induced pluripotent stem cells – eurostemcell.org

An important step in developing a therapy for a given disease is understanding exactly how the disease works: what exactly goes wrong in the body? To do this, researchers need to study the cells or tissues affected by the disease, but this is not always as simple as it sounds. For example, its almost impossible to obtain genuine brain cells from patients with Parkinsons disease, especially in the early stages of the disease before the patient is aware of any symptoms. Reprogramming means scientists can now get access to large numbers of the particular type of neurons (brain cells) that are affected by Parkinsons disease. Researchers first make iPS cells from, for example, skin biopsies from Parkinsons patients. They then use these iPS cells to produce neurons in the laboratory. The neurons have the same genetic background (the same basic genetic make-up) as the patients own cells. Thus scientist can directly work with neurons affected by Parkinsons disease in a dish. They can use these cells to learn more about what goes wrong inside the cells and why. Cellular disease models like these can also be used to search for and test new drugs to treat or protect patients against the disease.

iPS cells - derivation and applications:Certain genes can be introduced into adult cells to reprogramme them. The resulting iPS cells resemble embryonic stem cells and can be differentiated into any type of cell to study disease, test drugs or-after gene correction-develop future cell therapies

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induced pluripotent stem cells - eurostemcell.org

Induced Pluripotent Stem Cell Repository | California’s …

The Induced Pluripotent Stem Cell (iPSC) Repository is a major effort from CIRM to create a collection of stem cells developed from thousands of individuals.

CIRM is creating the iPSC bank so that scientists can use the cells, either in a petri dish or transplanted into animals, to study how disease develops and progresses and develop and test new drugs or other therapies. The iPSC bank is now open and cell lines are available at catalog.coriell.org/CIRM.

The large size of the collection will provide researchers with a powerful tool for studying genetic variation between individuals, helping scientists understand how disease and treatment may vary in a diverse population like Californias.

What is the iPSCRepository? How does it work? Why iPS cells? Who is generating the cells? Which diseases will be represented? How many samples are being collected for each condition?

What is the iPSCRepository? The Human Induced Pluripotent Stem Cell (hiPSC) Repositoryis one of the California stem cell agencys major efforts to provide valuable resources to the research community. The goal is to create a bank of high quality stem cell lines developed from thousands of individuals for use in research.

How does it work? Blood or skin samples collected from approximately 3,000 individuals will be turned into stem cell lines. These lines will be made available to researchers throughout California and around the world.

Why iPS cells? iPS cells are generated from cells easily obtained from living humans, i.e. blood or a small piece of skin; they have unlimited expansion potential in the petri dish, so huge numbers of cells can be generated for research studies or drug development; and they can be coaxed into the types of cells affected in various diseases, such as heart or brain disorders. This provides an unprecedented opportunity to study the cell types from patients that are affected in disease but cannot otherwise be easily obtained in large quantities from them.

Who is generating the cells? Seven clinician scientists from four California institutions recruit tissue donors who suffer from one of the included diseases or are healthy controls. Some blood or a small piece of skin is collected from those donors, and these samples are shipped to the company Cellular Dynamics International (CDI). CDI generates iPS cells from the samples, and then transfers the iPS cells to the Coriell Institute for Biomedical Research. Coriell operates a cell bank that will distribute the iPS cells to interested researchers at academic and other non-profit institutions, and also to pharmaceutical companies that may want to use them to find new drugs for the diseases that are included in this bank. While CDI and Coriell are located outside California, they have set up facilities at the Buck Institute in Novato, CA, where they generate and bank the iPS cells for this Initiative.

Which diseases will be represented? The stem cell lines created will represent a variety of diseases or conditions that affect brain, heart, lung, liver or eyes. Grantees come from a variety of California-based institutions:

How many samples are being collected? Below is a table that outlines CIRM's collection goalsfor each condition, along with control samples.

* these control donors will be specifically tested for the absence of lung disease

CIRM's New Stem Cell Bank Up, Running (California Healthline)

iPSC Repository Brochure [PDF] Stem Cell FAQ How do scientists model disease with iPSC's

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Induced Pluripotent Stem Cell Repository | California's ...

Embryonic stem (ES) cells and induced pluripotent stem …

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mhirarin@nips.ac.jp

Over the past 25 years, the reverse genetic approach including precise and conditional replacement or loss of gene function at a specific locus was considered possible only in mice due to the absence of embryonic stem (ES) or induced pluripotent stem (iPS) cell lines in other species. Recently, however, stem cell technology in rats has become available for biomedical research. In this paper we overview the recent progress of rat ES and iPS cell technology. Starting from the establishment of rat ES cells, the use of ES cells for foreign gene transfer and endogenous gene knock-out is discussed, followed by the successful establishment of rat iPS cells and the generation of an iPS cell-derived organ via interspecific blastocyst complementation. Finally, the possible contribution of rat stem cell technology to reproductive medicine is described.

Rats (Rattus norvegicus) have been used more extensively than mice in the research fields of neuroscience, pharmacology and toxicology. There are more than 100 rat strains with various genetic backgrounds, including some useful models for human diseases (e.g., SHR and BB for hypertension and diabetes, respectively), as well as the uncountable number of transgenic rat strains. Rats have the advantage of being a reasonably well-characterized and intermediate-sized rodent that can be maintained more cheaply than larger animals and can often be manipulated more easily than smaller rodents.

Although several technologies have been applied to modify rat genomes [13], the reverse genetic approach (precise and conditional replacements [knock-in] or loss of gene function [knock-out] at a specific locus) was considered impossible in rats because any protocols to establish stem cell lines conventionally used in mice [4, 5] were not applicable to rats. Recently, however, functional germline-competent embryonic stem (ES) cell lines [6, 7] and induced pluripotent stem (iPS) cell lines [8, 9] have been reported for this species. General advantages and disadvantages of ES and iPS cells are summarized in Table 1. In this paper we discuss the recent progress of rat ES/iPS cell technology.

The breakthrough in establishing rat ES cell lines was at the end of 2008. Functional germline-competent ES cell lines were reported by using a few inhibitors for fibroblast growth factor (FGF) receptor, mitogen activated protein kinase kinase (MEK) and glycogen synthase kinase 3 (GSK3) in differentiation-related signaling pathways [6, 7]. This protocol, the so-called 3i/2i culture system, originated from ES cell research in mice [10] and proved reproducible even after slight modifications were added to the culture system [11, 12]. The modification made by Hirabayashi et al. [11] is to replace MEK activation inhibitor PD1843521 with MEK inhibitor PD325901 and to add rat leukemia inhibitory factor (LIF) (ESGRO) instead of LIF-secreting feeder cells to the 3i culture system, while that made by Kawamata and Ochiya [12] is to add fetal bovine serum (FBS), -mercaptoethanol, rat LIF, and inhibitors for Rho-associated coiled-coil kinase and transforming growth factor- type-I receptor ALK5 kinase (Y-27632 and A-83-01, respectively) to the 2i culture medium containing MEK and GSK3 inhibitors (PD325901 and CHIR99021, respectively).

The rat ES cell lines established by Hirabayashi et al. [11] are described here in detail. Blastocysts at E4.5 were recovered from Wistar females copulated with a homogenous CAG/venus transgenic male rat (green fluorescence of the venus gene was used as the transgenic marker). Zona-free blastocysts were placed on mitomycin C (MMC)-treated mouse embryonic fibroblasts (MEF). The culture medium consisted of 2 M FGF receptor inhibitor (SU5402), 1 M MEK inhibitor (PD0325901), 3 M GSK3 inhibitor (CHIR99021) and 1,000 U/ml rat LIF (ESGRO) in N2B27 medium. After 7 days of culture, the outgrowths of the blastocysts (Fig. 1a) were disaggregated by gentle pipetting and transferred to the same MEF/3i conditions (first passage). When ES cell-like colonies emerged (Fig. 1b), they were trypsinized and then expanded (Fig. 1c). The tentative ES cell lines were maintained in MEF/3i conditions, with medium exchange every other day and trypsinization/expansion (passage) every 3 days. Otherwise, the ES cell lines were cryopreserved to prevent senescence.

Establishment of rat ES cell line [11]. a Outgrowth of a blastocyst on MEF feeders 7 days after plating. b Formation of colonies 3 days after the first passage. c Expanded ES cell colonies 2 days after fourth passage. d Alkaline phosphatase-positive colony at passage 12. e ES cells at passage 17. Under the daily exchange of culture medium, very few cells showed any signs of differentiation. f ES cells at passage 17, maintained with medium exchange every other day. Differentiated extraembryonic cells were observed. Scale barsa, e, f 100 m, b c, d 500 m

It was checked whether the delivered cells were alkaline phosphatase (AP)-positive (Fig. 1d). Nine ES cell lines (69.2%) were established from 13 transgenic blastocysts. Among them, two lines with excellent growth rate (rESWIv3i-1 and rESWIv3i-5) were selected, and both lines were found to be female by PCR analysis to detect rat Sry gene. Attachment of ES cell colonies with the feeder cells was not as strong, and the morphological appearance of the rat ES cell colonies was similar to that of mouse ES cells. As the passage number of the ES cells increased, signs of differentiation into extraembryonic cell-like cells were observed in cultures, especially when the culture medium was exchanged every other day rather than every day (Fig. 1e, f). In addition, expression of stem cell marker genes, such as Oct4, Nanog, Fgf4 and Rex1, was confirmed by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis in rESWIv3i-1 and rESWIv3i-5 lines. Furthermore, multipotency of the tentative ES cells was investigated by subcutaneous transplantation of the rESWIv3i-1 cells into an adult male F344 nude rat. Five weeks after the transplantation, a tumor was observed. By histological analysis, the tumor was found to be a teratoma with various tissues including gut-like epithelium or hepatic cells (endoderm), bone, cartilage or muscle (mesoderm), and neural tissues (ectoderm).

To generate ES cell-derived chimeras, host blastocysts at E4.5 derived from Wistar females or Wistar Dark Agouti (DA) F1 females were each microinjected with 10 ES cells at passages 68 (Fig. 2a, b). Collapsed blastocysts (Fig. 2c) were re-blasturated 12 h after the microinjection, and allowed to develop to fetus (E15.5) or full-term pups in pseudopregnant Wistar recipients. All of the E15.5 fetuses (100%; Fig. 2d) and the majority of the newborn pups (81.8100%) were chimeric and expressed the venus gene. The characteristics of the ES cells was successfully transmitted to their next generation in both lines. The ability of the ES cells to participate in chimeras was still high (78.6100%) at advanced passage numbers (17 or 18). Overall efficiency of producing chimeric rats (50.3%, 94 chimeras/187 injected embryos) was higher than 8.2% (20/245) as reported in Buehr et al. [6] and 11.0% (26/237) in Li et al. [7]. This higher efficiency of chimera rat production is probably due to the rat strain combination used for donor (ES cells) and host (blastocysts) and/or modification of culture medium.

Production of chimeric rats with ES cells [11]. a Microinjection of 10 ES cells into a blastocoele of E4.5 blastocyst. b Semi-bright, fluorescent image of venus-positive ES cells in the blastocyst. c Collapsed blastocysts immediately after microinjection. d Venus-positive fetal rat at E15.5

Functional germline-competent rat ES cell lines were established by applying the 2i/3i culture system, and the minimal essential materials for conducting transgenic studies including reverse genetic approaches, were now ready to use in rats. We now describe the production efficiency of chimeric rats by blastocyst injection of ES cells electroporated with a humanized Kusabira-Orange (huKO) gene and the germline transmission of the huKO gene from the chimeras to the next generation [13].

Rat ES cell lines were established from E4.5 blastocysts derived from BrownNorway (BN) females copulated with BN males, as described above, by applying the 2i (SU5402-free) culture system. One of the established lines, named as rESBN2i-4, was derived from a male embryo, based on PCR analysis. At passage 8, the ES cells in the N2B27 medium supplemented with 10% FBS (1 106 cells/0.5 ml) were electroporated with 25 g huKO gene (CAG/huKO-neo plasmid; 7.5 kb, Fig. 3a). The electroporated ES cells were placed in 3 ml of 2i medium + 5% FBS (passage 9), and the next day the medium was changed to serum-free 2i medium. Two days after the electroporation, G418 (200 g/ml) was added to the medium. The number of neomycin-resistant colonies (passage 10) was counted 8 days after electroporation. The huKO-positive ES cells were passaged twice in G418-free medium. Host blastocysts derived from Wistar/ST or WistarHannover females were each microinjected with 10 of the G418-resistant huKO-positive ES cells, and allowed to develop to full-term in pseudopregnant Wistar recipients. Transfer of 116 Wistar/ST blastocysts and 97 WistarHannover blastocysts resulted in 31 and 44 new-born offspring (26.7% and 45.4%), and 22 (70.9%; male 12, female 10) and 34 (77.3%; male 15, female 17, not-identified 2) out of the offspring were judged as chimeras by their coat color, respectively (Fig. 3b). Using non-electroporated control ES cells, a similar offspring rate (37.5%, 9/24) and chimera production efficiency (88.9%, 8/9) were obtained. Rat strain for host blastocysts may be a factor influencing the overall efficiency of chimera production, due to different preference for full-term development. Germline transmission of the CAG/huKO-neo gene was confirmed in 6 out of 25 G1 offspring (Fig. 3c) derived from 1 chimeric male with > 95% brown-colored coat. Thus, integration of exogenous DNA into rat ES cells did not affect the production efficiency of chimera offspring. The result described in this section [13] achieved the first successful production of transgenic rats via electroporated ES cells, followed by the work of Kawamata and Ochiya [12].

Rat transgenesis with ES cells [13]. a The construction of CAG/huKO-neo plasmid; 7.5 kb. b Chimeric rats with different contribution of brown-colored coat at G0 generation, 17 days old. c A huKO transgenic offspring (brown-colored) with huKO-negative littermates, 3 weeks old

Although knock-out rats were also successfully produced by N-ethyl-N-nitrosourea-induced transgenesis [2], sleeping beauty transposon-tagged mutagenesis [1], or zinc-finger nuclease-based transgenesis [3, 14], such successes may not have an impact on creating rat models for human diseases due to limitations in genome modification by these technologies. In this section, we describe the successful production of endogenous p53 gene knock-out rats by homologous recombination in ES cells [15].

p53, consisting of ten exons with the translation start codon located within exon 2, is a tumor suppressor gene located on rat chromosome 10, and mutations in the p53 gene are highly associated with genetic lesions in human cancers. Two male lines of ES cells were established from DA blastocysts by the 2i culture system and named as DAc4 and DAc8, respectively. After electroporation with CAG-EGFP-IRES-Pac vector (6.7 kb 5 and 1.6 kb 3 homology arms) to replace exons 25 of p53, successful targeting of the p53 gene occurred at 1.1% (4/356) and 3.7% (10/270) in the DAc4 line at passage 32 and the DAc8 line at passage 14, respectively. One correctly targeted ES cell colony, named as DAc8-p53-1, was microinjected into E4.5 F344 blastocysts (n = 79) and resulted in the birth of 24 live pups (30.4%). Among these 24 pups, 16 (75.0%; male 10, female 6) were chimeras with agouti coat color and all the male chimeras were fertile. However, examination of their >600 G1 offspring failed to show production of p53 targeted rats. The authors [15] suggest that this failure was caused by chromosomal abnormalities in the ES cells, because over 65% of the DAc8-p53-1 rat ES cells were found to be polyploid by karyotype analysis. Subcloning of the DAc8-p53-1 rat ES cells resulted in the appearance of round and compact colonies (approximately 10%). Among 20 subclones harvested, two (10.0%) were identified to carry euploid chromosome numbers (2n = 42). After microinjection of the subcloned ES cells into 39 F344 blastocysts and transfer to pseudopregnant SpragueDawley (SD) recipients, two male chimeras were produced and one of the two was germline chimera. Germline transmission of the GFP gene was confirmed in 6 out of the 76 G1 offspring. Three of the germline pups (male 1, female 2) were identified by genotyping and Southern blot analysis to be p53 heterozygotic rats carrying one wild-type allele and one targeted p53 allele. Intercrossing of the three p53 heterozygous rats resulted in the production of 12 pups, of which 9 (75.0%) were GFP-positive. Genotyping showed that 2 of the 9 pups were p53 homozygous (knock-out) rats, with further confirmation by Northern and Western blot analyses (absence of p53 mRNA and protein, respectively). Thus, gene targeting via homologous recombination in rat ES cells and the production of knock-out rats were achieved for the first time.

iPS cells are a type of pluripotent stem cell, similar to ES cells, that are artificially delivered from a non-pluripotent somatic cell by inducing a forced expression of specific genes. iPS cells can be established without the controversial use of blastocyst-stage embryos. iPS cells also have the advantage of avoiding the issue of graft-versus-host disease and immune rejection, if they are delivered entirely from the patient. The genes to be transfected into a somatic cell with a viral vector, the so-called Yamanaka factors, include Oct4, Sox2, c-Myc, and Klf4 [16, 17]. The Oct4 and Sox2 have been identified as crucial transcriptional regulators, both of which play a key role in maintaining pluripotency. The Klf4 is also the transcriptional regulator gene and is involved in cell proliferation, differentiation and survival. The c-Myc is the oncogene encoding for protein that binds to the DNA of other genes, and thus acts as a transcription factor. The induction efficiency of iPS cells (reprogramming efficiency of differentiated somatic cells) was 10-times higher when embryonic fibroblasts rather than tail tissue-derived fibroblasts (adult somatic cells) were used for transfection, and the morphology of iPS cell colonies was similar to that of ES cell colonies [18]. Once the iPS cells have been established, the retroviral or lentiviral transgenes become silenced theoretically, and the endogenous genes encoding these factors become activated. However, for the future therapeutic application of iPS cells, the viral transfection system and the use of oncogene c-Myc with the risk of tumor formation need to be replaced by alternative induction methods. The disadvantages of the original protocol have already been overcome by the use of adenovirus [19] or plasmid [20] as the vector, the direct introduction of proteins encoded by Yamanaka factors [21], and the elimination of the c-Myc gene from the cocktails [22], but the induction efficiency of iPS cells by such alternative methods may be unsatisfactory and should be improved further.

The first successful establishment of rat iPS cell lines was published from two independent laboratories in 2009 [8, 9]. Both protocols employed to establish the iPS cells were theoretically the same as, but slightly modified from, that reported by Takahashi and Yamanaka [16] where Yamanaka factors (Oct4, Sox2, c-Myc, and Klf4) were designed in a retroviral vector for transfection into adult somatic cells. The commercially available construction of the pMXs retroviral vector typical for the transfection of the factors [18] is shown in Fig. 4. The protocol by Li et al. [8] includes the construction of a retroviral vector with Oct4, Sox2 and Klf4 (c-Myc-free Yamanaka factors) and the transfection into liver progenitor cells prepared from WB-F344 rats. On the other hand, Liao et al. [9] used the lentiviral vector constructed with all four Yamanaka factors for transfection into primary ear fibroblasts prepared from 10-week-old SD rats, because transfection with retroviral vectors resulted in the failure of harvesting the iPS cell-like colonies. Both groups identified rat iPS cell-like colonies from the cultures in ES medium (Knockout; DMEM medium supplemented with 1020% KnockOut serum replacer, 0.1 mM non-essential amino acids, 1 mM l-glutamine, and 0.1 mM -mercaptoethanol) until 10 days after the viral transfection. The expression of stem cell marker genes (such as rat Oct4, Sox2, Nanog) in the iPS cells and the contribution to teratoma formation from the iPS cells were confirmed. Liao et al. [9] did not describe the production of chimeric rats using their iPS cells that were maintained on MEF in FBS-supplemented ES medium. In contrast, Li et al. [8] reported the successful production of chimeric rats by blastocyst injection of their iPS cells that were maintained in the presence of 0.5 M PD325901 (MEK inhibitor), 0.5 M A-83-01 (ALK5 inhibitor) and 3 M CHIR99021 (GSK3 inhibitor). The overall efficiency of producing chimeric rats was 16.7% (3 chimeras/18 injected embryos). The modified 3i culture system was known to support the self-renewal of mouse ES cells in a more robust manner [23, 24], but even after short-term culture a considerable amount of rat iPS cells exhibited spontaneous differentiation [8]. Transmission of the iPS cell genetic characteristics through the germline was not investigated in their studies [8, 9].

Retroviral vectors (pMXs) designed to transfect four transcriptional genes, the so-called Yamanaka factors (Oct4, Sox2, c-Myc, and Klf4), into somatic cells [18]. SD splicing donor, SA splicing acceptor, LTR long terminal repeats, MMLV Moloney murine leukemia virus

Therapeutic application of iPS cells includes the transplantation of differentiated cells, tissues or organs that can be regenerated from the stem cells. However, the in vitro production of tissue or organ with a three-dimensional structure from iPS cells is still difficult to achieve, despite the recent progress on the interactions of stem cells with growth factors and scaffolds. The idea for organ regeneration under an in vivo condition by blastocyst complementation was originally derived from Chen et al. [25]. Mouse blastocysts from a Rag2/ mutant strain lacking matured lymphocytes were microinjected with wild-type mouse ES cells and the resultant Rag2//ES chimera offspring produced fully matured T- and B-lymphocytes that were originated from the ES cells (Fig. 5). This strategy of intraspecies blastocyst complementation was expanded to interspecies approach. If the rescue of organ deficiency in mouse by rat iPS cells was proven effective, the interspecies blastocyst complementation may be applicable for a combination of pig (host animal) and human (iPS cell origin).

Intraspecies and interspecies blastocyst complementation for organ regeneration from pluripotent stem cells. Chen et al. [25] reported an intraspecies assay that enabled mouse ES cells to differentiate into mature lymphocytes in Rag2/ mice (mature lymphocyte-deficient). Kobayashi et al. [26] reported an interspecies assay in which rat iPS cells formed a fully functional rat pancreas when injected into mouse blastocysts lacking the Pdx1 gene required for pancreas formation

The rat iPS cell line (named as riPS#3) used for the interspecies blastocyst complementation [26] was established from primary embryonic fibroblasts prepared from E14.5 Crlj:Wistar fetuses. Ten to 15 iPS cells were microinjected into each of the blastocysts derived from Pdx1+/ Pdx1/ mice. The Pdx1 gene is responsible for the formation of functional pancreas, and the pancreas in the Pdx1/ male founder arose principally from exogenous mouse iPS cells. Therefore, half of the mice born from the blastocysts were expected to be Pdx1/. Transfer of 139 blastocysts into recipient mice resulted in the birth of 34 offspring (24.5%), and among these offspring, 5 and 10 neonates were found to be Pdx1+/ and Pdx1/ chimeric mice with rat iPS cell-derived pancreatic epithelial cells, respectively, by the enhanced green fluorescence protein (EGFP). Function of the pancreas derived from the rat iPS cells was confirmed by immunohistological analyses for the presence of EGFP, -amylase, insulin, glucagon, and somatostatin. Development into adulthood (8 weeks old) of the wild-type Pdx1/ mice was uncommon, but two Pdx1/ chimeras complemented with the rat iPS cells survived to the adulthood stage. They secreted insulin in response to glucose loading, and maintained a normal serum glucose level. Thus, Kobayashi et al. [26] demonstrated that rat iPS cells can rescue organ deficiency in mice, as the rat iPS cells formed a fully functional pancreas when injected into mouse blastocysts lacking the Pdx1 gene required for pancreas formation.

A few approaches are promising to produce offspring exclusively derived from stem cells, in addition to the conventional, time-consuming method via chimera (Table 2). Tetraploid embryos are able to implant in the endometrium and form extraembryonic tissues such as placenta, without participating into fetal development. In mice, tetraploid blastocysts complemented with ES/iPS cells can develop into full-term offspring with a phenotype of the ES/iPS characteristics [27, 28]. This approach, once found reproducible and applicable to other species, would allow the generation of different types of organs from the stem cells at the same time (showing ultimate pluripotency of the stem cells). The attempt at tetraploid blastocyst complementation with rat ES/iPS cells is, however, a challenging endeavor at the present stage [29]. Transplantation of stem cell nuclei into enucleated oocytes (cloning) is an alternative approach to produce animals derived from stem cells alone. Wakayama et al. [30] reported that 29% of mouse oocytes microinjected with ES cell nuclei developed into morulae/blastocysts and 8% of these embryos developed to full-term. Successful production of cloned rats with somatic cells was first reported by Zhou et al. [31]. However, the production of rat offspring exclusively derived from ES/iPS cells via nuclear transplantation has not been very practical, because the reproducibility of the data on rat cloning remained questionable [32].

Stem cell technology in rats can also contribute to the field of reproductive medicine, because germ cells derived from the stem cells were detected in xenogenic chimeras (mouserat chimeras). Isotani et al. [33] recently reported that rat ES cells injected into nu/nu mouse blastocysts could contribute to form not only thymus but also sperm-like germ cells. Although the minimal essential techniques for microinsemination, such as intracytoplasmic sperm injection and round spermatid injection, are almost available in the rat [34], the functional normality of the rat germ cell-like cells observed in such xenogenic chimeras has not yet been confirmed. Nevertheless, the interspecies blastocyst complementation system using mutant mice or experimentally nitch-induced mice and pluripotent stem cells of the rats would provide an appropriate model for generation of human germ cells in the body of non-human species.

Functional germline-competent rat ES/iPS cell lines have been established and successfully applied to the production of gene-modified rats as well as whole organ regeneration with a three-dimensional structure. Culturing of blastocysts with a few inhibitors for FGF receptor, MEK and GSK3 in differentiation-related signaling pathways (2i/3i system) was the key essential for ES cell establishment, and a forced expression of transcription-regulating genes as Yamanaka factors (Oct4, Sox2, Klf4 and/or c-Myc) in somatic cells played an important role in iPS cell establishment. The widespread use of the rat ES/iPS cells would provide a practical breakthrough for a variety of biomedical research in the rats. The accumulation of basic and practical knowledge in this system may be useful in improving the ultimate therapeutic performance against the most severe forms of male infertility in humans.

The Japan Society for Reproductive Medicine

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Embryonic stem (ES) cells and induced pluripotent stem ...

Induced pluripotent stem cell Wikipedia StemCell 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.[4]

iPSCs are typically derived by introducing products of 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 transcription factors 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 Yamanakas team at Kyoto University, Japan, in 2006.[1] They hypothesized that genes important to embryonic stem cell (ESC) function might be able to induce an embryonic state in adult cells. They chose twenty-four genes previously identified as important in ESCs and used retroviruses to deliver these genes to mouse fibroblasts. The fibroblasts were engineered so that any cells reactivating the ESC-specific gene, Fbx15, could be isolated using antibiotic selection.

Upon delivery of all twenty-four factors, ESC-like colonies emerged that reactivated the Fbx15 reporter and could propagate indefinitely. To identify the genes necessary for reprogramming, the researchers removed one factor at a time from the pool of twenty-four. By this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which were each necessary and together sufficient to generate ESC-like colonies under selection for reactivation of Fbx15.

Similar to ESCs, these iPSCs had unlimited self-renewal and were pluripotent, contributing to lineages from all three germ layers in the context of embryoid bodies, teratomas, and 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 failed to produce viable chimeras when injected into developing embryos.

In June 2007, three separate research groups, including that of Yamanakas, a Harvard/University of California, Los Angeles collaboration, and a group at MIT, published studies that substantially improved on the reprogramming approach, giving rise to iPSCs that were indistinguishable from ESCs. Unlike the first generation of iPSCs, these second generation iPSCs produced viable chimeric mice and contributed to the mouse germline, thereby achieving the gold standard for pluripotent stem cells.

These second-generation iPSCs were derived from mouse fibroblasts by retroviral-mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4). However, instead of using Fbx15 to select for pluripotent cells, the researchers used Nanog, a gene that is functionally important in ESCs. By using this different strategy, the researchers created iPSCs that were functionally identical to ESCs.[5][6][7][8]

Reprogramming of human cells to iPSCs was reported in November 2007 by two independent research groups: Shinya Yamanaka of Kyoto University, Japan, who pioneered the original iPSC method, and James Thomson of University of Wisconsin-Madison who was the first to derive human embryonic stem cells. With the same principle used in mouse reprogramming, Yamanakas group successfully transformed human fibroblasts into iPSCs with the same four pivotal genes, OCT4, SOX2, KLF4, and C-MYC, using a retroviral system,[9] while Thomson and colleagues used a different set of factors, OCT4, SOX2, NANOG, and LIN28, using a lentiviral system.[10]

Obtaining fibroblasts to produce iPSCs involves a skin biopsy, and there has been a push towards identifying cell types that are more easily accessible.[11][12] In 2008, iPSCs were derived from human keratinocytes, which could be obtained from a single hair pluck.[13][14] In 2010, iPSCs were derived from peripheral blood cells,[15][16] and in 2012, iPSCs were made from renal epithelial cells in the urine.[17]

Other considerations for starting cell type include mutational load (for example, skin cells may harbor more mutations due to UV exposure),[11][12] time it takes to expand the population of starting cells,[11] and the ability to differentiate into a given cell type.[18]

[citation needed]

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

Oct-3/4 and certain products 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.

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).[32] 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.[33] 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.[34][35]

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. [36][37]

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.[38] The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).

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.[39] In 2009, Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells.[40] 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.[41] 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. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving footprint mutations in the host cell genome. The PiggyBac Transposon System involves the re-excision of exogenous genes, which eliminates the issue of insertional mutagenesis. [42]

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.[43]

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.[44] On 4 June 2014, the lead author, Obokata agreed to retract both the papers [45] after she was found to have committed research misconduct as concluded in an investigation by RIKEN on 1 April 2014.[46]

MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Measuring variations in microRNA expression in iPS cells can be used to predict their differentiation potential.[47] Addition of microRNAs can also be used to enhance iPS potential. Several mechanisms have been proposed.[47] ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhance the efficiency of induced pluripotency by acting downstream of c-Myc.[48]microRNAs can also block expression of repressors of Yamanakas four transcription factors, and there may be additional mechanisms induce reprogramming even in the absence of added exogenous transcription factors.[47]

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.[49]

Gene expression and genome-wide H3K4me3 and H3K27me3 were found to be extremely similar between ES and iPS cells.[50][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.[62]

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.[63] 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[64]

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.[65][66] 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.[67] 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.[68] Furthermore, combining hiPSC technology and genetically-encoded voltage and calcium indicators provided a large-scale and high-throughput platform for cardiovascular drug safety screening.[69]

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.[70][71] 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.[72]

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.[73][74]

Labelled iPSCs-derived NSCs injected into laboratory animals with brain lesions were shown to migrate to the lesions and some motor function improvement was observed.[75]

Although a pint of donated blood contains about two trillion red blood cells and over 107 million blood donations are collected globally, there is still a critical need for blood for transfusion. 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. Human clinical trials were not expected to begin before 2016.[76]

The first human clinical trial using autologous iPSCs was approved by the Japan Ministry Health and was to be conducted in 2014 in Kobe. However the trial was suspended after Japans new regenerative medicine laws came into effect last November.[77] iPSCs derived from skin cells from six patients suffering from wet age-related macular degeneration were to be reprogrammed to differentiate into retinal pigment epithelial (RPE) cells. The cell sheet would be transplanted into the affected retina where the degenerated RPE tissue was excised. Safety and vision restoration monitoring would last one to three years.[78][79] 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.[79]

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Induced pluripotent stem cell Wikipedia StemCell Therapy

Induced stem cells – Wikiversity

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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Induced stem cells - Wikiversity

Clinical potential of human-induced pluripotent stem cells …

The recent establishment of induced pluripotent stem (iPS) cells promises the development of autologous cell therapies for degenerative diseases, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by retroviral transduction of somatic cells with core reprogramming genes. To avoid potential genotoxic effects associated with retroviral transfection, more recently, alternative non-viral gene transfer approaches were developed. Before a potential clinical application of iPS cell-derived therapies can be planned, it must be ensured that the reprogramming to pluripotency is not associated with genome mutagenesis or epigenetic aberrations. This may include direct effects of the reprogramming method or "off-target" effects associated with the reprogramming or the culture conditions. Thus, a rigorous safety testing of iPS or iPS-derived cells is imperative, including long-term studies in model animals. This will include not only rodents but also larger mammalian model species to allow for assessing long-term stability of the transplanted cells, functional integration into the host tissue, and freedom from undifferentiated iPS cells. Determination of the necessary cell dose is also critical; it is assumed that a minimum of 1 billion transplantable cells is required to achieve a therapeutic effect. This will request medium to long-term in vitro cultivation and dozens of cell divisions, bearing the risk of accumulating replication errors. Here, we review the clinical potential of human iPS cells and evaluate which are the most suitable approaches to overcome or minimize risks associated with the application of iPS cell-derived cell therapies.

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Clinical potential of human-induced pluripotent stem cells ...

Stem Cell Glossary – stemcells.nih.gov

Adult stem cellSee somatic stem cell.

AstrocyteA type of supporting (glial) cell found in the nervous system.

BlastocoelThe fluid-filled cavity inside the blastocyst, an early, preimplantation stage of the developing embryo.

BlastocystApreimplantationembryo consisting of a sphere made up of an outer layer of cells (thetrophoblast), a fluid-filled cavity (theblastocoel), and a cluster of cells on the interior (theinner cell mass).

Bone marrow stromal cellsA population of cells found in bone marrow that are different from blood cells.

Bone marrow stromal stem cells (skeletal stem cells)A multipotent subset of bone marrow stromal cells able to form bone, cartilage, stromal cells that support blood formation, fat, and fibrous tissue.

Cell-based therapiesTreatment in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cells or tissues.

Cell cultureGrowth of cells in vitro in an artificial medium for research.

Cell divisionMethod by which a single cell divides to create two cells. There are two main types of cell division depending on what happens to the chromosomes: mitosis and meiosis.

ChromosomeA structure consisting of DNA and regulatory proteins found in the nucleus of the cell. The DNA in the nucleus is usually divided up among several chromosomes.The number of chromosomes in the nucleus varies depending on the species of the organism. Humans have 46 chromosomes.

Clone (v) To generate identical copies of a region of a DNA molecule or to generate genetically identical copies of a cell, or organism; (n) The identical molecule, cell, or organism that results from the cloning process.

CloningSee Clone.

Cord blood stem cellsSee Umbilical cord blood stem cells.

Culture mediumThe liquid that covers cells in a culture dish and contains nutrients to nourish and support the cells. Culture medium may also include growth factors added to produce desired changes in the cells.

DifferentiationThe process whereby an unspecialized embryonic cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. Differentiation is controlled by the interaction of a cell's genes with the physical and chemical conditions outside the cell, usually through signaling pathways involving proteins embedded in the cell surface.

Directed differentiationThe manipulation of stem cell culture conditions to induce differentiation into a particular cell type.

DNADeoxyribonucleic acid, a chemical found primarily in the nucleus of cells. DNA carries the instructions or blueprint for making all the structures and materials the body needs to function. DNA consists of both genes and non-gene DNA in between the genes.

EctodermThe outermost germ layer of cells derived from the inner cell mass of the blastocyst; gives rise to the nervous system, sensory organs, skin, and related structures.

EmbryoIn humans, the developing organism from the time of fertilization until the end of the eighth week of gestation, when it is called a fetus.

Embryoid bodiesRounded collections of cells that arise when embryonic stem cells are cultured in suspension. Embryoid bodies contain cell types derived from all threegerm layers.

Embryonic germ cellsPluripotent stem cells that are derived from early germ cells (those that would become sperm and eggs). Embryonic germ cells are thought to have properties similar to embryonic stem cells.

Embryonic stem cellsPrimitive (undifferentiated) cells that are derived from preimplantation-stageembryos, are capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers.

Embryonic stem cell lineEmbryonic stem cells, which have been cultured under in vitro conditions that allow proliferation without differentiation for months to years.

EndodermThe innermost layer of the cells derived from the inner cell mass of the blastocyst; it gives rise to lungs, other respiratory structures, and digestive organs, or generally "the gut."

EnucleatedHaving had its nucleus removed.

EpigeneticThe process by which regulatory proteins can turn genes on or off in a way that can be passed on during cell division.

Feeder layerCells used in co-culture to maintain pluripotent stem cells. For human embryonic stem cell culture, typical feeder layers include mouse embryonic fibroblasts (MEFs) or human embryonic fibroblasts that have been treated to prevent them from dividing.

FertilizationThe joining of the male gamete (sperm) and the female gamete (egg).

FetusIn humans, the developing human from approximately eight weeks after conception until the time of its birth.

GameteAn egg (in the female) or sperm (in the male) cell. See also Somatic cell.

GastrulationThe process in which cells proliferate and migrate within the embryo to transform the inner cell mass of the blastocyst stage into an embryo containing all three primary germ layers.

GeneA functional unit of heredity that is a segment of DNA found on chromosomes in the nucleus of a cell. Genes direct the formation of an enzyme or other protein.

Germ layersAfter the blastocyst stage of embryonic development, the inner cell mass of the blastocyst goes through gastrulation, a period when the inner cell mass becomes organized into three distinct cell layers, called germ layers. The three layers are the ectoderm, the mesoderm, and the endoderm.

Hematopoietic stem cellA stem cell that gives rise to all red and white blood cells and platelets.

Human embryonic stem cell (hESC)A type of pluripotent stem cell derived from early stage human embryos, up to and including the blastocyststage. hESCs are capable of dividing without differentiating for a prolonged period in culture and are known to develop into cells and tissues of the three primary germ layers.

Induced pluripotent stem cell (iPSC)A type of pluripotent stem cell, similar to an embryonic stem cell, formed by the introduction of certain embryonic genes into a somatic cell.

In vitroLatin for "in glass;" in a laboratory dish or test tube; an artificial environment.

In vitro fertilizationA technique that unites the egg and sperm in a laboratory instead of inside the female body.

Inner cell mass (ICM)The cluster of cells inside the blastocyst. These cells give rise to the embryo and ultimately the fetus. The ICM may be used to generate embryonic stem cells.

Long-term self-renewalThe ability of stem cells to replicate themselves by dividing into the same non-specialized cell type over long periods (many months to years) depending on the specific type of stem cell.

MeiosisThe type of cell division a diploid germ cell undergoes to produce gametes (sperm or eggs) that will carry half the normal chromosome number. This is to ensure that when fertilization occurs, the fertilized egg will carry the normal number of chromosomes rather than causing aneuploidy (an abnormal number of chromosomes).

Mesenchymal stem cellsA term that is currently used to define non-blood adult stem cells from a variety of tissues, although it is not clear that mesenchymal stem cells from different tissues are the same.

MesodermMiddle layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to bone, muscle, connective tissue, kidneys, and related structures.

MicroenvironmentThe molecules and compounds such as nutrients and growth factors in the fluid surrounding a cell in an organism or in the laboratory, which play an important role in determining the characteristics of the cell.

MitosisThe type of cell division that allows a population of cells to increase its numbers or to maintain its numbers. The number of chromosomes in each daughter cell remains the same in this type of cell division.

MultipotentHaving the ability to develop into more than one cell type of the body. See also pluripotent and totipotent.

Neural stem cellA stem cell found in adult neural tissue that can give rise to neurons and glial (supporting) cells. Examples of glial cells include astrocytes and oligodendrocytes.

NeuronsNerve cells, the principal functional units of the nervous system. A neuron consists of a cell body and its processesan axon and one or more dendrites. Neurons transmit information to other neurons or cells by releasing neurotransmitters at synapses.

OligodendrocyteA supporting cell that provides insulation to nerve cells by forming a myelin sheath (a fatty layer) around axons.

ParthenogenesisThe artificial activation of an egg in the absence of a sperm; the egg begins to divide as if it has been fertilized.

PassageIn cell culture, the process in which cells are disassociated, washed, and seeded into new culture vessels after a round of cell growth and proliferation. The number of passages a line of cultured cells has gone through is an indication of its age and expected stability.

PluripotentThe state of a single cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development.

Scientists demonstrate pluripotency by providing evidence of stable developmental potential, even after prolonged culture, to form derivatives of all three embryonic germ layers from the progeny of a single cell and to generate a teratoma after injection into an immunosuppressed mouse.

Polar bodyA polar body is a structure produced when an early egg cell, or oogonium, undergoes meiosis. In the first meiosis, the oogonium divides its chromosomes evenly between the two cells but divides its cytoplasm unequally. One cell retains most of the cytoplasm, while the other gets almost none, leaving it very small. This smaller cell is called the first polar body. The first polar body usually degenerates. The ovum, or larger cell, then divides again, producing a second polar body with half the amount of chromosomes but almost no cytoplasm. The second polar body splits off and remains adjacent to the large cell, or oocyte, until it (the second polar body) degenerates. Only one large functional oocyte, or egg, is produced at the end of meiosis.

PreimplantationWith regard to an embryo, preimplantation means that the embryo has not yet implanted in the wall of the uterus. Human embryonic stem cells are derived from preimplantation-stage embryos fertilized outside a woman's body (in vitro).

ProliferationExpansion of the number of cells by the continuous division of single cells into two identical daughter cells.

Regenerative medicineA field of medicine devoted to treatments in which stem cells are induced to differentiate into the specific cell type required to repair damaged or destroyed cell populations or tissues. (See also cell-based therapies).

Reproductive cloningThe process of using somatic cell nuclear transfer (SCNT) to produce a normal, full grown organism (e.g., animal) genetically identical to the organism (animal) that donated the somatic cell nucleus. In mammals, this would require implanting the resulting embryo in a uterus where it would undergo normal development to become a live independent being. The firstmammal to be created by reproductive cloning was Dolly the sheep, born at the Roslin Institute in Scotland in 1996. See also Somatic cell nuclear transfer (SCNT).

SignalsInternal and external factors that control changes in cell structure and function. They can be chemical or physical in nature.

Somatic cellAny body cell other than gametes (egg or sperm); sometimes referred to as "adult" cells. See also Gamete.

Somatic cell nuclear transfer (SCNT)A technique that combines an enucleated egg and the nucleus of a somatic cell to make an embryo. SCNT can be used for therapeutic or reproductive purposes, but the initial stage that combines an enucleated egg and a somatic cell nucleus is the same. See also therapeutic cloning and reproductive cloning.

Somatic (adult) stem cellA relatively rare undifferentiated cell found in many organs and differentiated tissues with a limited capacity for both self renewal (in the laboratory) and differentiation. Such cells vary in their differentiation capacity, but it is usually limited to cell types in the organ of origin. This is an active area of investigation.

Stem cellsCells with the ability to divide for indefinite periods in culture and to give rise to specialized cells.

Stromal cellsConnective tissue cells found in virtually every organ. In bone marrow, stromal cells support blood formation.

SubculturingTransferring cultured cells, with or without dilution, from one culture vessel to another.

Surface markersProteins on the outside surface of a cell that are unique to certain cell types and that can be visualized using antibodies or other detection methods.

TeratomaA multi-layered benign tumor that grows from pluripotent cells injected into mice with a dysfunctional immune system. Scientists test whether they have established a human embryonic stem cell (hESC) line by injecting putative stem cells into such mice and verifying that the resulting teratomas contain cells derived from all three embryonic germ layers.

Therapeutic cloningThe process of using somatic cell nuclear transfer (SCNT) to produce cells that exactly match a patient. By combining a patient's somatic cell nucleus and an enucleated egg, a scientist may harvest embryonic stem cells from the resulting embryo that can be used to generate tissues that match a patient's body. This means the tissues created are unlikely to be rejected by the patient's immune system. See also Somatic cell nuclear transfer (SCNT).

TotipotentThe state of a cell that is capable of giving rise to all types of differentiated cells found in an organism, as well as the supporting extra-embryonic structures of the placenta. A single totipotent cell could, by division in utero, reproduce the whole organism. (See also Pluripotent and Multipotent).

TransdifferentiationThe process by which stem cells from one tissue differentiate into cells of another tissue.

TrophoblastThe outer cell layer of the blastocyst. It is responsible for implantation and develops into the extraembryonic tissues, including the placenta, and controls the exchange of oxygen and metabolites between mother and embryo.

Umbilical cord blood stem cellsStem cells collected from the umbilical cord at birth that can produce all of the blood cells in the body. Cord blood is currently used to treat patients who have undergone chemotherapy to destroy their bone marrow due to cancer or other blood-related disorders.

UndifferentiatedA cell that has not yet developed into a specialized cell type.

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Stem Cell Glossary - stemcells.nih.gov

Why Induced Pluripotent Stem Cells Are Vital for Glaucoma …

One of the most significant discoveries in regeneration research occurred when scientists learned that mature stem cells could be reprogrammedturned back into young stem cellsthen used to grow any type of new tissue. This revelation changed everything the experts thought they knew about cell development. Until then, they hadnt dreamed they could turn back the hands of time in old stem cells.

Now stem cells are poised to make a similar impact on glaucoma research and treatment, as adult stem cells can be taken from the eye or skin, and used to try to replace damaged cells in your eye. Stem cell treatment may one day restore vision lost to glaucoma, so this is a topic glaucoma patients and research supporters will want to know about.

At the start of life, embryonic stem cells are pluripotent, which means they have the remarkable ability to become any type of cell in the body. Following conception, stem cells rapidly reproduce to form clusters of cells that begin to specialize, or differentiate. Each cluster follows a different path, developing into the heart, brain, lungs, skin and every other tissue needed to build the human body.

Fully mature, adult stem cells continue to generate new cells, but only for the specific tissues where they live. For example, hair follicles have adult stem cells that regrow hair and adult stem cells in bone marrow give rise to blood cells, but they cant fill in for one another. These mature stem cells are the sentinels that guard your health, as they replace cells that are damaged due to normal wear-and-tear, injury and disease.

As long as theyre alive and thriving, adult stem cells continue to self-renew indefinitely, dividing and replicating as often as needed. Even if theyre inactive for a long time, they can jump back into action at a moments notice. But theres one thing they cant do: they cant reverse back into their pluripotent state. At least, they cant do that in their natural environment.

In the early 1960s, Sir John Bertrand Gurdon was a young developmental biologist searching for the answer to one question: Is it possible for adult stem cells to return to their immature state? He experimented with frog cells, transplanting mature stem cells into eggs that had their stem cells removed. After many trials, an astonishing thing happenedthe eggs grew into normal tadpoles. With that success, Gurdon proved that fully-differentiated adult stem cells retained the genetic information found in pluripotent embryonic cells.1

Nearly 50 years later, Shinya Yamanaka, MD, PhD and his co-workers published a stunning study. In a long series of experiments, he isolated 24 genes responsible for pluripotency. Then he reintroduced these genes into mature stem cells, individually and in various combinations, until he narrowed it down to four key genes. When used together, the four genes, now dubbed Yamanaka factors, accomplished the unbelievablethey reprogrammed adult stem cells, making them convert back into embryonic stem cells. The induced pluripotent stem cell had been discovered.2

Gurdon and Yamanaka were jointly awarded the 2012 Nobel Prize in Physiology or Medicine for these two discoveries.3 Of course, they both continued to study stem cells and, combined with results from other experts in the field, significant progress has been made. Now induced pluripotent stem cellsor iPS cells for shortcan be formed from human cells and they have a leading role in glaucoma research. Glaucoma Research Foundation gave our 2015 Visionary Award to Dr. Yamanaka to honor his pioneering work to improve global healthcare and treat blinding eye disease.

You may begin to hear a lot about iPS cells being used to develop treatments for glaucoma. When damaged cells in an area called the trabecular meshwork are replaced with iPS cells, intraocular pressure is normalized. If iPS cells could be used to restore parts of the retina, like photoreceptor, ganglion and Muller cells, vision could be restored. Heres one version of how the process might look:

A doctor takes a sample of cells called fibroblasts from a small area of skin on your arm. The fibroblasts are sent to a lab, put into a glass petri dish and injected with Yamanaka factors that convert them into induced pluripotent stem cells. Then substances known to trigger differentiation are added to the cells. They may be directed to become retinal ganglion cells, trabecular meshwork cells or another targeted cell in the eye. When a sufficient number of specialized cells are ready, theyre injected into the damaged eye, where they continue to grow and facilitate healing.4

This scenario isnt entirely hypothetical. Research using iPS cells to treat glaucoma is still in the early stages, but the European Commission has already authorized stem cell treatment for injured corneas. Their decision was based on clinical trials showing that healthy limbal stem cells could be taken from the cornea, expanded in the lab and transplanted back into the damaged part of the eye. The new iPS cells safely and effectively repaired the cornea and restored vision.5

Researchers also use iPS cells to create models of human cells and use them to learn how glaucoma progresses and to test emerging pharmaceutical treatments. Some of the most promising research uses iPS cells to develop models of retinal ganglion cells.6 Ganglion cells collect visual input and send it to the brainmore than a million ganglion cells are bundled together to form the optic nerveand theyre preferentially vulnerable among all retinal cells to glaucoma-inflicted damage.

Beyond their versatile uses, iPS cells have two other critical benefits. They allow glaucoma researchers to pursue new treatments while avoiding ethical concerns related to using embryonic stem cells. And best of all, when mature stem cells come from the same person who will use them for treatment, they dont have to worry about rejection by the immune system because the cells are already a genetic match. This is personalized medicine at a whole new level.

Much research has yet to be done, and clinical trials to test stem cell procedures on people with glaucoma are still down the road, yet the work accomplished so far shines enough light to show that answers are within reach. Glaucoma Research Foundation is determined to support research that will one day make the promise of restored vision come true.

Glaucoma Research Foundation depends on your donation to support research and patient education. Learn about the many ways you can join our cause.

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Why Induced Pluripotent Stem Cells Are Vital for Glaucoma ...

Live Cell Imaging of Induced Pluripotent Stem Cell …

Our live cell image program supports the advancement of iPSC technology in three ways:

1) Identification of process control measurements: A critical component to the translation of iPSCs into therapeutic applications is to design principles for predictably and reproducibly culturing cells and efficiently differentiating them into cell types of interest. Live cell imaging provides high-resolution measurements in the sense that we collect time-dependent data from large numbers of individual cells. We then use this data to discover lower resolution measurements, such as the activity of a biomarker at a single point in time, that can serve as critical process control points during processing of pluripotent stem cells.

2) Interpreting biomarkers: Cells are stochastic and dynamic and may interconvert between states and the expression of biomarkers can change over time. The predictive power of a biomarker or a set of biomarkers the indicate the differentiated state of a cell can be evaluated by examining the history of that cell by tracking forward and backward in time through a time lapse image set.

3) Predictive modeling: We have shown that fluctuations in promoter activity can be used in combination with appropriate models to predict rates of state change in cell populations. Similar mathematical models that can inform bioprocessing decisions during scale-up will be critical to obtaining iPSC populations with a desired set of characteristics.

Over the past several years, we have developed tools for measuring parameters related to size, shape and intensity from single cells over time (Halter Cytometry 2011). We have also developed modeling tools for using the temporal information to model the stochastic and deterministic components of gene expression (Sisan PNAS 2012; Lund Phys Chem B 2014).

We are now applying these live cell imaging tools to the study of stem cell pluripotency and differentiation (Bhadriraju Stem Cell Research 2016). Induced pluripotent stem cell technologies are a powerful new tool for biomedical research and have the potential to revolutionize medicine.

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Live Cell Imaging of Induced Pluripotent Stem Cell ...

Induced Pluripotent Stem Cells – cellapplications.com

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Human Induced Pluripotent Stem Cells (HiPSC) Top: HiPSC express pluriotency markers OCT4, Nanog, LIN28 and SSEA-4. Bottom: HiPSC differentiate into cell derivatives from the 3 embryonic layers: Neuronal marker beta III tubulin (TUJ1), Smooth Muscle Actin (SMA) and Hepatocyte Nuclear Factor 3 Beta (HNF3b).

Cell Applications, Inc. has a deep, rich history in HiPSC

Human Dermal Fibroblasts (HDF) from Cell Applications were used by Nobel Laureate S. Yamanaka to establish iPSC in his groundbreaking publications in 2007, unleashing a revolution in stem cell biology. Yamanaka and collaborators demonstrated that expression of four transcription factors widely prevalent in embryonic stem cells is sufficient to trigger the transition of somatic cells towards a pluripotent state that resembles embryonic stem cells in many aspects, such as the expression of classic pluripotency markers and the ability to generate cell derivatives from the three embryonic germ layers.

HiPSC are generated from somatic cells, eliminating ethical considerations associated with scientific work based on embryonic stem cells. Furthermore, being donor/patient-specific, they open possibilities for a wide variety of studies in biomedical research. Donor somatic cells carry the genetic makeup of the diseased patient, hence HiPSC can be used directly to model disease on a dish.

Thus, one of the main uses of HiPSC has been in genetic disease modeling in organs and tissues, such as the brain (Alzheimers, Autism Spectrum Disorders), heart (Familial Hypertrophic, Dilated, and Arrhythmogenic Right Ventricular Cardiomyopathies), and skeletal muscle (Amyotrophic Lateral Sclerosis, Spinal Muscle Atrophy). The combination of HiPSC technology and gene editing strategies such as the CRISPR/Cas9 system creates a powerful platform in which disease-causing mutations can be created on demand and sets of isogenic cell lines (with and without mutations) serve as convenient tools for disease modeling studies.

Other applications of HiPSC and iPSC-differentiated cells include drug screening, development, efficacy and toxicity assessment. As an example, through the FDA-backed CiPA (Comprehensive in vitro Pro-Arrhythmia Assessment) initiative, HiPSC-derived cardiac muscle cells (cardiomyocytes) are poised to constitute a new standard model for the evaluation of cardiotoxicity of new drugs, which is the main reason of drug withdrawal from the market. Finally, HiPSC-differentiated cells are being used in early stage technology development for applications in regenerative medicine. Bio-printing and tissue constructs have also been considered as attractive applications for HiPSC.

StemoniX

Our partner StemoniX is a cutting-edge biotechnology company that is leading the development and manufacturing of HiPSC. They generate biologically accurate miniaturized organ microtissue for academic and industrial pharmaceutical research and discovery. StemoniX, a licensee of Academia Japans iPS patent portfolio, provides high quality HiPSC cells to researchers around the world. StemoniX HiPSC are thoroughly characterized for pluripotency with established pluripotency markers. Proven technology incorporating the latest innovations is able to provide cardiomyocytes with in vitro-like features. Confirmative tests show the HiPSC differentiate into derivatives from the 3 embryonic layers.

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