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].

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.

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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 ...

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 ...

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.

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

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.

More here:
Induced pluripotent stem-cell therapy - Wikipedia, the ...

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

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 ...

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

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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

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

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 ...

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.

Read this article:
MDC researchers greatly increase precision of new genome editing tool

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.

More here:
Researchers greatly increase precision of new genome editing tool

Steve Perrin, Ph.D., CEO and CSO, discusses why iPS technology is ready for drug discovery for today's ALS patients. Click here to learn why Steve believes TDI is uniquely suited to implement this technology in ALS research.

Fernando Vieira, M.D., director of research operations, discusses how iPS technology can be used to model sporadic ALS, help to identify sub-types of ALS patients and accelerate drug development as part of a comprehensive translational research program at ALS TDI.

Jessie St. Martin, associate scientist, talks about induced pluripotent stem cells (iPS cells) and their importance in ALS research. Jessie, a recent addition to the translational research team, will play an integral part in developing this program at ALS TDI. Click here to learn more about iPS cells.

Jenny Dwyer, board member, explains why your support of the iPS program at ALS TDI may have the ability to rapidly accelerate treatments for today's patients. Jenny was a longtime ALS caregiver of her husband, Pat. Together, they were advocates for ALS research. Click here to listen to her message.

Continued here:
Invest in iPS @ TDI | ALS Therapy Development Institute

1 Control of Pluripotency Laboratory, Department of Physiological Sciences I, Faculty of Medicine, University of Barcelona, Hospital Clinic, Casanova 143, 08036, Barcelona, Spain 2 Faculty of Medicine, University of Sydney Medical School, Division of Pediatrics and Child Health, Westmead Children's Hospital, Locked Bag 4001, Westmead NSW 2145, Sydney, Australia 3 School of Anatomy Physiology & Human Biology and The Harry Perkins Institute for Medical Research (CCTRM), the University of Western Australia, 6 Verdun St, Nedlands WA 6009, Perth, Australia

* Author to whom correspondence should be addressed.

Received: 1 October 2014 / Accepted: 3 December 2014 / Published: 29 January 2015

Abstract: The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP) made from induced pluripotent stem cells (iPSCs) are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries.

Roca, I.; Requena, J.; Edel, M.J.; Alvarez-Palomo, A.B. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy. J. Clin. Med. 2015, 4, 243-259.

Roca I, Requena J, Edel MJ, Alvarez-Palomo AB. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy. Journal of Clinical Medicine. 2015; 4(2):243-259.

Roca, Isart; Requena, Jordi; Edel, Michael J.; Alvarez-Palomo, Ana B. 2015. "Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy." J. Clin. Med. 4, no. 2: 243-259.

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JCM | Free Full-Text | Myogenic Precursors from iPS Cells ...

IMAGE:Induced pluripotent stem cell-derived motor neurons from an ALS patient (left) compared with normal cells (right). The cells are being used to study the role of the genes TBK1 and... view more

NEW YORK, NY (February 19, 2015)--Using advanced DNA sequencing methods, researchers have identified a new gene that is associated with sporadic amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease. ALS is a devastating neurodegenerative disorder that results in the loss of all voluntary movement and is fatal in the majority of cases. The next-generation genetic sequencing of the exomes (protein-coding portions) of 2,874 ALS patients and 6,405 controls represents the largest number of ALS patients to have been sequenced in a single study to date.

Though much is known about the genetic underpinnings of familial ALS, only a handful of genes have been definitively linked to sporadic ALS, which accounts for about 90 percent of all ALS cases. The newly associated gene, called TBK1, plays a key role at the intersection of two essential cellular pathways: inflammation (a reaction to injury or infection) and autophagy (a cellular process involved in the removal of damaged cellular components). The study, conducted by an international ALS consortium that includes scientists and clinicians from Columbia University Medical Center (CUMC), Biogen Idec, and HudsonAlpha Institute for Biotechnology, was published today in the online edition of Science.

"The identification of TBK1 is exciting for understanding ALS pathogenesis, especially since the inflammatory and autophagy pathways have been previously implicated in the disease," said Lucie Bruijn, PhD, Chief Scientist for The ALS Association. "The fact that TBK1 accounts for one percent of ALS adds significantly to our growing understanding of the genetic underpinnings of the disease. This study, which combines the efforts of over two dozen laboratories in six countries, also highlights the global and collaborative nature of ALS research today.

"This study shows us that large-scale genetic studies not only can work very well in ALS, but that they can help pinpoint key biological pathways relevant to ALS that then become the focus of targeted drug development efforts," said study co-leader David B. Goldstein, PhD, professor of genetics and development and director of the new Institute for Genomic Medicine at CUMC. "ALS is an incredibly diverse disease, caused by dozens of different genetic mutations, which we're only beginning to discover. The more of these mutations we identify, the better we can decipher--and influence--the pathways that lead to disease." The other co-leaders of the study are Richard M. Myers, PhD, president and scientific director of HudsonAlpha, and Tim Harris, PhD, DSc, Senior Vice President, Technology and Translational Sciences, Biogen Idec.

"These findings demonstrate the power of exome sequencing in the search for rare variants that predispose individuals to disease and in identifying potential points of intervention. We are following up by looking at the function of this pathway so that one day this research may benefit the patients living with ALS," said Dr. Harris. "The speed with which we were able to identify this pathway and begin our next phase of research shows the potential of novel, focused collaborations with the best academic scientists to advance our understanding of the molecular pathology of disease. This synergy is vital for both industry and the academic community, especially in the context of precision medicine and whole-genome sequencing."

"Industry and academia often do things together, but this is a perfect example of a large, complex project that required many parts, with equal contributions from Biogen Idec. Dr. Tim Harris, our collaborator there, and his team, as well as David Goldstein and his team, now at Columbia University, as well as our teams here at HudsonAlpha, said Dr. Myers. "I love this research model because it doesn't happen very frequently, and it really shows how industry, nonprofits, and academic laboratories can all work together for the betterment of humankind. The combination of those groups with a large number of the clinical collaborators who have been seeing patients with this disease for many years and providing clinical information, recruiting patients, as well as collecting DNA samples for us to do this study, were all critical to get this done."

Searching through the enormous database generated in the ALS study, Dr. Goldstein and his colleagues found several genes that appear to contribute to ALS, most notably TBK1 (TANK-Binding Kinase 1), which had not been detected in previous, smaller-scale studies. TBK1 mutations appeared in about 1 percent of the ALS patients--a large proportion in the context of a complex disease with multiple genetic components, according to Dr. Goldstein. The study also found that a gene called OPTN, previously thought to play a minor role in ALS, may actually be a major player in the disease.

"Remarkably, the TBK1 protein and optineurin, which is encoded by the OPTN gene, interact physically and functionally. Both proteins are required for the normal function of inflammatory and autophagy pathways, and now we have shown that mutations in either gene are associated with ALS," said Dr. Goldstein. "Thus there seems to be no question that aberrations in the pathways that require TBK1 and OPTN are important in some ALS patients."

The researchers are currently using patient-derived induced pluripotent embryonic stem cells (iPS cells) and mouse models with mutations in TBK1 or OPTN to study ALS disease mechanisms and to screen for drug candidates. Several compounds that affect TBK1 signaling have already been developed for use in cancer, where the gene is thought to play a role in tumor-cell survival.

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New ALS gene and signaling pathways identified

CHICAGO -- The International Society for Stem Cell Research's 13th annual meeting will take place June 24-27, 2015 at the Stockholmsmssan Exhibition and Convention Center in Stockholm, Sweden. The meeting will bring together approximately 4,000 stem cell scientists, bioethicists, clinicians and industry professionals from over 50 countries to present and discuss the latest discoveries and technologies within the field.

"The ISSCR is excited to bring its annual meeting to Stockholm, a city that shares our passion and reputation for great scientific research and collaboration," said ISSCR President Rudolf Jaenisch, M.D., Whitehead Institute for Biomedical Research. "We look forward to learning more about the strong work being done in Sweden and across Europe."

The meeting will open with the Presidential Symposium on June 24 from 1:15-3:15 p.m. local time. The symposium sets the stage for the meeting with world renowned speakers, including Nobel Prize winner Shinya Yamanaka. It is also the platform for the formal recognition of the 2015 recipients of the McEwen Award for Innovation and the ISSCR Public Service Award. Another prestigious award, the ISSCR-BD Biosciences Outstanding Young Investigator Award, will be presented during Plenary VI on June 27 from 9-11:20 a.m. and followed by an award lecture.

"I look forward to the Presidential Symposium setting the tone for the entire program," Jaenisch said. "A thread throughout will be the use of stem cells to drive our understanding of development and disease, as we explore disease modeling, gene and tissue engineering technologies and other important advances that are bringing stem cells into the clinic."

Presidential Symposium speakers will include:

Fred H. Gage, Ph.D., Salk Institute for Biological Sciences, U.S.

Jrgen Knoblich, Ph.D., Institute of Molecular Biotechnology, Austria

Shinya Yamanaka, M.D., Ph.D., Center for iPS Cell Research & Application, Japan

Jeannie Lee, M.D., Ph.D., Massachusetts General Hospital, U.S.

The McEwen Award for Innovation award winners (Presidential Symposium):

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The International Society for Stem Cell Research announces annual meeting details

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Newswise NEW YORK, NY (February 27, 2015) Researchers have for the first time successfully converted adult human skin cells into neurons of the type that regulate appetite, providing a patient-specific model for studying the neurophysiology of weight control and testing new therapies for obesity. The study, led by researchers at Columbia University Medical Center (CUMC) and at the New York Stem Cell Foundation (NYSCF), was published last month in the online issue of the Journal of Clinical Investigation.

In a separate study, which appeared in the February 10 issue of the journal Development, Kevin Eggan, PhD, Florian Merkle, and Alexander Schier of Harvard University have also succeeded in creating hypothalamic neurons from iPS cells. These neurons help to regulate behavioral and basic physiological functions in the human body, including, in addition to appetite, hypertension, sleep, mood, and some social disorders. The investigators at Columbia and Harvard shared ideas during the course of the research, and these studies are co-validating.

Mice are a good model for studying obesity in humans, but it would better to have human cells for testing. Unfortunately, the cells that regulate appetite are located in an inaccessible part of the brain, the hypothalamus. So, until now, weve had to make do with a mouse model or with human cells harvested at autopsy. This has greatly limited our ability to study fundamental aspects of human obesity, said senior author Rudolph L. Leibel, MD, the Christopher J. Murphy Memorial Professor of Diabetes Research, professor of pediatrics and medicine, and co-director of the Naomi Berrie Diabetes Center at CUMC.

To make the neurons, human skin cells were first genetically reprogrammed to become induced pluripotent stem (iPS) cells. Like natural stem cells, iPS cells are capable of developing into any kind of adult cell when given a specific set of molecular signals in a specific order. The iPS cell technology has been used to create a variety of adult human cell types, including insulin-producing beta cells and forebrain and motor neurons. But until now, no one has been able to figure out how to convert human iPS cells into hypothalamic neurons, said co-author Dieter Egli, PhD, assistant professor of pediatrics (in developmental cell biology), a member of the Naomi Berrie Diabetes Center, and a senior research fellow at NYSCF.

This is a wonderful example of several institutions coming together to collaborate and advance research in pursuit of new therapeutic interventions. The ability to make this type of neuron brings us one step closer to the development of new treatments for obesity, said Susan L. Solomon, CEO of NYSCF.

The CUMC/NYSCF team determined which signals are needed to transform iPS cells into arcuate hypothalamic neurons, a neuron subtype that regulates appetite. The transformation process took about 30 days. The neurons were found to display key functional properties of mouse arcuate hypothalamic neurons, including the ability to accurately process and secrete specific neuropeptides and to respond to metabolic signals such as insulin and leptin.

We dont think that these neurons are identical to natural hypothalamic neurons, but they are close and will still be useful for studying the neurophysiology of weight control, as well as molecular abnormalities that lead to obesity, said Dr. Leibel. In addition, the cells will allow us to evaluate potential obesity drugs in a way never before possible.

This shows, said Dr. Eggan, how improved understanding of stem cell biology is making an impact on our ability to study, understand, and eventually treat disorders of the nervous system. Because there are so few hypothalamic neurons of a given type, they have been notoriously difficult to study. The successful work by both groups shows that this problem has been cracked.

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Neurons Controlling Appetite Made From Skin Cells

Jeanne Loring holds a petri dish with induced pluripotent stem cells from a Parkinsons patient.

A nine-year legal challenge to human embryonic stem cell patents ended Tuesday, when the Supreme Court declined to hear the case.

The decision means the Wisconsin Alumni Research Foundation, or WARF, will get to keep its patent rights for the cells, which were discovered in 1998 by University of Wisconsin - Madison scientist James Thompson.

However, the challengers succeeded in preventing WARF from gaining rights over another important type of stem cells called induced pluripotent stem cells, said Jeanne Loring, a stem cell scientist at The Scripps Research Institute in La Jolla who was part of a coalition contesting the WARF patents.

IPS cells act much like human embryonic stem cells, and are being researched as an alternative for stem cell therapy. Loring is working with a group that seeks to use them to treat Parkinson's disease.

WARF maintains it has the right to license use of human embryonic stem cells, because Thompson developed the methods to isolate them from embryos, which had not been previously done. Loring said the derivation is an obvious extension of methods used to derive non-primate embryonic stem cells, and therefore not patentable.

Loring and two public interest groups, Consumer Watchdog and the Public Patent Foundation, challenged the patents in 2006, and in 2007 succeeded in narrowing WARF's claims to exclude the IPS cells. Loring and the groups continued the challenge on the grounds that as a product of nature, human embryonic stem cells are not patentable.

The U.S. Patent and Trade Office turned down that challenge, and the case reached the Supreme Court last year. By not hearing the case, the Supreme Court let that decision stand.

"They still own human embryonic stem cells," Loring said. "But the way their patents were originally written, they would have also been able to own IPS cells. If there's one success that I would point to, that was worth all the effort, it's that they can't. And the reason they can't is because we challenged the patent."

Calls and an email sent Tuesday to WARF headquarters in Madison, Wis., were not immediately returned.

Read more:
Supreme Court rejects stem cell patent case

Kyoto University Hospital says it will open a center to conduct clinical studies on induced pluripotent stem cell therapies in 2019 year.

Officials said the 30-bed ward will test the efficacy and safety of the therapies on volunteer patients.

The hospital aims to break ground at the site next February and complete construction by September 2019.

As an iPS cell research hub, we hope to apply (the cells) to groundbreaking therapies and make developments in the field of drug discovery, the hospital said in a statement Monday.

Ongoing research on iPS cells at Kyoto University includes turning the cells into dopamine-releasing neurons for transplant into patients with Parkinsons disease, and creating a formulation of platelets that helps blood to clot.

Professor Shinya Yamanaka, who shared the 2012 Nobel Prize in medicine, leads the existing iPS cell research center at Kyoto University.

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Kyoto University Hospital to open iPS cell therapy center in 2019