Induced stem cells – Wikiversity

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Dec 17 2016

Welcome to the Wikiversity learning project for Induced stem cells (Guide to publications). This project provides learning resources that help participants learn about Induced stem cells and efforts to produce useful stem cells and obtaining their derivatives for medical therapies. Participants should feel free to ask questions on discuss page and explore related topics.

Induced stem cells (iSC) are stem cells artificially derived from somatic, reproductive, pluripotent or other cell types by deliberate w:epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor (multipotentiMSC, also called an induced multipotent progenitor celliMPC) or unipotent -- (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.

Three techniques are widely recognized:[1]

Back in 1895, Thomas Morgan remove one of the two frog w:blastomeres and found that w:amphibians are able to form whole w:embryo from the remaining part. This meant that the cells can change their differentiation pathway. Later, in 1924, Spemann and Mangold demonstrated the key importance of cellcell inductions during animal development.[20] The reversible transformation of cells of one differentiated cell type to another is called w:metaplasia.[21] This transition can be a part of the normal maturation process, or caused by an inducing stimulus. For example: transformation of iris cells to lens cells in the process of maturation and transformation of w:retinal pigment epithelium cells into the neural retina during regeneration in adult w:newt eyes. This process allows the body to replace cells not suitable to new conditions with more suitable new cells. In w:Drosophila imaginal discs, cells have to choose from a limited number of standard discrete differentiation states. The fact that transdetermination (change of the path of differentiation) often occurs for a group of cells rather than single cells shows that it is induced rather than part of maturation.[22]

The researchers were able to identify the minimal conditions and factors that would be sufficient for starting the cascade of molecular and cellular processes to instruct pluripotent cells to organize the w:embryo. They show that opposing gradients of w:bone morphogenetic protein (BMP) and Nodal, two w:transforming growth factor family members that act as w:morphogens, are sufficient to induce molecular and cellular mechanisms required to organize, w:in vivo or w:in vitro, uncommitted cells of the w:zebrafish w:blastula animal pole into a well-developed w:embryo.[23]

Some types of mature, specialized adult cells can naturally revert to stem cells. For example, differentiated airway epithelial cells can revert into stable and functional stem cells in vivo after the ablation of airway[24]. Another example, "chief" cells express the stem cell marker Troy. While they normally produce digestive fluids for the stomach, they can revert into stem cells to make temporary repairs to stomach injuries, such as a cut or damage from infection. Moreover, they can make this transition even in the absence of noticeable injuries and are capable of replenishing entire gastric units, in essence serving as quiescent reserve stem cells.[25]

After injury, mature terminally differentiated kidney cells dedifferentiate into more primordial versions of themselves, and then differentiate into the cell types needing replacement in the damaged tissue[26] Macrophages can self-renew by local proliferation of mature differentiated cells.[27] In newts, muscle tissue is regenerated from specialized muscle cells that dedifferentiate and forget the type of cell they had been. This capacity to regenerate does not decline with age and may be linked to their ability to make new stem cells from muscle cells on demand.[28]

A variety of nontumorigenic stem cells display the ability to generate multiple cell types. For instance, multilineage-differentiating stress-enduring (Muse) cells are stress-tolerant adult human stem cells that can self-renew. They form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency and can differentiate into w:endodermal, ectodermal and mesodermal cells both in vitro and in vivo.[29][30][31][32][33][34]

Other well-documented examples of w:transdifferentiation and their significance in development and regeneration were described in detail.[35]

Induced totipotent cells usually can be obtained by reprogramming somatic cells by w:somatic-cell nuclear transfer (SCNT) to the recipient eggs or oocytes.[3][5][36][37]

Using an approach based on the protocol outlined by Tachibana et al.,[3] hESCs can be generated by SCNT using dermal fibroblasts nuclei from both a middle-aged 35-year-old male and an elderly, 75-year-old male, suggesting that age-associated changes are not necessarily an impediment to SCNT-based nuclear reprogramming of human cells.[38] Such reprogramming of somatic cells to a pluripotent state holds huge potentials for w:regenerative medicine. Unfortunately, the cells generated by this technology, potentially are not completely protected from the immune system of the patient (donor of nuclei), because they have the same w:mitochondrial DNA, as a donor of oocytes, instead of the patients mitochondrial DNA. This reduces their value as a source for w:autologous stem cell transplantation therapy, as for the present, it is not clear whether it can induce an immune response of the patient upon treatment.

Induced androgenetic haploid embryonic stem cells can be used instead of sperm for cloning. These cells, synchronized in M phase and injected into the oocyte can produce viable offspring.[39]

These developments, together with data on the possibility of unlimited oocytes from mitotically active reproductive stem cells,[40] offer the possibility of industrial production of transgenic farm animals.

Repeated recloning of viable mice through a SCNT method that includes a w:histone deacetylase inhibitor, trichostatin, added to the cell culture medium,[41] show that it may be possible to reclone animals indefinitely with no visible accumulation of reprogramming or genomic errors[42]

Concerns still exist regarding telomere length resetting in cloned embryos and nuclear transfer ES cells, and possibilities of premature aging of cloned animals achieved by SCNT. It was shown that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however trichostatin A significantly increases telomere lengths in cloned pigs and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with trichostatin.[43]

However, research into technologies to develop sperm and egg cells from stem cells raises bioethical issues.[44]

Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes.[3][45] For example, the technology could prevent inherited w:mitochondrial disease from passing to future generations. Mitochondrial genetic material is passed from mother to child. Mutations can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia and other neurological diseases. The nucleus from one human egg has been transferred to another, including its mitochondria, creating a cell that could be regarded as having two mothers. The eggs were then fertilised, and the resulting embryonic stem cells carried the swapped mitochondrial DNA.[46] As evidence that the technique is safe author of this method points to the existence of the healthy monkeys that are now more than four years old and are the product of mitochondrial transplants across different genetic backgrounds.[47]

In late-generation w:telomerase-deficient (Terc/) mice, SCNT-mediated reprogramming mitigates telomere dysfunction and mitochondrial defects to a greater extent than iPSC-based reprogramming.[48]

Other cloning and totipotent transformation achievements have been described.[49]

Recently some researchers succeeded to get the totipotent cells without the aid of SCNT. Totipotent cells were obtained using the epigenetic factors such as oocyte germinal isoform of histone.[50] Reprogramming in vivo, by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice, confers totipotency features. Intraperitoneal injection of such in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic (w:trophectodermal) markers.[51]

iPSc were first obtained in the form of transplantable w:teratocarcinoma induced by grafts taken from mouse embryos.[52] Teratocarcinoma formed from somatic cells.[53]Genetically mosaic mice were obtained from malignant teratocarcinoma cells, confirming the cells' pluripotency.[54][55][56] It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent w:embryonic stem cell in an undifferentiated state, by supplying the culture medium with various factors.[57] In the 1980s, it became clear that transplanting pluripotent/embryonic stem cells into the body of adult mammals, usually leads to the formation of w:teratomas, which can then turn into a malignant tumor teratocarcinoma.[58] However, putting teratocarcinoma cells into the embryo at the blastocyst stage, caused them to become incorporated in the w:inner cell mass and often produced a normal chimeric (i.e. composed of cells from different organisms) animal.[59][60][61] This indicated that the cause of the teratoma is a dissonance - mutual miscommunication between young donor cells and surrounding adult cells (the recipient's so-called "niche").

In August 2006, Japanese researchers circumvented the need for an oocyte, as in SCNT. By reprograming mouse embryonic w:fibroblasts into pluripotent stem cells via the ectopic expression of four transcription factors, namely w:Oct4, w:Sox2, w:Klf4 and w:c-Myc, they proved that the overexpression of a small number of factors can push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.[7]

Later has been found a reprogramming factor BRD3R that increases the efficiency of creating human induced pluripotent stem cells (HiPSCs) from skin fibroblasts in xeno-free media more than 20-fold, speeds the reprogramming time by several days and enhances the quality of reprogramming.[62] Reprogramming mechanisms are thus linked, rather than independent and are centered on a small number of genes.[63] IPSC properties are very similar to ESCs[64]. iPSCs have been shown to support the development of all-iPSC mice using a w:tetraploid (4n) embryo,[65] the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to produce all-iPSC mice because of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster.[18]

An important advantage of iPSC over ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it became possible to obtain iPSC from adult and even elderly patients.[9][66][67]

Reprogramming somatic cells to iPSC leads to rejuvenation. It was found that reprogramming leads to telomere lengthening and subsequent shortening after their differentiation back into fibroblast-like derivatives.[68] Thus, reprogramming leads to the restoration of embryonic telomere length,[69] and hence increases the potential number of cell divisions otherwise limited by the w:Hayflick limit.[70]

However, because of the dissonance between rejuvenated cells and the surrounding niche of the recipient's older cells, the injection of his own iPSC usually leads to an w:immune response,[71] which can be used for medical purposes,[72] or the formation of tumors such as teratoma.[73] The reason has been hypothesized to be that some cells differentiated from ESC and iPSC in vivo continue to synthesize embryonic w:protein isoforms.[74] So, the immune system might detect and attack cells that are not cooperating properly.

A small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via w:cytochrome c release across the w:mitochondrial outer membrane) in human pluripotent stem cells, but not in differentiated cells. Shortly after differentiation, daughter cells became resistant to death. When MitoBloCK-6 was introduced to differentiated cell lines, the cells remained healthy. The key to their survival, was hypothesized to be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines has the potential to reduce the risk of teratomas and other problems in regenerative medicine.[75]

In 2012 other w:small molecules (selective cytotoxic inhibitors of human pluripotent stem cellshPSCs) were identified that prevented human pluripotent stem cells from forming teratomas in mice. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in w:oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule the undifferentiated cells can be selectively removed from culture.[76] An efficient strategy to selectively eliminate pluripotent cells with teratoma potential is targeting pluripotent stem cell-specific antiapoptotic factor(s) (i.e., w:survivin or Bcl10). A single treatment with chemical survivin inhibitors (e.g., w:quercetin or YM155) can induce selective and complete cell death of undifferentiated hPSCs and is claimed to be sufficient to prevent teratoma formation after transplantation.[77] However, it is unlikely that any kind of preliminary clearance,[78] is able to secure the replanting iPSC or ESC. After the selective removal of pluripotent cells, they re-emerge quickly by reverting differentiated cells into stem cells, which leads to tumors.[79] This may be due to the disorder of let-7 regulation of its target Nr6a1 (also known as w:Germ cell nuclear factor - GCNF), an embryonic transcriptional repressor of pluripotency genes that regulates gene expression in adult fibroblasts following w:micro-RNA miRNA loss.[80]

Yijie Geng et al., identified a small molecule, Displurigen, that potently disrupts pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8), which maintains pluripotency by facilitating the DNA-binding activity of OCT4[81]

Teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0,1-5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of w:Nanog as well as a propensity for increased glucose and cholesterol metabolism.[82] These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.[83] In connection with the above safety problems, the use iPSC for cell therapy is still limited.[84] However, they can be used for a variety of other purposes - including the modeling of disease,[85] screening (selective selection) of drugs, toxicity testing of various drugs.[86]

It is interesting to note that the tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation[87] At the same time, full reprogramming of adult cells in vivo within tissues by transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs.[51] Furthermore, partial reprogramming of cells toward pluripotency in vivo in mice demonstrates that incomplete reprogramming entails epigenetic changes (failed repression of w:Polycomb targets and altered w:DNA methylation) in cells that drive cancer development.[88]

Several methods have been reported that may increase the safety and eventually the efficacy of iPSC-based regenerative medicine. The first safety approach eliminates potential oncogenic factors, such as the expression of oncogene c-myc, or integrates the reprogramming transgenes into chromosomes. The latter would be eliminated by using so-called nonintegrating viral vectors. The second safety approach is based on the isolation of desired differentiated cells from other cell types and undifferentiated human pluripotent stem cells (hPSCs), such as the removal of the residual pluripotent cells using fluoresecent activated cell sorting or magnetic beads coated with antibodies against a particular antigen, including SSEA-5 and Claudin-6, and fucose-specific lectin UEA (Ulex europaeus agglutinin)-I. The third safety approach entails the direct targeting and killing of oncogenic cells by using cytotoxic antibody recognizing podocalyxin-like protein-1, a chemical inhibitor of stearoyl-coA desaturase, specific monoclonal antibodies, DNA topoisomerase II inhibitor, and suicide gene therapy under transcriptional control of a pluripotency-related promoter[90][91]. However, these strategies may not suffice to lower risk to acceptable levels, because the tumorigenic risk of iPSC-based cell therapy arises not just from contamination with undifferentiated iPSCs but also from other unexpected events associated with long-term culture for reprogramming and redifferentiation. There is always a chance of unexpected issues associated with first-in-human clinical studies. An efficient and reliable approach to provide safety for future regenerative therapy and first-in-human cell therapy can be a suicide gene engineered from human caspase-9, that is not immunogenic, and can kill transduced cells in a cell-cycle-independent manner[92][93][94][95].

Miki Ando et al.[96] demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 system in vivo with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. They introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that rejCTLs from iPSCs are expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. Upon induction, the iC9 system efficiently leads to apoptosis in rejuvenated CTLs. This safeguard system can eliminate contaminating iPSCs, debulk tumors originated from iPSCs, stop cytokine release syndrome associated with iPSC-derived CTL therapy, and control on-target, off-tumor toxicities. It should be applicable to other cell therapies using iPSC-derived cells.

The potential to develop patient-derived cells into any cell type makes human pluripotent stem cells one of the most promising sources for regenerative treatments. The proper differentiation of autologous iPSCs sometimes results in a loss of immunogenicity and leads to the induction of tolerance.[97] This differentiation of iPSCs to mature cell typesand ultimately to functional tissues and organsholds great promise for personalized disease modeling, drug screening, and the development of cell-based therapies. [98] However there are some problems that need to be solved previously:

The main steps for the production of human pluripotent stem cell-derived progenitor cells under safe and good manufacturing practice (GMP) conditions must include:

The data collected throughout such process already have led to approval for a first-in-man clinical trial of transplantation of SSEA-1+ progenitors in patients with severely impaired cardiac function. [102].

Lonza attempted to develop clinically compliant processes to generate cGMP-compliant human iPSC lines and have described a step-by-step cGMP-compliant process to generate clinically compliant cell lines[103].

Several works have reported evidence of genomic instability in iPSC, raising concerns on their biomedical use. The reasons behind the genomic instability observed in iPSC remain mostly unknown. Sergio Ruiz et al.[104] show that, similar to the phenomenon of oncogene-induced replication stress, the expression of reprogramming factors induces replication stress. Increasing the levels of the checkpoint kinase 1 (CHK1) reduces reprogramming-induced replication stress and increases the efficiency of iPSC generation. Similarly, nucleoside supplementation during reprogramming reduces the load of DNA damage and genomic rearrangements on iPSC. So, lowering replication stress during reprogramming, genetically or chemically, provides a simple strategy to reduce genomic instability on mouse and human iPSC.

By using solely w:small molecules, Deng Hongkui and colleagues demonstrated that endogenous master genes are enough for cell fate reprogramming. They induced a pluripotent state in adult cells from mice using seven small-molecule compounds.[17] The effectiveness of the method is quite high: it was able to convert 0.2% of the adult tissue cells into iPSCs, which is comparable to the gene insertion conversion rate. The authors note that the mice generated from CiPSCs were "100% viable and apparently healthy for up to 6 months.So. This chemical reprogramming strategy has potential use in generating functional desirable cell types for clinical applications.[105]

iPS-like cells (iPSLCs) were also generated from mouse somatic cells in two steps with small molecule compounds. In the first step, stable intermediate cells were generated from mouse astrocytes by Shh activators (oxysterol and purmorphamine) to replace Bmi1 function. These cells called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs) are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. In the second step, treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSLCs that were similar to mESCs, suggesting that Bmi1 is sufficient to reprogram astrocytes to partially reprogrammed pluripotency. So, combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSLCs. The chemically induced pluripotent stem cell-like cells (ciPSLCs) showed similar gene expression profiles, epigenetic status, and differentiation potentials to mESCs.[106]

The fact that human iPSCs capable of forming teratomas not only in humans but also in some animal body, in particular in mice or pigs, allowed to develop a method for differentiation of iPSCs in vivo. For this purpose, iPSCs with an agent for inducing differentiation into target cells are injected to genetically modified pig (such as biallelic RAG2 mutants[107]) or mouse that has suppressed immune system activation on human cells. The formed after a while teratoma is cut out and used for the isolation of the necessary differentiated human cells[108] by means of w:monoclonal antibody to tissue-specific markers on the surface of these cells. This method has been successfully used for the production of functional myeloid, erythroid, and lymphoid human cells suitable for transplantation (yet only to mice).[109] Mice engrafted with human iPSC teratoma-derived hematopoietic cells produced human B and T cells capable of functional immune responses. These results offer hope that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications. Using MitoBloCK-6 [75] and / or PluriSIn # 1 the differentiated progenitor cells can be further purified from teratoma forming pluripotent cells. The fact, that the differentiation takes place even in the teratoma niche, offers hope that the resulting cells are sufficiently stable to stimuli able to cause their transition back to the dedifferentiated (pluripotent) state, and therefore safe. A similar in vivo differentiation system, yielding engraftable hematopoietic stem cells from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis, was described by Suzuki et al.[110] They noted that neither leukemia nor tumors were observed in recipients after intravenous injection of iPSC-derived hematopoietic stem cells into irradiated recipients. Moreover, this injection resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Such system provides a useful tool for practical application of iPSCs in the treatment of hematologic and immunologic diseases.[111]

For further development of this method animal in which is grown the human cell graft, for example mouse, must have so modified genome that all its cells express and have on its surface human SIRP.[112] To prevent rejection after transplantation to the patient of the allogenic organ or tissue, grown from the pluripotent stem cells in vivo in the animal, these cells should express two molecules: CTLA4-Ig, which disrupts T cell costimulatory pathways, and w:PD-L1, which activates T cell inhibitory pathway.[113]

Methods based on the detection of reporter gene-GFP-positive cells in the teratoma derived from iPSCs, will help to identify different types of induced adult stem cells which were previously difficult to pick out and to grow from selected cells tissue cultures.[114]

See also: US 20130058900 patent.

In the near-future, clinical trials designed to demonstrate the safety of the use of iPSCs for cell therapy of the people with age-related macular degeneration, a disease causing blindness through retina damaging, will begin. There are several articles describing methods for producing retinal cells from iPSCs[115][116] and how to use them for cell therapy.[117] Reports of iPSC-derived retinal pigmented epithelium transplantation showed enhanced visual-guided behaviors of experimental animals for 6 weeks after transplantation.[118] However, clinical trials have been successful: ten patients suffering from retinitis pigmentosa have had their eyesight restoredincluding a woman who had only 17 percent of her vision left. [119]

Chronic lung diseases such as idiopathic pulmonary fibrosis and cystic fibrosis or w:chronic obstructive pulmonary disease and w:asthma are leading causes of morbidity and mortality worldwide with a considerable human, societal, and financial burden. So there is an urgent need for effective cell therapy and w:lung w:tissue engineering.[120][121] Several protocols have been developed for generation of the most cell types of the respiratory system, which may be useful for deriving patient-specific therapeutic cells.[122][123]

Some lines of iPSCs have the potentiality to differentiate into male germ cells and oocyte-like cells in an appropriate niche (by culturing in retinoic acid and porcine follicular fluid differentiation medium or seminiferous tubule transplantation). Moreover, iPSC transplantation make a contribution to repairing the testis of infertile mice, demonstrating the potentiality of gamete derivation from iPSCs in vivo and in vitro.[124]

Wu and his colleagues found that a combination of serum-free media plus fibroblast growth factor 2 (FGF2) and Wnt signaling inhibitors resulted in stable line of human rsPSCs (region-specific Induced stem cells)[125][126].

The transcriptomes of these cells resembled those of the posterior cells of the early mouse embryo, and grafting these cells into 7.5-day-old mouse embryos resulted in efficient incorporation in the posterior, but not the other parts of the embryo. After 36 hours of culturing these chimaeric embryos, the rsPSCs proliferated and could differentiate into the developing three germ layers, providing the first demonstration that human pluripotent cells can begin a differentiation program inside mice.

The region-specific cells could provide tremendous advantages -- the cells at this stage of an early embryo undergo dynamic changes to give rise to all cells, tissues and organs of the body. Each germ layer was theoretically capable of giving rise to specific tissues and organs. Whether human rsPSCs can generate more complicated tissue structures within mice or other animals requires further study[127].

These cells also have a lot of favorable characteristics for laboratory manipulation, including high cloning efficiency, stable passage in culture, and ease of genetic engineering.

The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications.

The risk of cancer and tumors creates the need to develop methods for safer cell lines suitable for clinical use. An alternative approach is so-called "direct reprogramming" - transdifferentiation of cells without passing through the pluripotent state.[128][129][130][131][132][133] The basis for this approach was that 5-azacytidine - a DNA demethylation reagent - can cause the formation of w:myogenic, chondrogenic and adipogeni] clones in the immortal cell line of mouse embryonic fibroblasts[134] and that the activation of a single gene, later named MyoD1, is sufficient for such reprogramming.[135] Compared with iPSC whose reprogramming requires at least two weeks, the formation of induced progenitor cells sometimes occurs within a few days and the efficiency of reprogramming is usually many times higher. This reprogramming does not always require cell division.[136] The cells resulting from such reprogramming are more suitable for cell therapy because they do not form teratomas.[133]

Originally only early embryonic cells could be coaxed into changing their identity. Mature cells are resistant to changing their identity once they've committed to a specific kind. However, brief expression of a single transcription factor, the ELT-7 GATA factor, can convert the identity of fully differentiated, specialized non-endodermal cells of the w:pharynx into fully differentiated intestinal cells in intact w:larvae and adult roundworm w:Caenorhabditis elegans with no requirement for a dedifferentiated intermediate.[137]

Determining the unique set of cellular factors that is needed to be manipulated for each cell conversion is a long and costly process that involved much trial and error. As a result, this first step of identifying the key set of cellular factors for cell conversion is the major obstacle researchers face in the field of cell reprogramming. An international collaboration of researchers from the Duke-NUS Medical School in Singapore, the University of Bristol in the United Kingdom, Monash University in Australia, and RIKEN in Japan have developed an algorithm, called Mogrify(1), that can predict the optimal set of cellular factors required to convert one human cell type to another. That will drastically reduce the time and effort needed to create induced stem cells When tested, Mogrify was able to accurately predict the set of cellular factors required for previously published cell conversions correctly. To further validate Mogrify's predictive ability, the team conducted two novel cell conversions in the laboratory using human cells, and these were successful in both attempts solely using the predictions of Mogrify.[138][139][140]

The future medical implications of this novel breakthrough in cellular reprogramming are not hard to imagine. A bewildering range of diseases and disorders could be relegated to the dustbin of medical historyfrom arthritis to macular degeneration, from lost limbs to cancer itself. Mogrify has been made available online for other researchers and scientists.

Another way of reprogramming is the simulation of the processes that occur during w:amphibian limb regeneration. In w:urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates into limb tissue. However, sequential small molecule treatment of the muscle fiber with myoseverin, w:reversine (the w:aurora B kinase inhibitor) and some other chemicals: BIO (glycogen synthase-3 kinase inhibitor), w:lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), w:SB203580 (w:p38 MAP kinase inhibitor), or w:SQ22536 (adenylyl cyclase inhibitor) causes the formation of new muscle cell types as well as other cell types such as precursors to fat, bone and nervous system cells.[141]

The researchers discovered that GCSF-mimicking w:antibody can activate a growth-stimulating receptor on marrow cells in a way that induces marrow stem cells that normally develop into white blood cells to become neural progenitor cells. The technique[142] enables researchers to search large libraries of antibodies and quickly select the ones with a desired biological effect.[143]

Schlegel and Liu[144] demonstrated that the combination of feeder cells[145][146][147] and a w:Rho kinase inhibitor (Y-27632) [148][149] induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro. This process occurs without the need for transduction of exogenous viral or cellular genes. These cells have been termed "Conditionally Reprogrammed Cells (CRC)". The induction of CRCs is rapid and results from reprogramming of the entire cell population. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible and removal of Y-27632 and feeders allows the cells to differentiate normally.[144][150][151] CRC technology can generate 2106 cells in 5 to 6 days from needle biopsies and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal w:karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.[144][152][153]

The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.[144][152][153] Using CRC technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.[154] In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host. While initial studies revealed that co-culturing epithelial cells with Swiss 3T3 cells J2 was essential for CRC induction, with transwell culture plates, physical contact between feeders and epithelial cells is not required for inducing CRCs, and more importantly that irradiation of the feeder cells is required for this induction. Consistent with the transwell experiments, conditioned medium induces and maintains CRCs, which is accompanied by a concomitant increase of cellular telomerase activity. The activity of the conditioned medium correlates directly with radiation-induced feeder cell apoptosis. Thus, conditional reprogramming of epithelial cells is mediated by a combination of Y-27632 and a soluble factor(s) released by apoptotic feeder cells.[155]

A different approach to CRC is to inhibit w:CD47 - a w:membrane protein that is the w:thrombospondin-1 receptor. Loss of CD47 permits sustained proliferation of primary w:murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent w:embryoid body-like clusters. CD47 knockdown acutely increases w:mRNA levels of c-Myc and other stem cell transcription factors in cells in vitro and in vivo. Thrombospondin-1 is a key environmental signal that inhibits stem cell self-renewal via CD47. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.[156] In vivo blockade of CD47 using an antisense w:morpholino increases survival of mice exposed to lethal total body irradiation due to increased proliferative capacity of bone marrow-derived cells and radioprotection of radiosensitive gastrointestinal tissues.[157]

Indirect lineage conversion is a reprogramming methodology in which somatic cells transition through a plastic intermediate state of partially reprogrammed cells (pre-iPSC), induced by brief exposure to reprogramming factors, followed by differentiation in a specially developed chemical environment (artificial niche).[158]

This method could be both more efficient and safer, since it does not seem to produce tumors or other undesirable genetic changes, and results in much greater yield than other methods. However, the safety of these cells remains questionable. Since lineage conversion from pre-iPSC relies on the use of iPSC reprogramming conditions, a fraction of the cells could acquire pluripotent properties if they do not stop the de-differentation process in vitro or due to further de-differentiation in vivo.[159]

A common feature of pluripotent stem cells is the specific nature of protein w:glycosylation of their outer membrane. That distinguishes them from most nonpluripotent cells, although not w:white blood cells.[160] The w:glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many w:stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81.[161] Suila Heli et al.[162] speculate that in human stem cells extracellular O-GlcNAc and extracellular O-LacNAc, play a crucial role in the fine tuning of w:Notch signaling pathway - a highly conserved cell signaling system, that regulates cell fate specification, differentiation, leftright asymmetry, apoptosis, somitogenesis, angiogenesis, and plays a key role in stem cell proliferation (reviewed by Perdigoto and Bardin[163] and Jafar-Nejad et al.[164])

Changes in outer membrane protein glycosylation are markers of cell states connected in some way with pluripotency and differentiation.[165] The glycosylation change is apparently not just the result of the initialization of gene expression, but perform as an important gene regulator involved in the acquisition and maintenance of the undifferentiated state.[166]

For example, activation of w:glycoprotein ACA,[167] linking glycosylphosphatidylinositol on the surface of the progenitor cells in human peripheral blood, induces increased expression of genes Wnt, w:Notch-1, w:BMI1 and w:HOXB4 through a signaling cascade w:PI3K/w:Akt/mTor/PTEN, and promotes the formation of a self-renewing population of hematopoietic stem cells.[168]

Furthermore, dedifferentiation of progenitor cells induced by ACA-dependent signaling pathway leads to ACA-induced pluripotent stem cells, capable of differentiating in vitro into cells of all three w:germ layers.[169] The study of w:lectins' ability to maintain a culture of pluripotent human stem cells has led to the discovery of lectin w:Erythrina crista-galli (ECA), which can serve as a simple and highly effective matrix for the cultivation of human pluripotent stem cells.[170]

w:Cell adhesion protein E-cadherin is indispensable for a robust pluripotent w:phenotype.[171] During reprogramming for iPS cell generation, N-cadherin can replace function of E-cadherin.[172] These functions of cadherins are not directly related to adhesion because sphere morphology helps maintaining the "stemness" of stem cells.[173] Moreover, sphere formation, due to forced growth of cells on a low attachment surface, sometimes induces reprogramming. For example, neural progenitor cells can be generated from fibroblasts directly through a physical approach without introducing exogenous reprogramming factors.

Physical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells epigenetic state. Specifically, "decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)a subunit of H3 methyltranferaseby microgrooved surfaces lead to increased histone H3 acetylation and methylation". Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state.[174]

Substrate rigidity is an important biophysical cue influencing neural induction and subtype specification. For example, soft substrates promote neuroepithelial conversion while inhibiting w:neural crest differentiation of hESCs in a BMP4-dependent manner. Mechanistic studies revealed a multi-targeted mechanotransductive process involving mechanosensitive Smad w:phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and w:actomyosin w:cytoskeleton integrity and w:contractility.[175]

An initial sensing event of tissue and extracellular matrix (ECM) stiffness includes a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas - Crk-associated substrates), and the guanosine triphosphatase Rac which selectively transduce ECM stiffness into stable intracellular stiffness, to increase the abundance of the cell cycle protein cyclin D1, and to promote S-phase entry. Rac-dependent intracellular stiffening involve its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Such mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling.[176]

Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the w:cytokine w:leukemia inhibitory factor (LIF). Following LIF withdrawal, mESCs differentiate, accompanied by an increase in cellsubstratum w:adhesion and cell spreading. Restricted cell spreading in the absence of LIF by either culturing mESCs on chemically defined, weakly adhesive biosubstrates, or by manipulating the w:cytoskeleton allowed the cells to remain in an undifferentiated and pluripotent state. The effect of restricted cell spreading on mESC self-renewal is not mediated by increased intercellular adhesion, as inhibition of mESC adhesion using a function blocking anti E-cadherin antibody or w:siRNA does not promote differentiation.[177] Possible mechanisms of stem cell fate predetermination by physical interactions with the extracellular matrix have been described.[178]

Cells involved in the reprogramming process change morphologically as the process proceeds. This results in physical difference in adhesive forces among cells. Substantial differences in 'adhesive signature' between pluripotent stem cells, partially reprogrammed cells, differentiated progeny and somatic cells allowed to develop separation process for isolation of pluripotent stem cells in w:microfluidic devices,[179][180] which is: fast (separation takes less than 10 minutes); efficient (separation results in a greater than 95 percent pure iPS cell culture); innocuous (cell survival rate is greater than 80 percent and the resulting cells retain normal transcriptional profiles, differentiation potential and karyotype).

Discussion on potential future applications of lab-on-a-chips for stem cell research, see in[181]

A novel method for cell reprogramming and fully automating stem cell cultures entire process is been developed by using smart surfaces that make cell adhesion and de-adhesion possible depending on changes in the environment.[182] This iterative method of cell culture enables to completely automate and remove the need for human involvement in the cell separation and washing stages, without using any additives that increase the toxicity level (such as trypsin).[183]

Stem cells possess mechanical memory (they remember past physical signals)with the w:Hippo signaling pathway factors:[184] Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) acting as an intracellular mechanical rheostatthat stores information from past physical environments and influences the cells fate.[185][186]

Stroke and many neurodegenerative disorders such as Parkinson's disease, Alzheimers disease, amyotrophic lateral sclerosis need cell replacement therapy. The successful use of converted neural cells (cNs) in transplantations open a new avenue to treat such diseases.[187] Nevertheless, induced neurons (iNs), directly converted from fibroblasts are terminally committed and exhibit very limited proliferative ability that may not provide enough autologous donor cells for transplantation.[188] Self-renewing induced neural stem cells (iNSCs) provide additional advantages over iNs for both basic research and clinical applications.[131][132][133][189][190]

For example, under specific growth conditions, mouse fibroblasts can be reprogrammed with a single factor, Sox2, to form iNSCs that self-renew in culture and after transplantation can survive and integrate without forming tumors in mouse brains.[191] INSCs can be derived from adult human fibroblasts by non-viral techniques, thus offering a safe method for autologous transplantation or for the development of cell-based disease models.[190]

Neural chemicaly-induced progenitor cells (ciNPCs) can be generated from mouse tail-tip fibroblasts and human urinary somatic cells without introducing exogenous factors, but - by a chemical cocktail, namely VCR (V, VPA, an inhibitor of HDACs; C, CHIR99021, an inhibitor of GSK-3 kinases and R, RepSox, an inhibitor of w:TGF beta signaling pathways), under a physiological hypoxic condition.[192] Alternative cocktails with inhibitors of histone deacetylation, glycogen synthase kinase, and TGF- pathways (where: w:sodium butyrate (NaB) or w:Trichostatin A (TSA) could replace VPA, w:Lithium chloride (LiCl) or lithium carbonate (Li2CO3) could substitute CHIR99021, or Repsox may be replaced with w:SB-431542 or w:Tranilast) show similar efficacies for ciNPC induction.[192]

Multiple methods of direct transformation of somatic cells into induced neural stem cells have been described.[193]

Proof of principle experiments demonstrate that it is possible to convert transplanted human fibroblasts and human w:astrocytes directly in the brain that are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes (Ascl1, Brn2a and w:Myt1l) are activated after transplantation using a drug.[194]

w:Astrocytesthe most common w:neuroglial brain cells, which contribute to w:scar formation in response to injurycan be directly reprogrammed in vivo to become functional neurons that formed networks in mice without the need of cell transplantation.[195] The researchers followed the mice for nearly a year to look for signs of tumor formation and reported finding none. The same researchers have turned scar-forming astrocytes into progenitor cells called neuroblasts that regenerated into neurons in the injured adult spinal cord.[196]

Without w:myelin to insulate neurons, nerve signals quickly lose power. Diseases that attack myelin, such as multiple sclerosis, result in nerve signals that cannot propagate to nerve endings, and as a consequence lead to cognitive, motor and sensory problems. Transplantation of w:oligodendrocyte precursor cells (OPCs), which can successfully create myelin sheaths around nerve cells, is a promising potential therapeutic response. Direct lineage conversion of mouse and rat fibroblasts into oligodendroglial cells provides a potential source of OPCs. Conversion by forced expression of both eight[197] or of the three[198] transcription factors Sox10, Olig2 and Zfp536, may provide such cells.

Cell-based in vivo therapies may provide a transformative approach to augment vascular and muscle growth and to prevent non-contractile scar formation by delivering transcription factors[128] or microRNAs[14] to the heart.[199] Cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of cardiac core transcription factors ( GATA4, MEF2C, TBX5 and for improved reprogramming plus ESRRG, MESP1, Myocardin and ZFPM2) after coronary ligation.[128][200] These results implicated therapies that can directly remuscularize the heart without cell transplantation. However, the efficiency of such reprogramming turned out to be very low and the phenotype of received cardiomyocyte-like cells does not resemble those of a mature normal cardiomyocyte. Furthermore, transplantation of cardiac transcription factors into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes.[201]

Meanwhile, advances in the methods of obtaining cardiac myocytes in vitro occurred.[202][203] Efficient cardiac differentiation of human iPS cells gave rise to progenitors that were retained within infarcted rat hearts, and reduced remodeling of the heart after ischemic damage.[204]

Furthermore, w:ischemic cardiomyopathy in the murine infarction model was targeted by iPS cell transplantation. It synchronized failing ventricles, offering a regenerative strategy to achieve resynchronization and protection from w:decompensation by dint of improved left ventricular conduction and contractility, reduced scarring and reversal of structural remodelling.[205] One protocol generated populations of up to 98% cardiomyocytes from hPSCs simply by modulating the canonical w:Wnt signaling pathway at defined time points in during differentiation, using readily accessible small molecule compounds.[206]

Discovery of the mechanisms controlling the formation of cardiomyocytes led to the development of the drug ITD-1, which effectively clears the cell surface from TGF- receptor type II and selectively inhibits intracellular TGF- signaling. It thus selectively enhances the differentiation of uncommitted w:mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells.[207]

One project seeded decellularized mouse hearts with human iPSC-derived multipotential cardiovascular progenitor cells. The introduced cells migrated, proliferated and differentiated in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the hearts. In addition, the heart's extracellular matrix (the substrate of heart scaffold) signalled the human cells into becoming the specialised cells needed for proper heart function. After 20 days of perfusion with growth factors, the engineered heart tissues started to beat again and were responsive to drugs.[208]

See also: review[209]

w:Tbx18 transduction is a method of turning on genes in heart muscle cells as a treatment for certain w:cardiac arrhythmias. Tbx18 gene therapy is aimed at treating a group of arrhythmias known as sick sinus syndrome. In a healthy heart, w:sinoatrial node (SAN) cells act as the hearts pacemaker and cause the heart to beat in a regular rhythm. Approximately 10 thousand of the 10 billion cells in the heart are SAN cells.[210] The Tbx18 gene is required for development of pacemaker cells in the heart during fetal development but is normally not functional after birth[211] Tbx18 transduction converts atrial muscle cells into SAN cells that initiate the heartbeat. An engineered virus carrying the Tbx18 gene is injected into animals and infects atrial muscle cells. Inside atrial muscle cells the Tbx18 gene is expressed. Tbx18 turns on genes that drive SA node cell development, simultaneously turning off genes that create atrial muscle cells. Tbx18 gene therapy has been successful in rodent hearts, converting atrial muscle cells into SAN cells by expression of the Tbx18 transcription factor. Tbx18 expression in atrial myocytes was shown to convert them into functional SAN cells in an experiment done in rodents. These converted SAN cells are able to respond to the nervous system, allowing the heart to be regulated as normal. Adenoviral TBX18 gene transfer could create biological pacemaker activity in vivo in a large-animal model of complete heart block. Biological pacemaker activity, originating from the intramyocardial injection site, was evident in TBX18-transduced animals starting at day 2 and persisted for the duration of the study (14 days) with minimal backup electronic pacemaker use. Relative to controls transduced with a reporter gene, TBX18-transduced animals exhibited enhanced autonomic responses and physiologically superior chronotropic support of physical activity. Induced sinoatrial node cells could be identified by their distinctive morphology at the site of injection in TBX18-transduced animals, but not in controls. No local or systemic safety concerns arose. Thus, minimally invasive TBX18 gene transfer creates physiologically relevant pacemaker activity in complete heart block, providing evidence for therapeutic somatic reprogramming in a clinically relevant disease model.[212]

The elderly often suffer from progressive w:muscle weakness and regenerative failure owing in part to elevated activity of the p38 and p38 mitogen-activated kinase pathway in senescent skeletal muscle stem cells. Subjecting such stem cells to transient inhibition of p38 and p38 in conjunction with culture on soft w:hydrogel substrates rapidly expands and rejuvenates them that result in the return of their strength.[213]

In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of w:p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions.[214]

Myogenic progenitors for potential use in disease modeling or cell-based therapies targeting skeletal muscle could also be generated directly from induced pluripotent stem cells using free-floating spherical culture (EZ spheres) in a culture medium supplemented with high concentrations (100ng/ml) of fibroblast growth factor-2 (w:FGF-2) and w:epidermal growth factor.[215]

Unlike current protocols for deriving w:hepatocytes from human fibroblasts, Saiyong Zhu et al., (2014)[216] did not generate iPSCs but, using small molecules, cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) were efficiently differentiated. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of human primary adult hepatocytes. iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level.[217]

These results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Complications of Diabetes mellitus such as w:cardiovascular diseases, retinopathy, neuropathy, nephropathy, and peripheral circulatory diseases depend on sugar dysregulation due to lack of w:insulin from pancreatic w:beta cells and can be lethal if they are not treated. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs).[218] Unfortunately, human PSC-derived insulin-expressing cells resemble human fetal cells rather than adult cells. In contrast to adult cells, fetal cells seem functionally immature, as indicated by increased basal w:glucose secretion and lack of glucose stimulation and confirmed by w:RNA-seq of whose transcripts.[219]

Overexpression of the three transcription factors, w:PDX1 (required for pancreatic bud outgrowth and beta-cell maturation), NGN3 (required for endocrine precursor cell formation) and MAFA (for beta-cell maturation) combination (called PNM) can lead to the transformation of some cell types into a beta cell-like state.[220] An accessible and abundant source of functional insulin-producing cells is intestine. PMN expression in human intestinal w:organoids stimulates the conversion of intestinal epithelial cells into -like cells possibly acceptable for transplantation.[221]

Adult proximal tubule cells were directly transcriptionally reprogrammed to w:nephron progenitors of the embryonic w:kidney, using a pool of six genes of instructive transcription factors (SIX1, SIX2, OSR1, Eyes absent homolog 1(EYA1), Homeobox A11 (HOXA11) and Snail homolog 2 (SNAI2)) that activated genes consistent with a cap mesenchyme/nephron progenitor phenotype in the adult proximal tubule cell line.[222] The generation of such cells may lead to cellular therapies for adult w:renal disease. Embryonic kidney organoids placed into adult rat kidneys can undergo onward development and vascular development.[223]

As blood vessels age, they often become abnormal in structure and function, thereby contributing to numerous age-associated diseases including myocardial infarction, ischemic stroke and atherosclerosis of arteries supplying the heart, brain and lower extremities. So, an important goal is to stimulate vascular growth for the w:collateral circulation to prevent the exacerbation of these diseases. Induced Vascular Progenitor Cells (iVPCs) are useful for cell-based therapy designed to stimulate coronary collateral growth. They were generated by partially reprogramming endothelial cells.[158] The vascular commitment of iVPCs is related to the epigenetic memory of endothelial cells, which engenders them as cellular components of growing blood vessels. That is why, when iVPCs were implanted into w:myocardium, they engrafted in blood vessels and increased coronary collateral flow better than iPSCs, mesenchymal stem cells, or native endothelial cells.[224]

Ex vivo genetic modification can be an effective strategy to enhance stem cell function. For example, cellular therapy employing genetic modification with Pim-1 kinase (a downstream effector of w:Akt, which positively regulates neovasculogenesis) of w:bone marrowderived cells[225] or human cardiac progenitor cells, isolated from failing myocardium[226] results in durability of repair, together with the improvement of functional parameters of myocardial hemodynamic performance.

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