in order to improve their suitability for pharmaceutical, agricultural or industrial applications. Many animals also help by serving as models of disease. If an animal gets a disease thats similar to humans, we can use that animal to test treatments. Animals are often used to help us understand how new drugs will work and whether or not theyll be safe for humans and effective in treating disease.

Related conferences

11th World Congress onBiotechnology and Biotech IndustriesMeet, July 28-29, 2016, Berlin, Germany; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 12thBiotechnology Congress, Nov 14-15, 2016, San Francisco, USA;BIO IPCC Conference, Cary, North Carolina, USA; World Congress onIndustrial Biotechnology, April 17-20, 2016, San Diego, CA; 6thBio based Chemicals: Commercialization & Partnering, November 16-17, 2015, San Francisco, CA, USA; The European Forum forIndustrial Biotechnology and Bio economy, 27-29 October 2015, Brussels, Belgium; 4thBiotechnology World Congress, February 15th-18th, 2016, Dubai, United Arab Emirates; International Conference on Advances inBioprocess Engineering and Technology, 20th to 22nd January 2016,Kolkata, India; GlobalBiotechnology Congress2016, May 11th 14th 2016, Boston, MA, USA

Biotechnology Applications

Biotechnology has application in four major industrial areas, including health care (medical), crop production and agriculture, nonfood (industrial) uses of crops and other products (e.g. biodegradable plastics, vegetable oil, biofuels), and environmental uses. AppliedMicrobiologyand Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins, applied genetics and molecular biotechnology,genomicsand proteomics, applied microbial and cell physiology, environmental biotechnology, process and products and more.

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3rd GlobalFood Safety Conference, September 01-03, 2016, Atlanta USA; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, Thailand; 11thEuro Biotechnology Congress, November 07-09,2016, Alicante Spain; 12thBiotechnology Congress, Nov 14-15, 2016, San Francisco, USA;Biologically Active Compoundsin Food, October 15-16 2015 Lodz, Poland; World Conference onInnovative Animal Nutrition and Feeding, October 15-17, 2015 Budapest, Hungary; 18th International Conference onFood Science and Biotechnology, November 28 29, 2016, Istanbul, Turkey; 18th International Conference on Agricultural Science, Biotechnology,Food and Animal Science, January 7 8, 2016, Singapore; International IndonesiaSeafood and Meat, 1517 October 2016, Jakarta, Indonesia.

Biotechnology Companies & Market Analysis

From agriculture to environmental science, biotechnology plays an important role in improving industry standards, services, and developing new products. Biotechnology involves the spectrum of life science-based research companies working ontransformative technologiesfor a wide range of industries. While agriculture, material science and environmental science are major areas of research, the largest impact is made in the field medicine. As a large player in the research and development of pharmaceuticals, the role ofbiotechnologyin the healthcare field is undeniable. From genetically analysis and manipulation to the formation of new drugs, many biotech firms are transforming into pharmaceutical and biopharmaceutical leaders.

Related conferences

10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok; 11thEuroBiotechnologyCongress, November 7-9, 2016 Alicante, Spain; 11th World Congress onBiotechnology and Biotech IndustriesMeet, July 28-29, 2016, Berlin, Germany; 13thBiotechnologyCongress, November 28-30, 2016 San Francisco, USA; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, UAE;BioInternational Convention, June 6-9, 2016 | San Francisco, CA;BiotechJapan, May 11-13, 2016, Tokyo, Japan;NANO BIOEXPO 2016, Jan. 27 29, 2016, Tokyo, Japan;ArabLabExpo2016, March 20-23, Dubai; 14thInternational exhibition for laboratory technology,chemical analysis, biotechnology and diagnostics, 12-14 Apr 2016, Moscow, Russia

Biotechnology Capital & Grants

Every new business needs some startup capital, for research, product development and production, permits and licensing and other overhead costs, in addition to what is needed to pay your staff, if you have any. Biotechnology products arise from successfulbiotechcompanies. These companies are built by talented individuals in possession of a scientific breakthrough that is translated into a product or service idea, which is ultimately brought into commercialization. At the heart of this effort is the biotech entrepreneur, who forms the company with a vision they believe will benefit the lives and health of countless individuals. Entrepreneurs start biotechnology companies for various reasons, but creatingrevolutionary productsand tools that impact the lives of potentially millions of people is one of the fundamental reasons why all entrepreneurs start biotechnology companies.

10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok; 11thEuroBiotechnologyCongress, November 7-9, 2016 Alicante, Spain; 11th World Congress onBiotechnology and Biotech IndustriesMeet, July 28-29, 2016, Berlin, Germany; 13thBiotechnologyCongress, November 28-30, 2016 San Francisco, USA; 10thAsia Pacific Biotech CongressJuly 25-27, 2016, Bangkok, UAE;BioInternational Convention, June 6-9, 2016 | San Francisco, CA;BiotechJapan, May 11-13, 2016, Tokyo, Japan;NANO BIOEXPO 2016, Jan. 27 29, 2016, Tokyo, Japan;ArabLabExpo2016, March 20-23, Dubai; 14thInternational exhibition for laboratory technology,chemical analysis, biotechnology and diagnostics, 12-14 Apr 2016, Moscow, Russia

Scope and Importance

From the simple facts of brewing beer and baking bread has emerged a field now known asBiotechnology. Over the ages the meaning of the word biotechnology has evolved along with our growing technical knowledge. Biotechnology began by using cultured microorganisms to create a variety of food and drinks, despite in early practitioners not even knowing the existence of microbial world. Today, biotechnology is still defined as many application of living organisms or bioprocesses to create new products. Although the underlying idea is unchanged, the use of genetic engineering and other modern scientific techniques has revolutionized the area.

The field of genetics, molecular biology, microbiology, and biochemistry are merging their respective discoveries into the expanding applied field of biotechnology, and advances are occurring at a record pace. Traditional biotechnology goes back thousands of years.

Modern biotechnology applies not only modern genetics but also advances in other sciences. However, there is a third revolution that is just emergingnanotechnology. The development of techniques to visualize and manipulate atoms individually or in small clusters is opening the way to an ever-finer analysis of living systems. Nanoscale techniques are now beginning to play significant roles in many area of biotechnology.

This raises the question of what exactly defines biotechnology. To this there is no real answer. Today, the application of modern genetics or other equivalent modern technology is usually seen as application of modern genetics or equivalent modern technology is usually seen as necessary for a process to count as biotechnology. Thus, the definition of biotechnology has become partly a matter of fashion. Therefore, to classical terms, (modern) biotechnology as resulting in a broaden manner from the merger of classical biotechnology with modern genetics, molecular biology, computer technology, and nanotechnology.

Biotech Congress 2017covers mostly all the allied areas of biotechnology which embraces both the basic sciences, technology and as well as its applications in research, industry and academia. This conference will promote global networking between researchers, institutions, investors, industries, policy makers and students. The conference varied topics in biotechnology like healthcare, environmental, animal, plant, marine, genetic engineering, industrial aspects, food science and bio process.

Through this conference we can get all the relevant information regarding how we can use the advances in the biotechnology for building a better tomorrow by reducing the environmental impacts.

Why Italy?

Rome is the capital of Italy; it is also the countrys largest and most populated comune and fourth-most populous city in the European Union. The Metropolitan City of Rome has a population of 4.3 million residents. The city is located in the central-western portion of the Italian Peninsula, within Lazio (Latium), along the shores of Tiber River. Vatican City is an independent country within the city boundaries of Rome, the only existing example of a country within a city: for this reason Rome has been often defined as capital of two states. Roman mythology dates the founding of Rome at only around 753 BC; the site has been inhabited for much longer, making it one of the oldest continuously occupied cities in Europe. It is referred to as Roma Aeterna (The Eternal City) and Caput Mundi (Capital of the World), two central notions in ancient Roman culture. One of the most important city, Rome, was founded in 753 B.C. by Romulus.

The Apennine Mountains form its backbone and stretch from north to south, with the Tiber River cutting through them in central Italy. Along the northern border, the Alps serve as a natural boundary. The three major bodies of water surrounding Italy are the Adriatic Sea, the Ionian Sea, and the Mediterranean Sea. Ancient Rome is characterized by the seven hills and the Tiber River. The Tiber River flows from the Apennine Mountain, to the Tyrrhenian Sea.

Rome is a sprawling, cosmopolitan city with nearly 3,000 years of globally influential art, architecture and culture on display. In 2005, the city received 19.5 million global visitors, up of 22.1% from 2001. Rome ranked in 2014 as the 14thmos-visited city in the world, 3rd most visited in the European Union, and the most popular tourist attraction in Italy. Its historic center is listed by UNESCO as a World Heritage Site. Monuments and museums such as the Vatican Museums and the Colosseum are among the worlds most visited tourist destinations with both locations receiving millions of tourists a year. Rome hosted the 1960 Summer Olympics and is the seat of United Nations Food and Agriculture Organization (FAO).Rome is the city with the most monuments in the world.

The weather is fantastic in Rome in June, when the average temperature starts off at around 20C and gradually climbs up to 23C-24C as the month progresses.

Congress Highlights:

Biotech Congress 2017 emphasizes on:

Target Audience

CEO, Directors, Vice Presidents, Co-directors, Biotechnologists, Academicians, Biostatistician, Biotechnologists, Clinical Laboratory Scientist, Clinical Metabolomics Data Analyst, Clinicians, Commissioner of Health, Community health workers, CROs, Directors, Environmental Scientists, Food Scientists, Genetic Engineers, Health Economist, Health officials, Healthcare Analyst, Manager of Quality Assurance and Evaluation, Market Access Manager, Marketing Intelligence Associate, Master/PhD students, Medical professionals, Microbiologists, Pharmaceutical Scientists, Physicians, Plant Scientists, Postdoctoral Fellows, Public Health Officer, Public Health Policy Analyst, Research Associates, Research Coordinator, Research Data Analyst, Research Intern, Researchers and faculty, Scientific and Medical Information Assistant, Scientists, Food, Environmental & Plant Scientists, Clinicians, Professors, Health care industrialists, Post Doctorate Fellows, Brand Manufacturers of Consumer Products/ Managers, Pharmaceutical Scientists, Students.

Focusing areas to get more participations & Exhibitions

Why to attend?

Biotech Congress is a remarkable event which brings together a unique and international mix of Biotechnology Researchers, Industrial Biotechnologists, leading Universities and Research Institutions making the congress a perfect platform to share experience, foster collaboration across Industry and Academia, and evaluate emerging technologies across the globe.

Biotechnology in Europe

Only in March a market analysis by British researchers at the University of Cambridge had calculated a market potential of three billion euros for Europe.At present, such Crowd Investing platforms only have a market share of 6.5%, however, the growth forecasts are good. The biotech industry in Europe spends nearly $7.32 billion in R&D and $23.2 billion in revenue. Around 20% of the total marketed medicines, and as much as 50% of all drugs that are in the pipeline, are all healthcare biotech products. The European biotech industry provides employment to approximately 95,000 people. Biotechnology sector makes a substantial contribution to the fundamental EU policy objectives, such as job creation, economic growth, ageing society, public health, environmental protection and sustainable development.

Biotechnology in Italy

The Italian Biotechnology Report by Ernst&Young and Assobiotec, in cooperation with Farmindustria and Italian Trade Promotion Agency, shows that the Italian biotech companies are able to compete outstandingly on the international market, managing to grow despite continuing difficulties in the economic situation. With 394 companies, of which 248 pure biotech, Italy is third in Europe after Germany and the United Kingdom, for the number of pure biotech companies, with a growth trend (+2,5%) in clear contrast with that of the countries that occupy the top ranking positions. With 206 companies operating in the health-care field, the red biotech is the prevalent sector. Looking at the other sectors, 43 green biotech, 34 white biotech, 61 GPET (Genomics, Proteomics and Enabling Technologies) and 50 multi core companies are operating in Italy. 77% of the companies are small (less than 50 employees) and micro (less than 10 employees) enterprises, mainly located in Science and Technology Parks or Incubators. Total revenues in the biotech field amount to 7 billion Euros (+4%). Investments in R&D amount to 1,8 billion Euros (+8%), equal to 25% of total revenues. Italian biotech revenues contributes to 0,7% of GDP and the sector is being considered more and more often as a meta-sector, able to create value and employment and with significant effects on various fields, ranging from textiles to detergents, cosmetics, polymers, paper and animal feed, from paints to food, from treatment of waste to leather treatment, and many others. The future trends of Italian red biotech are connected to a further specialization in oncology, neurology and infectious diseases and to new achievements in the fields of Advanced Therapies and personalized medicine. The analysis of the Italian biotech pipeline shows 319 products for therapeutic use, of which 80 in the preclinical phase, 43 in Phase I, 98 in Phase II and 98 in Phase III. Plant genomics and traceability, preservation and safety of foods, as well as bioremediation and biomasses, are the most promising applications in the green & white fields.

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research IPS Cell Therapy IPS Cell Therapy

IPS Cell Therapy Comments Off on research IPS Cell Therapy IPS Cell Therapy | August 25th, 2016

Progress in basic research, starting with the work on neural stem cells in the middle 1990's to embryonic stem (ES) cells and induced pluripotent stem (iPS) cells at present, will lead the cell therapy (regenerative medicine) of various organs, including the central nervous system to a big medical field in the future. The author's group transplanted iPS cell-derived retinal pigment epithelial (RPE) cell sheets to the eye of a patient with exudative age-related macular degeneration (AMD) in 2014 as a clinical research. Replacement of the RPE with the patient's own iPS cell-derived young healthy cell sheet will be one new radical treatment of AMD that is caused by cellular senescence of RPE cells. Since it was the first clinical study using iPS cell-derived cells, the primary endpoint was safety judged by the outcome one year after surgery. The safety of the cell sheet has been confirmed by repeated tumorigenisity tests using immunodeficient mice, as well as purity of the cells, karyotype and genetic analysis. It is, however, also necessary to prove the safety by clinical studies. Following this start, a good strategy considering cost and benefit is needed to make regenerative medicine a standard treatment in the future. Scientifically, the best choice is the autologous RPE cell sheet, but autologous cell are expensive and sheet transplantation involves a risky part of surgical procedure. We should consider human leukocyte antigen (HLA) matched allogeneic transplantation using the HLA 6 loci homozyous iPS cell stock that Prof. Yamanaka of Kyoto University is working on. As the required forms of donor cells will be different depending on types and stages of the target diseases, regenerative medicine will be accomplished in a totally different manner from the present small molecule drugs. Proof of concept (POC) of photoreceptor transplantation in mouse is close to being accomplished using iPS cell-derived photoreceptor cells. The shortest possible course for treatment is now being investigated in preclinical research. Among the mixture of rod and cone photoreceptors in the donor cells, the percentage of cone photoreceptors is still low. Donor cells with more. cone photoreceptors will be needed. If that will work well, photoreceptor transplantation will be the first example of neural network reconstruction in the central nervous system. These efforts will reach to variety of retinal cell transplantations in the future.

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[Retinal Cell Therapy Using iPS Cells].

IPS Cell Therapy Comments Off on [Retinal Cell Therapy Using iPS Cells]. | August 25th, 2016

In the past year, Japan has accelerated its position as a hub for regenerative medicine research, largely driven by support from Prime Minister Shinzo Abe who has identified regenerative medicine and cellular therapy as key to the Japans strategy to drive economic growth. The Prime Minister has encouraged a growing range of collaborations between private industry and academic partners through an innovative legal framework approved last fall. He has also initiated campaigns to drive technological advances in drugs and devices by connecting private companies with public funding sources. The result has been to drive progress in both basic and applied research involving induced pluripotent stem cells (iPS cells) and related stem cell technologies.

Indeed, 2013 represented a landmark year in Japan, as it saw the first cellular therapy involving transplant of iPS cells into humans initiated at the RIKEN Center in Kobe, Japan.[1] The RIKEN Center is Japans largest, most comprehensive research institution, backed by both Japans Health Ministry and government. To speed things along, RIKEN did not seek permission for a clinical trial involving iPS cells, but instead applied for a type of pretrial clinical research allowed under Japanese regulations.

As such, this pretrial clinical research allowed the RIKEN research team to test the use of iPS cells for the treatment of wet-type age-related macular degeneration (AMD) on a very small scale, in only a handful of patients. Unfortunately, this trial was paused in 2015 due to safety concerns and is currently on hold pending further investigation. Regardless, the trial has set a new international standard for considering iPS cells as a viable cell type to investigate for clinical purposes.

If this iPS cell trial is ultimately reinstated, it will help to accelerate the acceptance of cellular therapies within other countries. Currently, the main concern surrounding iPS cell-based cellular therapy isthe fear of creating multiplying cell populations within patients. Even treatments using embryonic stem cells, which have been cultured and studied for decades, are still in very early clinical trials, so it is not surprising that clinical trials using iPS cells are being conducted on a small-scale, experimental level.[2]

Japan has a unique affection for iPS cells, as the cells were originally discovered by the Japanese scientist, Shinya Yamanaka of Kyoto University. Mr. Yamanaka was awarded the Nobel Prize in Physiology or Medicine for 2012, an honor shared jointly with John Gurdon, for the discovery that mature cells can be reprogrammed to become pluripotent. In addition, Japans Education Ministry said its planning to spend 110 billion yen ($1.13 billion) on induced pluripotent stem cell research during the next 10 years, and the Japanese parliament has been discussing bills that would speed the approval process and ensure the safety of such treatments.[3] In April, Japanese parliament even passed a law calling for Japan to make regenerative medical treatments like iPSC technology available for its citizens ahead of the rest of the world.[4] If those forces were not enough, Masayo Takahashi of the RIKEN Center for Developmental Biology in Kobe, Japan, who is heading the worlds first clinical research using iPSCs in humans, was also chosen by the journal Natureas one of five scientists to watch in 2014.[5]

In summary, Japan is the clear global leader with regard to investment in iPS cell technologies and therapies. While progress with stem cells has not been without setbacks within Japan, including a recent scandal at the RIKEN Institute that involved falsely manipulated research findings and the aforementioned hold on the first clinical trial involving transplant of an iPS cell product into humans, Japan has emerged from these troubles to become the most liberalized and progressive nation pursuing the development of iPS cell products and services.

To learn more about induced pluripotent stem cell (iPSC)industry trends and events, view the Compete 2015-16 Induced Pluripotent Stem Cell (iPSC) Industry Report.

To receive future posts about the stem cell industry, sign-up here. We will never share your information with anyone, and you can opt-out at any time. No spam ever, just great stuff.

BioInformant is the only research firm that has served the stem cell sector since it emerged. Our management team comes from a BioInformatics background the science of collecting and analyzing complex genetic codes and applies these techniques to the field of market research. BioInformant has been featured on news outlets including the Wall Street Journal, Nature Biotechnology, CBS News, Medical Ethics, and the Center for BioNetworking.

Serving Fortune 500 leaders that include GE Healthcare, Pfizer, Goldman Sachs, Beckton Dickinson, and Thermo Fisher Scientific, BioInformant is your global leader in stem cell industry data.

Footnotes [1] Dvorak, K. (2014).Japan Makes Advance on Stem-Cell Therapy [Online]. Available at: http://online.wsj.com/news/articles/SB10001424127887323689204578571363010820642. Web. 14 Apr. 2015. [2] Note: In the United States, some patients have been treated with retina cells derived from embryonic stem cells (ESCs) to treat macular degeneration. There was a successful patient safety test for this stem cell treatment last year that was conducted at the Jules Stein Eye Institute in Los Angeles. The ESC-derived cells used for this study were developed by Advanced Cell Technology, Inc, a company located in Marlborough, Massachusetts. [3] Dvorak, K. (2014).Japan Makes Advance on Stem-Cell Therapy [Online]. Available at: http://online.wsj.com/news/articles/SB10001424127887323689204578571363010820642. Web. 8 Apr. 2015. [4] Ibid. [5] Riken.jp. (2014).RIKEN researcher chosen as one of five scientists to watch in 2014 | RIKEN [Online]. Available at: http://www.riken.jp/en/pr/topics/2014/20140107_1/. Web. 14 Apr. 2015.

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Japan Most Liberalized Market for iPS Cell Therapy ...

IPS Cell Therapy Comments Off on Japan Most Liberalized Market for iPS Cell Therapy … | October 28th, 2015

Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanakas lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells.[1] He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." [2]

Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.

The most well-known type of pluripotent stem cell is the embryonic stem cell. However, since the generation of embryonic stem cells involves destruction (or at least manipulation) [3] of the pre-implantation stage embryo, there has been much controversy surrounding their use. Further, because embryonic stem cells can only be derived from embryos, it has so far not been feasible to create patient-matched embryonic stem cell lines.

Since iPSCs can be derived directly from adult tissues, they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. While the iPSC technology has not yet advanced to a stage where therapeutic transplants have been deemed safe, iPSCs are readily being used in personalized drug discovery efforts and understanding the patient-specific basis of disease.[citation needed]

Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose significant risks that could limit their use in humans. For example, if viruses are used to genomically alter the cells, the expression of oncogenes (cancer-causing genes) may potentially be triggered. In February 2008, scientists announced the discovery of a technique that could remove oncogenes after the induction of pluripotency, thereby increasing the potential use of iPS cells in human diseases.[4] In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.[5] The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).

iPSCs are typically derived by introducing a specific set of pluripotency-associated genes, or reprogramming factors, into a given cell type. The original set of reprogramming factors (also dubbed Yamanaka factors) are the genes Oct4 (Pou5f1), Sox2, cMyc, and Klf4. While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non-related genes such as lineage specifiers.

iPSC derivation is typically a slow and inefficient process, taking 12 weeks for mouse cells and 34 weeks for human cells, with efficiencies around 0.01%0.1%. However, considerable advances have been made in improving the efficiency and the time it takes to obtain iPSCs. Upon introduction of reprogramming factors, cells begin to form colonies that resemble pluripotent stem cells, which can be isolated based on their morphology, conditions that select for their growth, or through expression of surface markers or reporter genes.

Induced pluripotent stem cells were first generated by Shinya Yamanaka's team at Kyoto University, Japan, in 2006.[1] Their hypothesis was that genes important to embryonic stem cell function might be able to induce an embryonic state in adult cells. They began by choosing twenty-four genes that were previously identified as important in embryonic stem cells, and used retroviruses to deliver these genes to fibroblasts from mice. The mouse fibroblasts were engineered so that any cells that reactivated the ESC-specific gene, Fbx15, could be isolated using antibiotic selection.

Upon delivery of all twenty-four factors, colonies emerged that had reactivated the Fbx15 reporter, resembled ESCs, and could propagate indefinitely. They then narrowed their candidates by removing one factor at a time from the pool of twenty-four. By this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which as a group were both necessary and sufficient to obtain ESC-like colonies under selection for reactivation of Fbx15.

Similar to ESCs, these first-generation iPSCs showed unlimited self-renewal and demonstrated pluripotency by contributing to lineages from all three germ layers in the context of embryoid bodies, teratomas, fetal chimeras. However, the molecular makeup of these cells, including gene expression and epigenetic marks, was somewhere between that of a fibroblast and an ESC, and the cells also failed to produce viable chimeras when injected into developing embryos.

In June 2007, the same group published a breakthrough study along with two other independent research groups from Harvard, MIT, and the University of California, Los Angeles, showing successful reprogramming of mouse fibroblasts into iPS cells. Unlike the first generation of iPS cells, these cells could produce viable chimeric mice and could contribute to the germline, the 'gold standard' for pluripotent stem cells. These cells were derived from mouse fibroblasts by retroviral-mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4), but the researchers used a different marker to select for pluripotent cells. Instead of Fbx15, they used Nanog, a gene that is functionally important in ESCs. By using this different strategy, the researchers were able to create iPS cells that were more similar to ESCs than the first generation of iPS cells, and independently proved that it was possible to create iPS cells that are functionally identical to ESCs.[6][7][8][9]

Unfortunately, two of the four genes used (namely, c-Myc and KLF4) are oncogenic, and 20% of the chimeric mice developed cancer. In a later study, Yamanaka reported that one can create iPSCs even without c-Myc. The process takes longer and is not as efficient, but the resulting chimeras didn't develop cancer.[10]

Induced pluripotent cells have been made from adult stomach, liver, skin cells, blood cells, prostate cells and urinary tract cells.[11]

In November 2007, a milestone was achieved[12][13] by creating iPSCs from adult human cells; two independent research teams' studies were released one in Science by James Thomson at University of WisconsinMadison[14] and another in Cell by Shinya Yamanaka and colleagues at Kyoto University, Japan.[15] With the same principle used earlier in mouse models, Yamanaka had successfully transformed human fibroblasts into pluripotent stem cells using the same four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc with a retroviral system. Thomson and colleagues used OCT4, SOX2, NANOG, and a different gene LIN28 using a lentiviral system.

On 8 November 2012, researchers from Austria, Hong Kong and China presented a protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine on Nature Protocols.[16] This method of acquiring donor cells is comparatively less invasive and simple. The team reported the induction procedure to take less time, around 2 weeks for the urinary cell culture and 3 to 4 weeks for the reprogramming; and higher yield, up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) were found to show good differentiation potential, and thus represent an alternative choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.[16]

Although the methods pioneered by Yamanaka and others have demonstrated that adult cells can be reprogrammed to iPS cells, there are still challenges associated with this technology:

The table at right summarizes the key strategies and techniques used to develop iPS cells over the past half-decade. Rows of similar colors represents studies that used similar strategies for reprogramming.

One of the main strategies for avoiding problems (1) and (2) has been to use small compounds that can mimic the effects of transcription factors. These molecule compounds can compensate for a reprogramming factor that does not effectively target the genome or fails at reprogramming for another reason; thus they raise reprogramming efficiency. They also avoid the problem of genomic integration, which in some cases contributes to tumor genesis. Key studies using such strategy were conducted in 2008. Melton et al. studied the effects of histone deacetylase (HDAC) inhibitor valproic acid. They found that it increased reprogramming efficiency 100-fold (compared to Yamanakas traditional transcription factor method).[25] The researchers proposed that this compound was mimicking the signaling that is usually caused by the transcription factor c-Myc. A similar type of compensation mechanism was proposed to mimic the effects of Sox2. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of calcium channels in the plasma membrane in order to increase reprogramming efficiency.[26] Deng et al. of Beijing University reported on July 2013 that induced pluripotent stem cells can be created without any genetic modification. They used a cocktail of seven small-molecule compounds including DZNep to induce the mouse somatic cells into stem cells which they called CiPS cells with the efficiency at 0.2% comparable to those using standard iPSC production techniques. The CiPS cells were introduced into developing mouse embryos and were found to contribute to all major cells types, proving its pluripotency.[27][28]

Ding et al. demonstrated an alternative to transcription factor reprogramming through the use of drug-like chemicals. By studying the MET (mesenchymal-epithelial transition) process in which fibroblasts are pushed to a stem-cell like state, Dings group identified two chemicals ALK5 inhibitor SB431412 and MEK (mitogen-activated protein kinase) inhibitor PD0325901 which was found to increase the efficiency of the classical genetic method by 100 fold. Adding a third compound known to be involved in the cell survival pathway, Thiazovivin further increases the efficiency by 200 fold. Using the combination of these three compounds also decreased the reprogramming process of the human fibroblasts from four weeks to two weeks. [29][30]

Another key strategy for avoiding problems such as tumor genesis and low throughput has been to use alternate forms of vectors: adenovirus, plasmids, and naked DNA and/or protein compounds.

In 2008, Hochedlinger et al. used an adenovirus to transport the requisite four transcription factors into the DNA of skin and liver cells of mice, resulting in cells identical to ESCs. The adenovirus is unique from other vectors like viruses and retroviruses because it does not incorporate any of its own genes into the targeted host and avoids the potential for insertional mutagenesis.[31] In 2009, Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells.[32] Another advantage of using adenoviruses is that they only need to present for a brief amount of time in order for effective reprogramming to take place.

Also in 2008, Yamanaka et al. found that they could transfer the four necessary genes with a plasmid.[33] The Yamanaka group successfully reprogrammed mouse cells by transfection with two plasmid constructs carrying the reprogramming factors; the first plasmid expressed c-Myc, while the second expressed the other three factors (Oct4, Klf4, and Sox2). Although the plasmid methods avoid viruses, they still require cancer-promoting genes to accomplish reprogramming. The other main issue with these methods is that they tend to be much less efficient compared to retroviral methods. Furthermore, transfected plasmids have been shown to integrate into the host genome and therefore they still pose the risk of insertional mutagenesis. Because non-retroviral approaches have demonstrated such low efficiency levels, researchers have attempted to effectively rescue the technique with what is known as the piggyBac transposon system. The lifecycle of this system is shown below. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving any footprint mutations in the host cell genome. As demonstrated in the figure, the piggyBac transposon system involves the re-excision of exogenous genes, which eliminates issues like insertional mutagenesis

In January 2014, two articles were published claiming that a type of pluripotent stem cell can be generated by subjecting the cells to certain types of stress (bacterial toxin, a low pH of 5.7, or physical squeezing); the resulting cells were called STAP cells, for stimulus-triggered acquisition of pluripotency.[34]

In light of difficulties that other labs had replicating the results of the surprising study, in March 2014, one of the co-authors has called for the articles to be retracted.[35] On 4 June 2014, the lead author, Obokata agreed to retract both the papers [36] after she was found to have committed research misconduct as concluded in an investigation by RIKEN on 1 April 2014.[37]

Studies by Blelloch et al. in 2009 demonstrated that expression of ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhances the efficiency of induced pluripotency by acting downstream of c-Myc .[38] More recently (in April 2011), Morrisey et al. demonstrated another method using microRNA that improved the efficiency of reprogramming to a rate similar to that demonstrated by Ding. MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Morriseys team worked on microRNAs in lung development, and hypothesized that their microRNAs perhaps blocked expression of repressors of Yamanakas four transcription factors. Possible mechanisms by which microRNAs can induce reprogramming even in the absence of added exogenous transcription factors, and how variations in microRNA expression of iPS cells can predict their differentiation potential discussed by Xichen Bao et al.[39]

[citation needed]

The generation of iPS cells is crucially dependent on the genes used for the induction.

Oct-3/4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.

Induced pluripotent stem cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability, but the full extent of their relation to natural pluripotent stem cells is still being assessed.[42]

Gene expression and genome-wide H3K4me3 and H3K27me3 were found to be extremely similar between ES and iPS cells.[43][citation needed] The generated iPSCs were remarkably similar to naturally isolated pluripotent stem cells (such as mouse and human embryonic stem cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity, authenticity, and pluripotency of iPSCs to naturally isolated pluripotent stem cells:

Recent achievements and future tasks for safe iPSC-based cell therapy are collected in the review of Okano et al.[55]

The task of producing iPS cells continues to be challenging due to the six problems mentioned above. A key tradeoff to overcome is that between efficiency and genomic integration. Most methods that do not rely on the integration of transgenes are inefficient, while those that do rely on the integration of transgenes face the problems of incomplete reprogramming and tumor genesis, although a vast number of techniques and methods have been attempted. Another large set of strategies is to perform a proteomic characterization of iPS cells. The Wu group at Stanford University has made significant progress with this strategy.[56] Further studies and new strategies should generate optimal solutions to the five main challenges. One approach might attempt to combine the positive attributes of these strategies into an ultimately effective technique for reprogramming cells to iPS cells.

Another approach is the use of iPS cells derived from patients to identify therapeutic drugs able to rescue a phenotype. For instance, iPS cell lines derived from patients affected by ectodermal dysplasia syndrome (EEC), in which the p63 gene is mutated, display abnormal epithelial commitment that could be partially rescued by a small compound[57]

An attractive feature of human iPS cells is the ability to derive them from adult patients to study the cellular basis of human disease. Since iPS cells are self-renewing and pluripotent, they represent a theoretically unlimited source of patient-derived cells which can be turned into any type of cell in the body. This is particularly important because many other types of human cells derived from patients tend to stop growing after a few passages in laboratory culture. iPS cells have been generated for a wide variety of human genetic diseases, including common disorders such as Down syndrome and polycystic kidney disease.[58][59] In many instances, the patient-derived iPS cells exhibit cellular defects not observed in iPS cells from healthy patients, providing insight into the pathophysiology of the disease.[60] An international collaborated project, StemBANCC, was formed in 2012 to build a collection of iPS cell lines for drug screening for a variety of disease. Managed by the University of Oxford, the effort pooled funds and resources from 10 pharmaceutical companies and 23 universities. The goal is to generate a library of 1,500 iPS cell lines which will be used in early drug testing by providing a simulated human disease environment.[61]

A proof-of-concept of using induced pluripotent stem cells (iPSCs) to generate human organ for transplantation was reported by researchers from Japan. Human liver buds (iPSC-LBs) were grown from a mixture of three different kinds of stem cells: hepatocytes (for liver function) coaxed from iPSCs; endothelial stem cells (to form lining of blood vessels) from umbilical cord blood; and mesenchymal stem cells (to form connective tissue). This new approach allows different cell types to self-organize into a complex organ, mimicking the process in fetal development. After growing in vitro for a few days, the liver buds were transplanted into mice where the liver quickly connected with the host blood vessels and continued to grow. Most importantly, it performed regular liver functions including metabolizing drugs and producing liver-specific proteins. Further studies will monitor the longevity of the transplanted organ in the host body (ability to integrate or avoid rejection) and whether it will transform into tumors.[62][63] Using this method, cells from one mouse could be used to test 1,000 drug compounds to treat liver disease, and reduce animal use by up to 50,000.[64]

Embryonic cord-blood cells were induced into pluripotent stem cells using plasmid DNA. Using cell surface endothelial/pericytic markers CD31 and CD146, researchers identified 'vascular progenitor', the high-quality, multipotent vascular stem cells. After the iPS cells were injected directly into the vitreous of the damaged retina of mice, the stem cells engrafted into the retina, grew and repaired the vascular vessels.[65][66]

In a study conducted in China in 2013, Superparamagnetic iron oxide (SPIO) particles were used to label iPSCs-derived NSCs in vitro. Labeled NSCs were implanted into TBI rats and SCI monkeys 1 week after injury, and then imaged using gradient reflection echo (GRE) sequence by 3.0T magnetic resonance imaging (MRI) scanner. MRI analysis was performed at 1, 7, 14, 21, and 30 days, respectively, following cell transplantation. Pronounced hypointense signals were initially detected at the cell injection sites in rats and monkeys and were later found to extend progressively to the lesion regions, demonstrating that iPSCs-derived NSCs could migrate to the lesion area from the primary sites. The therapeutic efficacy of iPSCs-derived NSCs was examined concomitantly through functional recovery tests of the animals. In this study, we tracked iPSCs-derived NSCs migration in the CNS of TBI rats and SCI monkeys in vivo for the first time. Functional recovery tests showed obvious motor function improvement in transplanted animals. These data provide the necessary foundation for future clinical application of iPSCs for CNS injury.[67]

In 2014, type O red blood cells were synthesized at the Scottish National Blood Transfusion Service from iPSC. The cells were induced to become a mesoderm and then blood cells and then red blood cells. The final step was to make them eject their nuclei and mature properly. Type O can be transfused into all patients. Each pint of blood contains about two trillion red blood cells, while some 107 million blood donations are collected globally every year. Human transfusions were not expected to begin until 2016.[68]

The first human clinical trial using autologous iPSCs is approved by the Japan Ministry Health and will be conducted in 2014 in Kobe. iPSCs derived from skin cells from six patients suffering from wet age-related macular degeneration will be reprogrammed to differentiate into retinal pigment epithelial (RPE) cells. The cell sheet will be transplanted into the affected retina where the degenerated RPE tissue has been excised. Safety and vision restoration monitoring is expected to last one to three years.[69][70] The benefits of using autologous iPSCs are that there is theoretically no risk of rejection and it eliminates the need to use embryonic stem cells.[70]

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Induced pluripotent stem cell - Wikipedia, the free ...

IPS Cell Therapy Comments Off on Induced pluripotent stem cell – Wikipedia, the free … | October 24th, 2015

As many of you know, the pioneering, first of its kind IPSC clinical study in Japan has been suspended as I first blogged about here.

In the comments section of that blog post there has been a helpful overall discussion that has involved Dr. Masayo Takahashi, the leader of the trial. It is great that Dr. Takahashi has been participating in this discussion and I commend her for that openness.

This comment stream has been particularly important because the media have only minimally reported on this important development. There have been only a few articles in Japanese (several months ago) and as far as I know only one in English, which was posted in the last day or so in The New Scientist. Unfortunately The New Scientist article, as many have noted here, used an inflammatory title invoking a supposed cancer scare and some over-the-top language. Although that article had some bits of important info, the negative bias in the article made it overall not very helpful. Some readers of that article were likely confused by how it was written and the title.

The clinical study in question is for macular degeneration and involves the use of sheets of retinal pigmented epithelial cells (RPE) made from IPSC (e.g. see image above from RIKEN).Several of us have been discussing the suspension of this trial over on Twitter too including Dr. Takahashi (@masayomasayo). Some tweets by the community have been constructive. Others not so much.

Two main possible issues have come up in the discussion of the reasons for the trial stopping: (1) six mutations were detected in the 2nd patients IPSC and (2) significant regulatory changes are on the way in Japan that apparently in some way will delimit IPSC research there. Dr. Takahashi has indicated that the latter reason was the dominant factor in their decision to suspend the trial. The fact that the 2nd patients IPSC reportedly had six mutations that were not present in the original somatic cells warrantsfurther discussion too. For example, when and how did these mutations arise? To be clear, however, I do not see (based on the information available) that there was a cancer scare by any stretch of the imagination as The New Scientist article had indicated.

At some point a restarted version of this study will likely focus on allogeneic use of IPSC perhaps via an IPSC bank being developed by Dr. Shinya Yamanaka. For many years the consensus, most exciting aspect of IPSCs in the field was considered to be their potential for use as the basis for powerful patient-specific autologous therapies. The apparent planned shift to non-autologous clinical use of IPSC in this case raises the question of how it would be superior or substantially different to the use of hESC, other than that making IPSC does not involve the use of a leftover IVF embryo.

This development also raises a 2nd question as to whether there will be a domino effect now of other clinical studies or trials that are in the works using IPSC switching to allogeneic paths as well. In other words, is this a historic, turning point moment for the IPSC field in Japan overall away from an autologous path?Or is the switch here to allogeneic just a one time, one study decision? More info on the regulatory changes is needed to help clarify the answer to this question and the path forward as well.

Hopefully the regulatory body in Japan (Ministry of Education?) that has made or is making the relevant regulatory changes will announce them publicly in detail soon. If that information is already out there (e.g. in Japanese on the web) perhaps someone can find it and well post it here.

. Bookmark the

.

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Historic turning point for IPS cell field in Japan ...

IPS Cell Therapy Comments Off on Historic turning point for IPS cell field in Japan … | October 16th, 2015

In 2006, Shinya Yamanaka of Kyoto University in Japan was the first to disprove the previous notion that reversible cell differentiation of mammals was impossible. He reprogrammed a fully differentiated mouse cell into a pluripotent stem cell by introducing four genes, Oct-4, SOX2, KLF4, and Myc, into the mouse fibroblast through gene-carrying viruses. With this method, he and his coworkers created induced pluripotent stem cells (iPS cells), the key component in this experiment.[1] Scientists have been able to conduct experiments that show the ability of iPS cells to treat and even cure diseases. In this experiment, tests were run on mice with inherited sickle-cell anemia. Skin cells were turned into cells containing genes that transformed the cells into iPS cells. These replaced the diseased sickled cells, curing the test mice. The reprogramming of the pluripotent stem cells in mice was successfully duplicated with human pluripotent stem cells within about a year of the experiment on the mice.[citation needed]

Sickle-cell anemia is a disease in which the body produces abnormally shaped red blood cells. Red blood cells are flexible and round, moving easily through the blood vessels. Infected cells are shaped like a crescent or sickle (the namesake of the disease). As a result of this disorder the hemoglobin protein in red blood cells is faulty. Normal hemoglobin bonds to oxygen, then releases it into cells that need it. The blood cell retains its original form and is cycled back to the lungs and re-oxygenated.

Sickle cell hemoglobin, however, after giving up oxygen, cling together and make the red blood cell stiff. The sickle shape also makes it difficult for the red blood cell to navigate arteries and causes blockages.[2] This can cause intense pain and organ damage. The sickled red blood cells are fragile and prone to rupture. When the number of red blood cells decreases from rupture (hemolysis), anemia is the result. Sickle cells die in 1020 days as opposed to the traditional 120-day lifespan of a normal red blood cell.

Sickle cell anemia is inherited as an autosomal (meaning that the gene is not linked to a sex chromosome) recessive condition.[2] This means that the gene can be passed on from a carrier to his or her children. In order for sickle cell anemia to affect a person, the gene must be inherited from both the mother and the father, so that the child has two recessive sickle cell genes (a homozygous inheritance). People who inherit one sickle cell gene from one parent and one normal gene from the other parent, i.e. heterozygous patients, have a condition called sickle cell trait. Their bodies make both sickle hemoglobin and normal hemoglobin. They may pass the trait on to their children.

The effects of sickle-cell anemia vary from person to person. People who have the disease suffer from varying degrees of chronic pain and fatigue. With proper care and treatment, the quality of health of most patients will improve. Doctors have learned a great deal about sickle cell anemia since its discovery in 1979. They know its causes, its effects on the body, and possible treatments for complications. Sickle cell anemia has no widely available cure. A bone marrow transplant is the only treatment method currently recognized to be able to cure the disease, though it does not work for every patient. Finding a donor is difficult and the procedure could potentially do more harm than good. Treatments for sickle cell anemia are generally aimed at avoiding crises, relieving symptoms, and preventing complications. Such treatments may include medications, blood transfusions, and supplemental oxygen.

During the first step of the experiment, skin cells (also known as fibroblasts) were collected from infected test mice and put in a culture. The fibroblasts were reprogrammed by infecting them with retroviruses that contained genes common to embryonic stem cells. These genes were the same four used by Yamanaka (Oct-4, SOX2, KLF4, and Myc) in his earlier study. The investigators were trying to produce cells with the potential to differentiate into any type of cell needed (i.e. pluripotent stem cells). As the experiment continued, the fibroblasts multiplied into identical copies of iPS cells. The cells were then treated to form the mutation needed to reverse the anemia in the mice. This was accomplished by restructuring the DNA containing the defective globin gene into DNA with the normal gene through the process of homologous recombination. The iPS cells then differentiated into blood stem cells, or hematopoietic stem cells. The hematopoietic cells were injected back into the infected mice, where they proliferate and differentiate into normal blood cells, curing the mice of the disease.[3][4][verification needed]

To determine whether the mice were cured from the disease, the scientists checked for the usual symptoms of sickle cell disease. They examined the blood for mean corpuscular volume (MCV) and red cell distribution width (RDW) and urine concentration defects. They also checked for sickled red blood cells. They examined the DNA through gel electrophoresis, checking for bands that display an allele that causes sickling. Compared to the untreated mice with the disease, which they used as a control, "the treated animals had marked increases in RBC counts, healthy hemoglobin, and packed cell volume levels".[5]

Researchers examined "the urine concentration defect, which results from RBC sickling in renal tubules and consequent reduction in renal medullary blood flow, and the general deteriorated systemic condition reflected by lower body weight and increased breathing."[5] They were able to see that these parts of the body of the mice had healed or improved. This indicated that "all hematological and systemic parameters of sickle cell anemia improved substantially and were comparable to those in control mice."[5] They cannot say if this will work in humans because a safe way to inject the genes for the induced pluripotent cells is still needed.[citation needed]

The reprogramming of the induced pluripotent stem cells in mice was successfully duplicated in humans within a year of the successful experiment on the mice. This reprogramming was done in several labs and it was shown that the iPS cells in humans were almost identical to original embryonic stem cells (ES cells) that are responsible for the creation of all structures in a fetus.[1] An important feature of iPS cells is that they can be generated with cells taken from an adult, which would circumvent many of the ethical problems associated with working with ES cells. These iPS cells also have potential in creating and examining new disease models and developing more efficient drug treatments.[6] Another feature of these cells is that they provide researchers with a human cell sample, as opposed to simply using an animal with similar DNA, for drug testing.

One major problem with iPS cells is the way in which the cells are reprogrammed. Using gene-carrying viruses has the potential to cause iPS cells to develop into cancerous cells.[1] Also, an implant made using undifferentiated iPS cells, could cause a teratoma to form. Any implant that is generated from using these iPS cells would only be viable for transplant into the original subject that the cells were taken from. In order for these iPS cells to become viable in therapeutic use, there are still many steps that must be taken.[5][7]

In the future, researchers hope that induced pluripotent cells may be used to treat other diseases. Pluripotency is a crucial part of disease treatment because iPS cells are capable of differentiation in a way that is very similar to embryonic stem cells which can grow into fully differentiated tissues. iPS cells also demonstrate high telomerase activity and express human telomerase reverse transcriptase, a necessary component in the telomerase protein complex. Also, iPS cells expressed cell surface antigenic markers expressed on ES cells. Also, doubling time and mitotic activity are cornerstones of ES cells, as stem cells must self-renew as part of their definition. As said, iPS cells are morphologically similar to embryonic stem cells. Each cell has a round shape, a large nucleolus and a small amount of cytoplasm. One day, the process may be used in practical settings to provide a fundamental way of regeneration.

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Induced pluripotent stem-cell therapy - Wikipedia, the ...

IPS Cell Therapy Comments Off on Induced pluripotent stem-cell therapy – Wikipedia, the … | October 4th, 2015

This is a sixth post of the series Not Lost in Translation.

If youre trying to develop a cellular product and just entering the field of cell therapy, you should be aware of existent standards. Why is it important? Knowing standards in your field allows to:

Even though, cell therapy filed relatively new, there are numerous related standards. Unfortunately, many professionals are unaware about organizations and standards in cell therapy field. The purpose of this post is to indicate few leadig organizations, providing standards and types of standards in cell products development. Significant part of this topic was summarized from the recent public FDA workshop Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products.

Type of standards in cell therapy:

Standards-developing organizations and examples: ISO International Organization for Standardization Developing and providing international standards, including medical devices, laboratory testing and some, related to cell therapy and tissue engineered products. Examples: ISO/TC 194/SC 1 Tissue product safety ISO/TC 150/SC 7 Tissue-engineered medical products

ASTM International American Society for Testing and Materials ASTM leading international standards organization. ASTM has Subcommittee F04.43 for developing standards in cell therapy and tissue engineering. Examples: ASTM F2210 Standard Guide for Processing Cells, Tissues, and Organs for Use in Tissue Engineered Medical Products ASTM F2739 Standard Guide for Quantitating Cell Viability Within Biomaterial Scaffolds ASTM F2315 Standard Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels ASTM F2944 Standard Test Method for Automated Colony Forming Unit (CFU) Assays

USP U.S. Pharmacopeial Convention Provides standards for use ancillary and raw materials for cellular and tissue products. Examples: Chapter 1046 Cell and Gene Therapies Products Chapter 1047 Gene Therapy Products Chapter 1043 Ancillary Materials for Cell, Gene and Tissue-Engineered Products Chapter 92 Growth Factors and Cytokines Used in Cell Therapy Manufacturing Chapter 90 Fetal Bovine SerumQuality Attributes and Functionality Tests

GBSI Global Biological Standard Institute Developing standards for life sciences, including biomedical research.

ATCC American Type Culture Collection Manufactures and provides reference material (including cells), developing biological standards for basic and translational research. Examples: ATCC Certified reference material ATCC Standards Development Organization

BSI British Standards Institution Has a project for developing regenerative medicine definitions and guidelines for clinical cell products characterization. Examples: PAS 93:2011 Characterization of human cells for clinical applications. Guide PAS 84:2012 Cell therapy and regenerative medicine. Glossary

FACT Foundation for the Accreditation of Cellular Therapy Provides standards for collection and processing cellular products. Accredits clinical stem cell labs, cord blood banks and more than minimal manipulation cell therapy facilities. Examples: FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration FACT-JACIE Cellular Therapy Accreditation Manual

AABB American Association of Blood Banks Center for Cellular Therapies In cell therapy field, AABB has very similar functions with FACT. Examples: Standards for Cellular Therapy Services

ICCBBA International Council for Commonality in Blood Bank Automation Management of the ISBT-128 Standard the terminology, identification, coding and labeling of medical products of human origin (including blood, cell, tissue, and organ products).

ISCT International Society for Cellular Therapy ISCT leverages expertise of cell therapy professionals to develop guidelines and recommendations for cellular products development, characterization, and quality. Examples: Minimal criteria for defining multipotent mesenchymal stromal cells Potency assay development for cellular therapy products Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells IFATS/ISCT statement

Coordination and harmonization As you can see, there are many organizations, involved in different aspects of cell therapy standardization. How can we make sure that there are no overlaps between them? How to coordinate and harmonize their activities? There are some good existent examples of such coordination:

*********************** This post is a part of Not Lost in Translation online community project. In this series we will try to bridge the translational gaps between scientific discovery in research labs and clinical cell applications for therapies. We will look at challenges in translation of cell product development and manufacturing in academic and industry settings. If you would like to contribute to this community project, please contact us!

Tagged as: cell therapy, reference material, standard, translation

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Standards in Cell Therapy

IPS Cell Therapy Comments Off on Standards in Cell Therapy | September 25th, 2015

Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers.

Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatment for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. In any case, this breakthrough discovery has created a powerful new way to "de-differentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

Previous|VI. What are induced pluripotent stem cells?|Next

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What are induced pluripotent stem cells? [Stem Cell ...

IPS Cell Therapy Comments Off on What are induced pluripotent stem cells? [Stem Cell … | September 18th, 2015

Recently, Ive written about transition from iPS cell research to iPS cell large-scale manufacturing and automation. Ive described iPS cell process development in Cellular Dynamics International and New York Stem Cell Foundation Research Institute. Today, Id like to share presentations of 2 more players in the field Lonza and Roslin Cells. Both presentations were recorded at Stem Cell Meeting on the Mesa, held on October 14-16, 2013.

What was especially interesting to see a cost comparison between research and clinical-grade GMP-produced iPS cell lines:

(Screenshot from Lonza presentation at Stem Cell Meeting on the Mesa, 2013)

Interestingly, the major cost contributor in GMP-grade iPS cell production is a facility cost. I think, this is a first estimation of cost difference, presented for public.

The framework for establishing clinical-grade iPS cell manufacturing, nicely outlined in the recent article. Id also recommend you to read the following open access articles:

Tagged as: cost, iPS, manufacturing

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Clinical GMP-grade iPS cell production - Stem Cell Assays

IPS Cell Therapy Comments Off on Clinical GMP-grade iPS cell production – Stem Cell Assays | July 23rd, 2015

Featured post

Stem Cell Research is an amazing field right now, and promises to be a powerful and potent tool to help us live longer and healthier lives. Just last month, for example, Stem Cell Therapy was used to restore sight in patients with severe retinal deterioration, allowing them to see clearer than they had in years, or even decades.

Now, there is another form of Stem Cell Treatment on the horizonthis one of a very different form. Stem Cells have now been used as a mechanism to deliver medical treatment designed to eliminate cancer cells, even in hard to reach places. One issue with current cancer treatments is that, treatments that are effective at treating tumors on the surface of the brain cannot be performed safely when the tumor is deeper within the brains tissues.

Stem Cells have the fantastic ability to transform into any other kind of cell within the human body, given the appropriate stimulation. As of today, most of these cells come from Embryonic Lines, but researchers are learning how to backwards engineer cells in the human body, reverting them back to their embryonic state. These cells are known as Induced Pluripotent Stem Cells.

How Does This Stem Cell Cancer Treatment Work?

Using genetic engineering, it is possible to create stem cells that are designed to release a chemical known as Pseudomonas Exotoxin, which has the ability to destroy certain tumor cells in the human brain.

What is Pseudomonas Exotoxin?

Pseudomonas Exotoxin is a compound that is naturally released by a form of bacteria known as Pseudomonas Aeruginosa. This chemical is toxic to brain tumor cells because it prevents polypeptides from growing longer, essentially preventing the polypeptides from growing and reproducing. When used in a specific manner, this toxin has the ability to destroy cancerous and malignant tissue without negatively impacting healthy tissue. In addition to its potential as a cancer treatment, there is also evidence that the therapy could be used for the treatment of Hepatitis B.

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IPS Cell Therapy

IPS Cell Therapy Comments Off on IPS Cell Therapy | July 2nd, 2015

SAN DIEGO(BUSINESS WIRE)Cytori Therapeutics, Inc. (NASDAQ: CYTX) today confirmed that two Japanese regenerative medicine laws, which went into effect on November 25, 2014, remove regulatory uncertainties and provide a clear path for the Company to commercialize and market Cytori Cell Therapy and its Celution System under the Companys existing and planned regulatory approvals.

Japans new regenerative medicine laws substantially clarify regulatory ambiguities of pre-existing guidelines and this news represents a significant event for Cytori, said Dr. Marc Hedrick, President & CEO of Cytori. We have a decade of operating experience in Japan and Cytori is nicely positioned to see an impact both on existing commercial efforts and on our longer-term efforts to obtain therapeutic claims and reimbursement for our products.

Under the two new laws, Cytori believes its Celution System and autologous adipose-derived regenerative cells (ADRCs) can be provided by physicians under current Class I device regulations and used under the lowest risk category (Tier 3) for many procedures with only the approval by accredited regenerative medicine committees and local agencies of the Ministry of Health, Labour and Welfare (MHLW). This regulatory framework is expected to streamline the approval and regulatory process and increase clinical use of Cytori Cell Therapy and the Celution System over the former regulations.

Before these new laws were enacted, the regulatory pathway for clinical use of regenerative cell therapy was one-size-fits-all, irrespective of the risk posed by certain cell types and approaches, said Dr. Hedrick. Now, Cytoris point-of-care Celution System can be transparently integrated into clinical use by providers under our Class I device status and the streamlined approval process granted to cell therapies that pose the lowest risk. Our technology is unique in that respect.

Cytoris Celution System Is in Lowest of Three Risk Categories

The Act on the Safety of Regenerative Medicines and an amendment of the 2013 Pharmaceutical Affairs Act (the PMD Act), collectively termed the Regenerative Medicine Laws, replace the Human Stem Cell Guidelines. Under the new laws, the cell types used in cell therapy and regenerative medicine are classified based on risk. Cell therapies using cells derived from embryonic, induced pluripotent, cultured, genetically altered, animal and allogeneic cells are considered higher risk (Tiers 1 and 2) and will undergo an approval pathway with greater and more stringent oversight due to the presumed higher risk to patients. Cytoris Celution System, which uses the patients own cells at the point-of-care, will be considered in the lowest risk category (Tier 3) for most cases, and will be considered in Tier 2 if used as a non-homologous therapy.

Streamlined Regulatory Approval for Certain Medical Devices

In the near future, Cytori intends to pursue disease-specific or therapeutic claims and reimbursement for Cytoris Celution System and the Company would, at that point, sponsor a clinical trial to obtain Class III device-based approval and reimbursement. The new laws include changes to streamline regulation of Class II and some Class III devices, which will now require the approval of certification bodies rather than the PMDA, similar to the European notified body model. To date, certification bodies have only been used for some Class II devices.

Conditional Regulatory Approval and Reimbursement Potential

As a supplementary benefit to Cytori, the Company may also choose to take advantage of the new conditional approval opportunities granted under the new laws. Once clinical safety and an indication of efficacy are shown, sponsors may apply for their cell product to receive conditional approval for up to seven years and may be eligible for reimbursement under Japans national insurance coverage. Under the conditional approval, the sponsor can then generate post-marketing data to demonstrate further efficacy and cost effectiveness.

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japanese | StemCell Therapy MD

IPS Cell Therapy Comments Off on japanese | StemCell Therapy MD | June 19th, 2015

There have been hundreds of science fiction stories and books written about growing organs in scientific laboratories as replacements for those that no longer function properly, or about injecting scientifically transmuted cells into ailing patients that can repair the broken cells within their bodies, bringing them back to robust health. In todays language what they were talking about was Induced Pluripotent Stem Cell (iPSC) Therapy.

Here, in the early 21st century, the gap between science fiction and science truth is closing at a record rate due to the rapid progress made in iPSC Therapy research, especially over the last three years.

After the virtual stop order placed on embryonic cell stem research in 2001, the race to find an alternative type of stem cell began in earnest, and in 2006 Shinya Yamanaka of Kyoto University in Japan announced his teams successful reprogramming of mouse cells into iPSCs. This was the breakthrough that made it possible for stem cell research to continue without the use of controversial embryonic stem cells.

The next major announcement came in 2007, again from Yamanaka in Japan, followed by one only a few weeks later by James A. Thompson from the University of Wisconsin, detailing the making of iPSC from adult human cells. Again, neither used embryos in their experiments.

From that time on the goal has been developing stem cell science that will eventually be safe iPS Cell Therapy modalities to be used in Regenerative or Reparative Medicine. What kinds of illnesses or diseases will iPSC Therapies be used to treat in the future? Only a partial list would include:

The world of iPSC Therapy research is wide open today and its on the move! This website is dedicated to bringing you first, the story of stem cell research, both embryonic and iPStem Cell, and the controversy surrounding them, as well as the most up to date information in the easiest to understand language about major milestone accomplishments in the field.

If you were to go back 100 years you would be amazed by how primitive medicine was. Even 60 years ago there were no organ transplants, no cystoscopic surgeries, and there was a massive polio outbreak in the United States that closed public swimming pools and beaches and other public gathering places across the country for the summer. Who can tell where medicine will be in 10 or 15 years? There is no predicting, but with the rapid advancement of the last few years and the bright promise shown so far, iPSC Therapy is sure to play a major role.

Continued here: iPSCTherapy.com: Induced Pluripotent Stem Cell therapy

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iPSCTherapy.com: Induced Pluripotent Stem Cell therapy ...

IPS Cell Therapy Comments Off on iPSCTherapy.com: Induced Pluripotent Stem Cell therapy … | June 8th, 2015

CRISPR-Cas9 is a powerful new tool for editing the genome. For researchers around the world, the CRISPR-Cas9 technique is an exciting innovation because it is faster and cheaper than previous methods. Now, using a molecular trick, Dr. Van Trung Chu and Professor Klaus Rajewsky of the Max Delbrck Center for Molecular Medicine (MDC) Berlin-Buch and Dr. Ralf Khn, MDC and Berlin Institute of Health (BIH), have found a solution to considerably increase the efficiency of precise genetic modifications by up to eightfold (Nature Biotechnology: doi:10.1038/nbt.3198)**.

"What we used to do in years, we can now achieve in months," said gene researcher and immunologist Klaus Rajewsky, indicating the power of this new genome-editing technology. CRISPR-Cas9 not only speeds up research considerably - at the same time it is much more efficient, cheaper and also easier to handle than the methods used so far.

The CRISPR-Cas9 technology allows researchers to transiently introduce DNA double-strand breaks into the genome of cells or model organisms at genes of choice. In these artificially produced strand breaks, they can insert or cut out genes and change the genetic coding according to their needs.

Mammalian cells are able to repair DNA damage in their cells using two different repair mechanisms. The homology-directed repair (HDR) pathway enables the insertion of preplanned genetic modifications using engineered DNA molecules that share identical sequence regions with the targeted gene and which are recognized as a repair template. Thus, HDR repair is very precise but occurs only at low frequency in mammalian cells.

The other repair system, called non-homologous end-joining (NHEJ) is more efficient in nature but less precise, since it readily reconnects free DNA ends without repair template, thereby frequently deleting short sequences from the genome. Therefore, NHEJ repair can only be used to create short genomic deletions, but does not support precise gene modification or the insertion and replacement of gene segments.

Many researchers, including Van Trung Chu, Klaus Rajewsky and Ralf Khn, are seeking to promote the HDR repair pathway to make gene modification in the laboratory more precise in order to avoid editing errors and to increase efficiency. The MDC researchers succeeded in increasing the efficiency of the more precisely working HDR repair system by temporarily inhibiting the most dominant repair protein of NHEJ, the enzyme DNA Ligase IV. In their approach they used various inhibitors such as proteins and small molecules.

"But we also used a trick of nature and blocked Ligase IV with the proteins of adeno viruses. Thus we were able to increase the efficiency of the CRISPR-Cas9 technology up to eightfold," Ralf Khn explained. For example, they succeeded in inserting a gene into a predefined position in the genome (knock-in) in more than 60 per cent of all manipulated mouse cells. Khn has just recently joined the MDC and is head of the research group for "iPS cell based disease modeling". Before coming to the MDC, he was on the research staff of Helmholtz Zentrum Mnchen. "The expertise of Ralf Khn is very important for gene research at MDC and especially for my research group," Klaus Rajewsky said.

Concurrent with the publication of the article by the MDC researchers, Nature Biotechnology published another, related paper on CRISPR-Cas9 technology. It comes from the laboratory of Hidde Ploegh of the Whitehead Institute in Cambridge, MA, USA.

Somatic gene therapy with CRISPR-Cas9 is a goal

The new CRISPR-Cas9 technology, developed in 2012, is already used in the laboratory to correct genetic defects in mice. Researchers also plan to modify the genetic set up of induced pluripotent stem cells (iPS), which can be differentiated into specialized cell types or tissues. That is, researchers are able to use the new tool to introduce patient-derived mutations into the genome of iPS cells for studying the onset of human diseases. "Another future goal, however, is to use CRISPR-Cas9 for somatic gene therapy in humans with severe diseases," Klaus Rajewsky pointed out.

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MDC researchers greatly increase precision of new genome editing tool

IPS Cell Therapy Comments Off on MDC researchers greatly increase precision of new genome editing tool | March 25th, 2015

CRISPR-Cas9 is a powerful new tool for editing the genome. For researchers around the world, the CRISPR-Cas9 technique is an exciting innovation because it is faster and cheaper than previous methods. Now, using a molecular trick, Dr. Van Trung Chu and Professor Klaus Rajewsky of the Max Delbrck Center for Molecular Medicine (MDC) Berlin-Buch and Dr. Ralf Khn, MDC and Berlin Institute of Health (BIH), have found a solution to considerably increase the efficiency of precise genetic modifications by up to eightfold.

"What we used to do in years, we can now achieve in months," said gene researcher and immunologist Klaus Rajewsky, indicating the power of this new genome-editing technology. CRISPR-Cas9 not only speeds up research considerably - at the same time it is much more efficient, cheaper and also easier to handle than the methods used so far.

The CRISPR-Cas9 technology allows researchers to transiently introduce DNA double-strand breaks into the genome of cells or model organisms at genes of choice. In these artificially produced strand breaks, they can insert or cut out genes and change the genetic coding according to their needs.

Mammalian cells are able to repair DNA damage in their cells using two different repair mechanisms. The homology-directed repair (HDR) pathway enables the insertion of preplanned genetic modifications using engineered DNA molecules that share identical sequence regions with the targeted gene and which are recognized as a repair template. Thus, HDR repair is very precise but occurs only at low frequency in mammalian cells.

The other repair system, called non-homologous end-joining (NHEJ) is more efficient in nature but less precise, since it readily reconnects free DNA ends without repair template, thereby frequently deleting short sequences from the genome. Therefore, NHEJ repair can only be used to create short genomic deletions, but does not support precise gene modification or the insertion and replacement of gene segments.

Many researchers, including Van Trung Chu, Klaus Rajewsky and Ralf Khn, are seeking to promote the HDR repair pathway to make gene modification in the laboratory more precise in order to avoid editing errors and to increase efficiency. The MDC researchers succeeded in increasing the efficiency of the more precisely working HDR repair system by temporarily inhibiting the most dominant repair protein of NHEJ, the enzyme DNA Ligase IV. In their approach they used various inhibitors such as proteins and small molecules.

"But we also used a trick of nature and blocked Ligase IV with the proteins of adeno viruses. Thus we were able to increase the efficiency of the CRISPR-Cas9 technology up to eightfold," Ralf Khn explained. For example, they succeeded in inserting a gene into a predefined position in the genome (knock-in) in more than 60 per cent of all manipulated mouse cells. Khn has just recently joined the MDC and is head of the research group for "iPS cell based disease modeling." Before coming to the MDC, he was on the research staff of Helmholtz Zentrum Mnchen. "The expertise of Ralf Khn is very important for gene research at MDC and especially for my research group," Klaus Rajewsky said.

Concurrent with the publication of the article by the MDC researchers, Nature Biotechnology published another, related paper on CRISPR-Cas9 technology. It comes from the laboratory of Hidde Ploegh of the Whitehead Institute in Cambridge, MA, USA.

Somatic gene therapy with CRISPR-Cas9 is a goal

The new CRISPR-Cas9 technology, developed in 2012, is already used in the laboratory to correct genetic defects in mice. Researchers also plan to modify the genetic set up of induced pluripotent stem cells (iPS), which can be differentiated into specialized cell types or tissues. That is, researchers are able to use the new tool to introduce patient-derived mutations into the genome of iPS cells for studying the onset of human diseases. "Another future goal, however, is to use CRISPR-Cas9 for somatic gene therapy in humans with severe diseases," Klaus Rajewsky pointed out.

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Researchers greatly increase precision of new genome editing tool

IPS Cell Therapy Comments Off on Researchers greatly increase precision of new genome editing tool | March 25th, 2015