Derivation of Human Induced Pluripotent Stem Cell

Stem Cell Rev and Rep DOI 10.1007/s12015-017-9766-9

Derivation of Human Induced Pluripotent Stem Cell (iPSC) Lines and Mechanism of Pluripotency: Historical Perspective and Recent Advances Arvind Chhabra1  

© Springer Science+Business Media, LLC 2017

Abstract Derivation of human embryonic stem cell (hES) lines in 1998 was not only a major technological breakthrough in the field of regenerative medicine; it also triggered a passionate debate about the ethical issues associated with the utilization of human embryos for derivation of hESC lines. Successful derivation of induced pluripotent stem cell (iPS) lines from human somatic cells with defined reprogramming factors by Shinya Yamanaka`s group in 2007 was another breakthrough that generated enormous excitement and hope for the development of donor-specific personalized cell replacement therapies (CRT) without the ethical dilemma associated with it. As we approach twentieth anniversary of derivation of hESC lines and the tenth anniversary of isolation of donor-specific iPSC lines, this manuscript summarizes the key advances in pluripotent stem cell (PSC) research field that led to derivation of human iPSC lines, different methodologies for derivation iPSC lines and characterization of the mechanism of reprogramming. We will also review progress towards generating donorspecific somatic cell lineages from iPSC lines, especially the functional immune cell lineages, and progress towards advancing these findings to the clinic. Finally, we will discuss the challenges, such as genome instability and inherent immunogenicity of hPSC lines that need to be addressed to develop safe and effective iPSC-based CRT.

Arvind Chhabra [email protected] 1

Department of Medicine, University of Connecticut Health Center (UConn Health), Farmington, CT 06030, USA

Keywords Human embryonic stem cells (hESC) · Human pluripotent stem cells (hPS) · Induced pluripotent stem cells (iPS). iPSC-based cell replacement therapies (CRT) Abbreviations hPS cells Human pluripotent stem cells hES cells Human embryonic stem cells iPS cells Induced pluripotent stem cells EB Embryoid bodies

Key Technological Advances Leading to Derivation of Human Pluripotent Stem Cell (PSC) Lines Although it was long recognized that donor-specific human pluripotent stem cells (hPSCs) would be a useful resource for personalized cell replacement therapies (CRT), technological challenges towards derivation of hPSC lines, coupled with the ethical issues associated with utilization of human embryos for derivation of human embryonic stem cells (hESCs) lines, limited our ability to develop these approaches. We here take a brief overview of the key technological advances that led to derivation of PSC lines in animal models and eventually to the derivation of hESC and induced pluripotent stem cell (iPSC) lines (Fig. 1). Embryonic Carcinoma (EC) Cultures Before development of technology to derive PSC lines from early stage embryos, teratocarcinomas were used as a model to study mammalian cell differentiation [1–4]. Teratocarcinomas are malignant tissues characterized by the presence of disorganized differentiated tissue representing three germ layers and undifferentiated stem cells. Progressive growth of

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Stem Cell Rev and Rep Fig. 1 Timeline of key breakthroughs leading to generation of human induced pluripotent stem cell (iPSC) lines

teratocarcinoma depends upon the presence of EC, and differentiation of these stem cells generates benign somatic derivatives. Tumor comprised of somatic tissue but lacking EC is not malignant and is called teratoma [2]. Klensmith and Pierce in 1964 showed that teratocarcinoma cells are capable of multipotential differentiation, as single cell grafts of mouse teratocarcinoma could produce clonal teratocarcinoma lines that exhibited varied degree of differentiation potential and could also form embryoid bodies (EB) [3]. The first pluripotent human EC lines were derived from teratocarcinoma in 1984, and were shown to exhibit multipotential differentiation profile [4]. With EC cells of teratocarcinoma exhibiting pluripotency features, these cells were used as a model to study the mechanism of cellular differentiation during embryonic development [1, 3, 4]. Derivation of Pluripotent Embryonic Stem Cell (ESC) Lines in Mouse Models Mouse ESC lines were derived in 1981 by two groups [5, 6]. In the first report, Evans and Kaufman retrieved mouse early blastocysts from the uterine lumen, following implantation delay induced by overiectomy that results in hatching of early blastocysts. The blastocysts when cultured adhered to the culture dishes and generated pluripotent stem cells lines that exhibited normal karyotype and formed EB in suspension cultures and teratomas in mice [5]. Authors utilized this approach to derive 15 PSC lines of different embryonic origins. Shortly thereafter, Gail Martin derived PSC lines from the inner cell mass (ICM) of late mouse blastocysts cultured on fibroblast feeder cells in the presence of EC lines derived condition medium, based on the hypothesis that the EC conditioned medium will provide necessary growth factors to the PSC progenitors until the PSC lines are established that can themselves produce these factors [6]. Early blastocysts flushed from the uterus of the impregnated mice were cultured in the presence of EC conditioned medium, facilitating adherence of the blastocysts and generation of PSC lines that did not require conditioned medium beyond the 15 passages, and could form EB in suspension cultures and teratoma when injected in mice [6].

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Mouse primordial germ cells (PGCs) were also used as a source of mouse pluripotent ESC lines in 1992 [7, 8]. PGCs originate from the epiblast and are found in 7.0 days post coitum mouse embryo as eight alkaline phosphatase positive cells that proliferate substantially in 6 days to generate 25,000 progeny cells [8]. It was known that Steel factor (SF), necessary for the survival of PGCs, in combination with leukemia inhibitory factor (LIF) could support proliferation of PGCs. The two groups showed that incorporation of basic fibroblast growth factor (bFGF) could help long term maintenance and proliferation of PGCs and generation of PSC lines that could form EB is suspension cultures and teratomas in mice [7, 8]. Derivation of Pluripotent ESC Lines in Primates While first mouse pluripotent ESC lines were derived in 1981, it was 1995 when first primate pluripotent ESC line was successfully generated by James Thomson from a 15 years old rhesus monkey [9]. Authors utilized 6 days post ovulation uterine flush retrieved azonal blastocysts in their study. The trophoectoderm was removed by immunosurgery by incubating the blastocysts with rabbit anti-rhesus anti-serum followed by exposure to gunea pig complement, and the intact inner cell mass (ICM) was then cultured on mouse embryonic fibroblasts (MEF) feeder cells in the presence of human LIF for 16 days, following which central mass of the clone was transferred on to fresh MEF to generate PSC line that resembled human EC cells. The PSC line generated was characterized by expression of pluripotency markers and teratoma formation assay [9]. Derivation of Human ESC Lines A major technological advance in human pluripotent stem cell research field was achieved by James Thomson group in 1998 by successful derivation of five human ESC lines from the ICM of cultured blastocysts from IVC clinic donated human embryos [10]. The cell lines qualified to be PSC by maintaining undifferentiated prolonged proliferation, expression of pluripotency markers, and by their ability to generate all the three

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embryonic germ layers [10]. Generation of hESC lines not only provided the opportunity to characterize the biology of hESC and develop methods to generate functional somatic cell lineages, it also fueled an ethical debate about utilization of human embryos for derivation of hESC lines, resulting in a moratorium on derivation of additional hESC lines. However, it also inspired efforts to identify the molecular factors that could program somatic cells into pluripotent state, resulting in technological breakthrough of derivation of donor-specific iPSC lines [11–14]. Derivation of PSC Lines by Somatic Cell Nuclear Transfer (SCNT) Approach Gurdon et al. pioneered the SCNT approach in amphibians in 1962 by showing that the nuclei derived from a somatic cell of frog, when transferred into an enucleated egg, could produce viable progeny [15]. However, it was 1996 when Ian Wilmut for the first time showed that the SCNT approach could be successfully utilized to produce a viable mammal [16]. Authors transferred the nuclei of an ESC line derived cell into an enucleated egg to produce a viable lamb [16]. Soon thereafter, authors reproduced their findings with the nuclear material derived from adult mammary gland, fetus and embryo-derived cell lines [17]. Their success proved that the genetic material of an adult mammalian somatic cell contains the complete genetic code and the enucleated egg contains all the accessory factors needed to successfully reprogram the somatic cell nuclei to advance through the developmental cycle, to not only produce viable offspring but also to derive donorspecific PSC lines to study disease mechanisms and develop personalized CRT. PSC lines were subsequently generated by SCNT approach in several other animal models, including mouse and the primates [18, 19], however, similar attempts in human system proved quite difficult, until 2013 when Tachibana et al. overcame underlying technical challenges [20]. Based on the observations that SCNT in nucleated human egg can generate blastocyst and the PSC lines, but the SCNT in enucleated egg cannot [21], and that the mitotic arrest of human metaphase II oocyte is unstable and removal of meiotic spindle results in completion of meiosis and thereby renders oocytes incapable of reprogramming the somatic cell nucleus to pluripotent state, authors slowed meiotic completion by adding caffeine and successfully derived nuclear transfer-PSC (ntPSC) lines [20]. Although SCNT had been reported in human cells before [22, 23], this was the first demonstration of ntPSC line derivation in humans. Reprogramming by ESC and Somatic Cell Fusion ESC have also been used to reprogram somatic cells by fusing them with somatic cells in mouse [24] as well as in human [25, 26]. These hybrid cells have been shown to differentiate into all the three germ layers, in-vitro [24–26], and

also contribute to development of chimeric embroys [24]. Despite having tetraploid chromosome, these hybrid cells could also differentiate into somatic cell lineage [26]. Although SCNT and ESC fusion approaches can be used to derive donor-specific PSC lines, it should be noted that since mitochondrial DNA comes from the enucleated egg, SCNT-derived PSC lines are not perfectly donor-specific. Requirement for human eggs for SCNT also makes it difficult for many laboratories to derive donor-specific PSC line with this approach. Ethical concerns associated with the utilization of human eggs for SCNT is another challenge. In this context, transcription factor-based somatic cell reprogramming is an effective approach to generate donor-specific iPSC lines. We will now discuss progress towards deriving iPSC lines by somatic cell reprogramming and derivation of somatic cell lineages from these iPSC lines. Identification of Reprogramming Factors and Derivation of Donor-Specific iPSC Lines Based on the findings showing that Oct3/4, Sox-2 and Nanog play essential roles in maintenance of early embryos as well as ES cells [27–30], findings showing that the genes associated with tumor cells, such as stat-3, E-Ras, c-myc and β-catenin, play critical role in proliferation and long-term maintenance of ES cells, and the identification of genes that are specifically expressed in ES cells [31–38], Shinya Yamanaka shortlisted a pool of 24 genes to identify the reprogramming factors that could induce pluripotency in somatic cells [39]. Authors expressed these genes in different permutation and combinations in a retroviral vector expressing the pluripotency-associated gene Fbx15 [39], with β geo insertion encoding for β-galactosidase and neomycin fusion genes. Since the ESC homozygous for β-geo knockin construct (Fbx15βgeo/βgeo) were G418 resistant while the somatic cells of Fbx15βgeo/βgeo mouse were sensitive to G418 selection, authors utilized this screening system to identify four genes, Oct3/4, Sox2, c-Myc and Klf-4, now known as “Yamanaka factors”, that could successfully reprogram mouse fibroblasts into iPSC lines that exhibited ESC like phenotype [11]. A year later, authors used the same four factors to successfully reprogram adult human fibroblasts to generate human iPSC lines [12], that was independently confirmed by Rudolf Jaenisch group [14]. Utilizing hESC engineered to express neomycin phosphotransferase gene under the control of endogenous Oct-4 promoter, providing gentamycin resistance to the reprogrammed cells expressing endogenous oct-4, James Thomson group independently identified a different combination of four reprogramming factors, Oct3/4, Sox2, Nanog and Lin-28, from a pool of 14 candidate genes, that could reprogram hESC-derived myeloid cells into iPSC lines [13]. Figure 2 outlines the technological differences between different methods of PSC line derivation. Derivation of human iPSC lines not only spearheaded efforts to generate donor-specific

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Fig. 2 Different routes to pluripotency. a Somatic cell nuclear transfer approach. b Human embryonic stem cell (hESC) line derivation. c Donor-specific induced pluripotent stem cell (iPSC) line derivation with defined reprogramming factors

somatic cell lineages of choice, it also provided tools to address questions of fundamental importance, such as the epigenetic profile of hPSC, mechanism of pluripotency development, and inherent immunogenicity of hiPSC lines, discussed in details below.

Methods for Derivation of Donor-Specific iPSC Lines Since the first report on iPSC line derivation from fibroblast cells different methods have been developed to reprogram several different types of somatic cells, including terminally differentiated human primary cells. We will now take a brief overview of some of these approaches, and discuss their relative advantages and disadvantages. Genome Integrating Vector-Based Methods to Derive iPSC Lines One of the key concerns in early studies on

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iPSC line generation studies was ensuring efficient delivery and expression of reprogramming factors in somatic cells that made genome integrating viral vectors, such as retro-viruses and lenti-viruses, preferred delivery vehicles. Yamanaka group utilized retrovirus-vectors to reprogram mouse and human fibroblasts [11, 12]. Authors introduced mouse receptor of retrovirus, Slc7a1, in human fibroblasts to improve transduction efficiency [12]. While primary requirement for retro-virus-mediated transdution is the proliferative state of target cells, lentiviral vectors can transduce somatic cells independent of their proliferative state. Thomson group utilized recombinant lentivirus to deliver the reprogramming factor genes in their study [13]. The need to use multiple viral vectors was overcome by the creation of lentiviral vector expressing a single “stem cell cassette” [40]. Interestingly, despite high infection rates, iPSC line derivation efficiency of both of these approaches was quite low, with ~ 0.001–0.03% of iPSC generation efficiency reported by Okita et al. [41] and ~ 0.01% by Yin et al. [13]. Besides

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the retrovirus and lenti-virus vectors, transposons have also been used to generate iPSC lines that can be subsequently excised out without leaving a trace in the genome. Transposon-mediated delivery of reprogramming factors involves inverted repeats flanking the transgene cassette, followed by transient expression of transposase enzyme for insertion and excision events. Utilizing the PiggyBac transposon system iPSC lines have been generated from both mouse and human fibroblasts, and the transposons were subsequently excised out [42–44]. Although genome integrating viral vector-based approaches provided proof of principal for iPSC generation technology [11–14], several concerns were raised with the clinical use of viral vector derived iPSC lines and their cellular derivatives. For example, retrovirus integration sites (RIS) in transduced cells is a random phenomenon and the integration is preferentially at the transcription initiation sites that could cause loss of function of gene at the integration site with unknown consequences, and reactivation of reprogramming factors with known oncogenic effect could also result in tumor development [41]. Takahashi et al. found ~ 20 RIS per iPS clone [11, 12] and Okita et al. reported c-myc reactivation in germline chimeras resulting in tumor development [41]. Although transposons can be excised out, multiple transposon integrations have been reported in transposon-derived iPSC lines [42–44] that could cause insertion-associated gene mutations with unknown effect. These safety concerns led to development of iPSC line derivation methods that do not utilize genome-integrating viral vectors. Non-genome Integrating Vector-Based iPSC Derivation Approaches Among these include methods utilizing recombinant plasmid, episome, RNA/DNA transfection, protein delivery, and methods utilizing non-integrating viruses, such as adenovirus and sendai virus, as delivery vehicles. Small molecules that can facilitate epigenetic remodeling have also been used for reprogramming. In addition, microRNAs (miRNA) have also been used to reprogram somatic cells. We here discuss some of these approaches, with their respective advantages and disadvantages. Plasmid-Based Methods Shinya Yamanaka group utilized recombinant plasmids to generate iPSC lines that met pluripotency quality control requirements, including pluripotency marker expression, teratoma formation and contribution to adult chimeras, however, the iPSC generation efficiency was quite low, as the authors reported 1–29 potential iPSC colonies from 1 × 106 infected cells against 100–1000 with retro-virus-based approach [45]. mRNA-Based Methods Synthetic mRNA encoding for Yamanaka factors have also been used to reprogram human

somatic cells [46]. A 5ƍ guanine cap was incorporated in the in-vitro transcribed mRNAs to improve their half life. The mRNAs were synthesized by incorporating modified ribonucleosides, for example by substitution of 5-methylcytidine (5mC) for cytidine or pseudouridine (psi) for uridine, to minimize the activation of innate immune mechanisms by these RNAs [46]. Comparison of reprogramming efficiencies by synthetic RNAs and retroviruses showed 0.04% reprogramming efficiency with retroviruses versus 1.4% efficiency with synthetic RNAs [46]. However, RNA modification requirements makes it a technically challenging approach for many laboratories. Episome-Based Methods Minicircle vectors are supercoiled DNA molecules that lack the bacterial origin of replication and the antibiotic resistance, and contain a eukaryotic expression casette. These vectors exhibit high transfection efficiencies and longer ectopic expression owing to lower activation of endogenous silencing mechanisms. While minicircle approach exhibited greater transfection efficiency than the plasmid-based approach, iPSC derivation yield was significantly lower (~ 0.005%) than the genome integrating virus-based methods (~ 0.01%) [47]. Adenovirus-Based Methods Towards developing iPSC line derivation methods that do not utilize genome integrating viruses but still offer efficient delivery and expression of the reprogramming factors, replication incompetent adenoviruses were used [48]. Although recombinant adenoviruses expressing reprogramming factors exhibited very high infection rates, with individual vectors infecting 70–80% of the target cells and 50–60% target cells expressing all the four reprogramming factors, overall iPS generation efficiency was quite low (0.0001–0.001%) in comparison to the integrating virus-based methods (0.01–0.1%). Interestingly adenovirusderived iPSC lines also contained tetraploid lines that was not reported for the retro- or the lenti-virus-derived iPSC lines. Sendai Virus-Based Methods Sendai virus (SeV) or the murine parainfluenza virus type I is a negative strand RNA virus that replicates exclusively in the cytoplasm without chromosomal integration, causes severe airway inflammation in mice, and is non-pathogenic in humans. SeV requires brief contact for cellular uptake and offers a strong expression of foreign genes. These features make it an attractive tool for programming somatic cells into iPSC lines [49]. We have recently used this method to successfully reprogram terminally differentiated human peripheral blood-derived dendritic cells, and have used these iPSC lines to characterize the inherent immunogenicity profile of human iPSC lines [50], discussed in details later.

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Protein Transduction-Based Methods It is well known now that certain short peptide tags, for example the HIV TAT and poly-arginine, can be used as carriers of tagged proteins to transduce target cells. Reprogramming factors tagged with poly-arginine have been successfully used to generate iPSC lines [51]. This approach is unique in a sense that it eliminates the need for any genetic material (DNA or RNA) to program somatic cells. MicroRNA-Mediated Reprogramming MicroRNAs are ~ 22 nucleotide long small RNAs that play crucial roles in post-transcriptional regulation. Dicer deficient mice fail to develop, and the ESC deficient in dicer are compromised in their ability to produce microRNAs and exhibit defects in proliferation and differentiation [52, 53]. A subset of microRNAs are preferentially expressed in the ESC [54]. Towards examining the role of microRNAs in pluripotency development, miR-294 was shown to substitute c-Myc in OSMKmediated reprogramming [55]. Interestingly, miR302/367 cluster in combination with HDAC blockade has been used to achieve direct reprogramming by activating Oct-4 expression [56]. Gurber et el. showed that ESC-specific microRNAs contribute to pluripotency by inhibiting regulators of differentiation pathways [57]. Small Molecule-Mediated Reprogramming As histone acetyltransferase (HAT) and DNA methyltransferase (DNMT) inhibitors were earlier shown to improve the efficiency of SCNT [58–60], effect of small molecule epigenetic modulators, in conjunction with the reprogramming factors, was examined on the efficiency of iPSC line derivation. Since neuronal stem cells (NSC) express higher levels of endogenous Sox-2 and c-Myc, Kim et al. [61] and Shi et al. [62] examined whether NSC could be reprogrammed without the exogenous SOX-2 and c-Myc, and showed that Oct-4 and Klf-4 were sufficient to reprogram these cells [61, 62]. In addition, Shi et al. found that the incorporation of BIX01294, a small molecular inhibitor of G9a histone methyltransferase that epigenetically inactivates Oct-4 [63], could significantly improve iPSC derivation efficiency of Oct-4 and Klf-4 [62, 64, 65]. It is interesting to notice that the iPSC derivation efficiency of all the above discussed methods is significantly low, despite ensuring efficient delivery and expression of reprogramming factors, which could be due to the requirement of complete epigenetic remodeling of the somatic cell genome for development of iPSC lines, as partial remodeling would result in genomic instability and cell death. Of the above discussed methods, non-genome integrating methods are recommended for derivation of donor-specific iPSC lines to avoid potential challenges associated genome integrating vector-derived iPSC lines. In this context, we have found sendai virus-based method to be quite efficient, as it ensures

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efficient delivery and expression of the reprogramming factors and easy removal of the vector during the course of iPSC passaging [50].

Methods for Characterization of PSC Lines Methods used to characterize the pluripotency profile of ESC lines have also been used to validate the quality of iPSC lines. Among these includes expression of pluripotency markers, ability to form EB and teratomas, and their ability to contribute to chimeric animal development, (Fig. 3). Pluripotency Markers Expression Expression of pluripotency markers such as nuclear transcription factors Oct4, Sox-2 and Nanog, keratin sulfate antigens Tra-1-61 and Tra-1-81, and the glycolipid antigens SSEA-3 and SSEA-4, is routinely used to characterize the pluripotency profile of hESC and iPSC lines, using several different techniques, such as RT-PCR, Q-RT-PCR, FACS, immuno-histochemistry and gene chip analysis etc. [50]. Since Oct-4, Sox-2 are also used for inducing reprogramming, expression of non-coding regions of these markers is used to verify that the expression of these biomarkers is due to endogenous expression, and not from the transgene encoded coding regions delivered to the cells by different delivery vehicles [50]. Updated versions of these methods examine expression of a panel of pluripotency and differentiation associated genes to validate the pluripotency profile of PSC lines, using Q-RT-PCR [50], gene chip analysis or next-generation sequencing approaches. Karyotype Analysis Genome integrity of the hESC and iPSC lines is characterized by their karyotpe analysis [50]. Karyotyping is the analysis of chromosome numbers and morphology. G-banding is a common technique for karyotype analysis that involves Geimsa staining of nuclear preparations of metaphase stage cells. Other methods utilized for karyotyping include R-banding (reverse Giemsa staining), C-banding (constitutive heterochromatin staining), Q-banding (quinacrine staining) and T-banding (telomeric staining) etc. Careful analysis of distinct banding patterns in different chromosomes of cells is used to identify genomic abnormalities. It is recommended that the PSC lines are karyotyped periodically over long term culture to verify their genome integrity. Alkaline Phosphatase Expression Analysis Expression of alkaline phosphatase is used as a marker of pluripotency [50, 66, 67]. However, it should be noted that human alkaline phosphatase has several isoforms that are expressed in different cells and tissues, and it is the placental human alkaline

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Fig. 3 Methodologies for characterization of PSC lines. a–d characterization of pluripotency profile. a Characterization of expression of pluripotency markers by immunofluorescence staining (i), FACS (ii) and RT-PCR (iii). Expression of endogenous pluripotency markers is also done in RT-PCR analysis to rule out amplification from exogenously introduced reprogramming factors. b Alkaline phosphatase staining in PSC lines. c Karyotype analysis of PSC lines. d Chimera complementation assay, whereby PSC lines are incorporated into early stage embryo and ability of PSC lines is examined for line-

age complementation in the progeny. e–h Characterization of differentiation potential of PSC lines. e Embryoid Bodies (EB) formation. f Characterization of EB by germ layer associate marker expression. g generation of hematopoietic stem cell (HSC) precursors in hPESC derived EB. Shown is FACS analysis of CD34 + CD38 + HSC precursors in hPSC-derived EB, before and after magnetic bead-mediated enrichment method. h Characterization of the differentiation potential of hematopoietic stem cell (HSC) precursors generated in hPESC derived EB in CFU assay

phosphatase that correlates with pluripotency. Therefore, expression of alkaline phosphatase is an indicative marker and not a confirmatory marker of pluripotency.

Embryonic Chimera Formation One of the criteria for determining the pluripotency of iPSC lines in animal models is the embryonic chimera formation, whereby ESC and iPSC lines are incorporated into pre-implantation embryos to confirm that these PSC lines can contribute to the development of chimeric animals [68]. During the iPSC derivation schema, phenotypic identification of PSC like colonies is indicative of successful reprogramming that should then be confirmed by alkaline phosphatase expression and pluripotency markers expression analysis. As mentioned before, several advanced versions of these basic methods have now been developed that examine

Embryoid Body and Teratoma Formation Analysis The differentiation potential of hESC and iPSC lines is characterized by their ability to form EB, in-vitro, and teratoma in mice [50]. PSC-derived EB are characterized for the expression of ectoderm, mesoderm and endoderm-associated biomarkers, and the teratomas are characterized by immuno-histochemistry analysis for characterization of tissues associated with three germ layers [50].

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the expression of a panel of genes, associated with pluripotency and differentiation state of cells, to verify complete reprogramming of iPSC lines generated [50]. While formation of EB, expressing three germ layer markers, and the formation of teratoma in animals are efficient methods to validate the differentiation potential of iPSC lines, contribution to chimeric animals should also be performed to further validate the pluripotency status of iPSC lines.

Molecular Mechanism of Reprogramming Successful reprogramming of somatic cell nuclear material by SCNT as well as by introduction of reprogramming factors has demonstrated that the somatic cell differentiation is not an irreversible process, and that the somatic cell nucleus has all the genetic information necessary for achieving pluripotency, provided a set of pluripotency initiators

Fig. 4 Molecular mechanism of reprogramming. a Changes in organization of chromatin in nucleus during cell cycle. The DNA of a cell is packaged inside nucleus in a DNA–protein complex called chromatin. The chromatin reorganizes in chromosomes before mitosis, and is faithfully copied into daughter cells. b Reprogramming results in silencing of lineage associated genes while activation of pluripotency

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are available. Although efficient delivery and expression of reprogramming factors was initially believed to be the key limiting factor for successful reprogramming of somatic cells, efficiency of iPSC line derivation from all the above methods is quite low, in comparison to the percentage of somatic cells showing expression of reprogramming factors. The reason for this is that reprogramming of somatic cells has to achieve complete reorganization of the nuclear material, whereby lineage-specific transcription factors and genes are silenced and the transcription factors associated with pluripotency development and maintenance are systematically turned on, as shown in Fig. 4. We will now discuss the genetic and epigenetic mechanisms involved in development and maintenance of pluripotency. Genetic Regulators of Pluripotency Development and Maintenance As discussed above, the two original reprogramming factor cocktails used to generate iPSC lines were a combination of Oct-4, Sox-2, Klf-4 and c-Myc [11, 12],

associated genes, by epigenetic rewiring mediated by site-specific acetylation and methylation of histones in the chromatin. The histone octamers in permissive regions of the genome are shown in green color while the histone octamers in silent regions of the genome are shown in red color

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or Oct-3/4, Sox-2, Nanog and Lin-28 [13]. We will now briefly discuss the roles these factors play in development and maintenance of pluripotency. Oct-4 Oct-4 is a member of octamer-binding family of transcription factors that is expressed in all totipotent cells in mouse and human early stage embryos, and its expression is subsequently restricted to the inner cell mass (ICM) that produces pluripotent stem cells and thereafter to the germ cells where it supports their survival [69, 70]. Oct-4 regulates the expression of pluripotency-associated genes [71], and Oct-4 knockout embryos fail to produce ICM and die at the blastocyst stage [27]. However, Oct-4 over-expression as well as low-expression leads to differentiation, as less than two-fold increase in Oct-4 expression results in differentiation of ESC into primitive endoderm and mesoderm, and its repression causes differentiation towards trophectoderm, suggesting that a tightly regulated level of Oct-4 is critical for development and maintenance of pluripotency [28]. While Oct-4 is part of the core pluripotency circuit, comprised of Oct-4, Sox-2 and Nanog [71], and it was an essential component of both the original iPSC derivation cocktails, small moleculemediated chemical and genetic reprogramming has been shown to generate iPSC lines without Oct-4, albeit at very low efficiency levels [62]. Sox-2 Sox-2 is a member of SRY-related HMG box family of transcription factors that plays a critical role in maintenance of pluripotency [29]. While Sox-2 is expressed in the ICM, germ cells and the epiblast of early embryos, it is also expressed in the extra-embryonic ectoderm as well as neural precursors [29]. Mouse embryos lacking Sox-2 die at the implantation stage, due to failure to form epiblast [29]. Sox-2 is also a part of the core transcriptional regulatory circuit of pluripotency, and it can regulate the expression of Oct-4 and also cooperate with Oct-4 to control the expression of pluripotency-associated genes [70–72]. Klf-4 Klf-4 is a member of Kruppel-like factor-4 family of zinc finger transcription factors that is expressed in several different tissues and it plays critical roles in a variety of physiological processes, including cell proliferation, differentiation and apoptosis [73]. Klf-4 can function as transcription activator or suppressor in a context dependent manner, and it can also function as a tumor suppressor or an oncogene. Mice lacking Klf-4 develop normally, but they die shortly after birth due to skin barrier function defect [74]. Klf-4 binds to Nanog promoter region and modulates its transcription [75, 76], however, Klf-4 is not indispensible for pluripotency development as Thomson group generated iPSC lines in the absence of Klf-4 [13].

c-Myc c-Myc is a multi-domain transcription factor that has been associated with regulation of around 15% genes from flies to humans, that include genes associated with cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis and mitochondrial function, and it plays a critical role in regulation of a wide variety of physiological processes, including cell proliferation, growth as well as differentiation [77]. In addition, it can also exert its effect by regulating microRNAs [78]. With regards to pluripotency development and maintenance, it is believed that c-Myc contributes by facilitating proliferation of somatic cells, as proliferation supports reprogramming via global chromatic architecture remodeling [79, 80]. It should also be noted that c-Myc is a well known oncogen that is up-regulated in ~ 70% of cancers, and concerns have been raised about its use in reprogramming cocktails, especially in methods that utilize genome integrating viral vectors. However, these concerns can be easily addressed as c-Myc is not indispensable for reprogramming, and iPSC lines can be derived by transient expression of c-Myc [50] or even without the use of c-Myc [13]. Nanog Nanog is present in mouse and human PSC lines, and it is absent in differentiated cells [30]. Nanog was shown to be critical for maintenance of pluripotency in mouse epiblast and ES cells, as it facilitates ES cell self-renewal in the absence of leukemia inhibitor factor (LIF) or Stat-3, Nanog deficient ICM fail to form epiblast, and Nanog deficient ES cells loose pluripotency and progress towards differentiation into extra-embryonic endoderm lineage [30, 81]. Although Nanog was not part of the reprogramming cocktail used by Yamanaka group, it is part of the core pluripotency transcription circuit. Nanog can activate the expression of Oct-4, and down-regulation of Nanog expression up-regulates the expression of trophoectoderm-associated transcription factors, GATA4, GATA6 and Cdx2, suggesting that Nanog is essential for the maintenance of pluriotency by regulating differentiation mechanisms [82]. Lin-28 Lin-28 and Nanog, instead of Klf-4 and c-Myc, were used in combination with Oct-4 and Sox-2 by Thomson group to generate iPSC lines. Lin-28 is expressed in nematodes during first larval stage and it is down-regulated thereafter by microRNAs to regulate the developmental timing [83]. Lin-28 harbors RNA-binding domains, and it is present in processing bodies or the P-bodies, the sites of mRNA degradation and microRNA regulation [84]. Lin-28 is believed to contribute to pluripotency by maintaining the stability of RNA transcripts. As discussed above, Oct-3/4 and Sox-2 are part of the the core pluripotency transcription factors, as they not only activate the expression of endogenous Oct-3/4 and Sox-2 genes, but also of other factors associated with pluripotency

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maintenance, such as Nanog. Klf-4, c-Myc contribute towards pluripotency development by facilitating cell proliferation, while Lin-28 participates either by fine-tuning the expression of these accessory molecules, modulating cellular proliferation or by facilitating chromatin reorganization [85]. It should also be noted that although the reprogramming factors identified in mouse are also sufficient to reprogram human cells, significant differences have been identified in human and mice pluripotency requirements. For example, BMP-4 in humans facilitates differentiation of hPSC lines into trophoblast lineages [86], while in mice it is required for pluripotency maintenance [87]. Therefore, additional work is required to fully comprehend the similarities and differences in the biology, pluripotency maintenance requirements, and the differentiation potential of human and mice iPSC lines. Epigenetic Regulation of Pluripotency and Differentiation The DNA of a cell accounts for more than six billion base-pairs that can stretch to about two meter in length; however, it is condensed inside the nucleus that measures merely a few micrometers, by means of coiling and super-coiling in a DNA–protein complex called chromatin. The basic unit of chromatin is nucleosome that is comprised of histone octamer, comprised of two copies each of H2A, H2B, H3 and H4 histones, wrapped around by 146 bp of DNA. Chromatin is further characterized as heterochromatin and euchromatin, for “silent” and “active” part of the genome. Histones play an essential role not only in the packaging of the DNA, but also regulate the accessibility of the transcription machinery to the DNA by means of post translational modifications, such as site-specific acetylation and methylation by DNA methyltransferase (DNMT) and histone acetyltransferase (HAT) enzymes. Histone acetylation is regarded as the permissive signal for gene expression, while methylation can be permissive, e.g. histone-3 lysine-4 trimethylation (H3K4me3) or repressive, e.g. histone-3 lysine-9 trimethylation (H3K9me3), histone-3 lysine-27 trimethylation (H3K27me3) and histone-4 lysine-20 trimethylation (H4K20me3), depending upon the methylation site. DNA methylation on the other hand is considered a repressive signal. The chromatin organization and the epigenetic profiles of PSC and differentiated cells are quite distinct [88–91]. Utilizing fluorescent recovery after bleaching (FRAP) Meshorer et al. found that the steady state chromatin of mESC is hyperdynamic, such that linker histone H1 and the core histones H2B, H3 and H3.3 are loosely attached to the chromatin with shorter residency times, unlike the differentiated neural progenitors [88]. Authors also found that the mESC cells lacking nucleosome assembly factor, HirA, form EB faster, while the mESC expressing H1 histone that exhibit stronger chromatin binding fail to differentiate into neurons, suggesting that the hyperdynamic chromatin is a

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sign of plasticity of PSC [88]. Localization of heterochromatin protein 1 (HIP1α) and H3K9me3 revealed that the mESC harbor large diffused heterochromatin foci, while differentiated cells have smaller but more tightly organized heterochromatin foci, a sign of global chromatin reorganization during differentiation of mESC [88]. Bernstein et al. characterized the status of H3K4 and H4K27 methylation patterns, the permissive and repressive methylation marks, in ~ 2.5% of the total genome, representing most highly conserved noncoding element (HCNE) regions that are gene poor but enriched in developmentally important transcription factors (TF), in mESC and differentiated cells using chromatin immunoprecipitation-sequencing (CHIP-Seq) technique [89]. Authors found both of these marks in three quarters of the K27 marked areas in mESC, while the differentiated cells were marked with either permissive K4 or repressive K27 marks. These marks were mostly comprised of large K27 marks harboring smaller K4 marks, named “bivalent marks”, that coincided with the differentiation genes that were expressed at very low levels, suggesting that the bivalent signatures keep these genes silent but poised for activation upon differentiation [89]. Simultaneous presence of permissive (H3K9 and H3K4) and repressive (H3K27) methylation marks in mouse ESC was independently confirmed by Azura et al. [90]. Polycomb proteins that are the key development regulators have been found localized at the development regulated genes marked by H3K27me3 marks, suggesting their involvement in pluripotency maintenance [92]. The importance of epigenetic remodeling in pluripotency development is also supported by findings that the epigenetic modulators can successfully facilitate reprogramming in the absence of one or more reprogramming factors [62, 64, 65]. Interestingly, iPSC lines derived from different somatic cells have been shown to inherit lineage-specific epigenetic marks termed as “epigenetic memory”, unlike the iPSC lines generated by nuclear transfer approach, and these iPSC lines have been shown to exhibit differential differentiation profiles [93, 94].

PSC-Based CRT: Recent Advances and Prevailing Challenges Several different types of functional somatic cells have been generated from hESC and iPSC lines, including neurons, blood cells, cardiomyocytes and the pancreatic beta cells [50, 95]. While reviewing progress in all these areas is beyond the scope of this manuscript, we here briefly discuss recent progress towards generating functional immune cells from hPSC lines, progress towards advancing PSC-based CRT to the clinic, and prevailing challenges towards developing safe and effective PSC-based CRT.

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Progress Towards Developing PSC-Based CRT DC, effector T cells and natural killer (NK) cells are some of the key immune cells that are essential for development of protective cellular immunity. A significant progress has been made towards generating functional immune cells from hESC and iPSC lines. While Zhan et al. were first to generate functional human DC-like cells from hESC-derived EB [96], we have also generated functional iPSC-APC from human DC-derived iPSC lines that exhibit potent phagocytic potential and can present antigenic peptides to human tumor antigen-specific as well as influenza-specific T cells [50]. Donor-specific immunogenic iPSC-APC would have significant translational implications for development of vaccines against life threatening diseases, such as cancers, while tolerogenic iPSC-APC will be useful for the treatment of autoimmune diseases. T cells are essential for providing protective cellular immunity and several different types of vaccines against infectious diseases as well as cancers have been developed for optimal priming of host T cell repertoire. Customized anti-tumor T cells have also been recently generated by engineering human peripheral blood-derived T cells with tumor antigen-specific T cell receptor (TCR) and chimeric antigen receptors (CAR), and these approaches have produced remarkable clinical outcomes [97–101]. Derivation of donor-specific naive T cells with customized functional profiles derived from donor-specific iPSC lines would have significant translational implications, since mature T cells express TCR of their primary functional specificities and engineered mature T cells might have undesired side effects in patients [98]. Galic et al. were first to show that hESCderived HSC engrafted in SCID-hu (thy/liv) mice can generate functional T cells [102]. Donor-specific iPSC lines have also been used to generate anti-tumor T cells with TCR and CAR-based approaches [103–105]. Functional NK cells have also been generated from hPSC lines [106–109] that express NK cell-associated inhibitory and activating receptors, exhibit cytolytic function and cytokine production [110], and also exhibit in-vivo anti-tumor response [111]. In addition, functional platelets have also been derived from the hESC and donor-specific iPSC lines [112–115]. Significant progress has also been made towards advancing hESC and iPSC-based CRT to clinical settings. Geron Corporation was first to initiate hESC-based clinical trial for the treatment of spinal cord injury in 2010, however, the study was prematurely terminated because of financial reasons [116]. Since eye is an immune privileged site, a significant progress has been made towards using PSC-derived cells for the treatment of macular degeneration conditions [117]. Among these, hESC-derived retinal pigment epithelium cell-based clinical trials in patients with age-related macular degeneration (AMD) and Stargardt`s macular dystrophy represent the first published work administering

hPSC-derived cells in nine patients, reporting no evidence of adverse response or rejection and improved vision-related quality of life measures [118, 119]. Pancreatic endoderm cells derived from hESC lines have been used to generate pancreatic cells, including insulin secreting pancreatic-beta cells that can regulate blood glucose levels in mouse [120], and a clinical trial utilizing hESC-derived pancreatic endoderm cells is also underway [116]. Patient-derived iPSC lines have also been used to develop drug screening platforms to identify new drug targets for diseases such as Alzheimer and Amyotrophic lateral sclerosis (ALS), that is quite challenging due to the unavailability of cells exhibiting disease phenotype [95, 121, 122]. The iPSC-derived motor neurons have helped identify the molecules that can inhibit microglial neurotoxicity [122]. Motor neurons generated from the iPSC lines of familial ALS patient with mutations in Tar binding protein-43 (TDP-43) have been shown to form cytosolic aggregates as observed in postmortem tissues from ALS patients and these neurons have been used to rescue the abnormal phenotype in a chemical screen [123]. Figure 5 schematically outlines some of the PSC-derived functional cells and PSC-based therapeutic strategies. Challenges Towards Developing PSC-Based CRT While these advances are encouraging, several challenges have alos been identified that need to be addressed to develop safe and effective PSC-based CRT. Among these, generation of iPSC lines with stable pluripotency profile is paramount. Although initial iPSC derivation studies utilized integrating viral vectors as delivery tools and fibroblasts remain preferred somatic cells for generating donor-specific iPSC lines, as discussed before, several different types of methods are now available to generate iPSC lines without the use of genome integrating vectors from different types of somatic cells, including terminally differentiated immune cells [50]. These iPSC lines offer opportunities to systematically characterize the differences in the genome stability, inherent immunogenicity, and the differentiation profiles of the iPSC lines derived from different somatic cell sources, since iPSC lines derived from different somatic cell sources have been shown to harbor somatic cell memory and exhibit differentiation bias [93, 94]. Incorporation of non human sialic acid in hESC lines cultured on MEF that could result in immunogenicity of these lines [124], led to development of feeder free culture methods, whereby hPSC lines are cultured on specific matrices, such as Matrigel. However, lotto-lot variability in its ability to support hPSC and reports of presence of mouse viruses in some Matrigel lots have highlighted the need to develop culture methods with defined chemical components [125]. Characterization of inherent immunogenicity of iPSC lines and iPSC-derived somatic cell lineages is also essential,

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Fig. 5 Schema showing some of the PSC-based therapeutic approaches. Functional somatic cell lineages, for example neurons and immune cells, derived from donor-specific iPSC lines can

be used for cell replacement therapies (CRT). Cells derived from patient-specific iPSC lines could be used for drug screening, for example neurons from Alzheimers and ALS patients

given that the mouse iPSC lines, especially the iPSC lines generated by integrating virus-based approaches, have been shown to be inherently immunogenic [126], although these findings have been subsequently challenged by reports showing no or minimal inherent immunogenicity in syngenic mouse iPSC lines [127, 128]. Towards characterizing the inherent immunogenicity of human iPSC lines, we have recently generated iPSC lines from human peripheral blood-derived terminally differentiated DC and found that the innate and adaptive immune mechanisms are effectively shut down in these iPSC lines [50, 129]. However, further work is needed to address the issue of inherent immunogenicity of human iPSC lines and their cellular derivatives. These studies are essential to identify the best cell source and the iPSC-derivation method to generate donor-specific iPSC lines exhibiting little or no inherent immunogenicity.

hESC lines and reprogramming of somatic cells into iPS lines. Significant progress has been made towards characterizing the underlying mechanism behind reprogramming, derivation of donor-specific somatic cell lineages, and characterization of their molecular, cellular and functional profiles. Progress towards advancing PSC-based CRT to the clinic is also quite encouraging. However, further studies are needed to identify best somatic cell source and iPSC-derivation strategy to generate iPSC lines with stable genomic and epigenomic profiles and exhibiting little of no inherent immunogenicity, since iPSC lines derived with different reprogramming methods and from different somatic cell sources can harbor somatic cell memory and exhibit differentiation bias. Detailed characterization of the molecular, cellular and functional profile of cellular derivatives derived from these iPSC lines is also essential to develop best strategy to derive functional somatic cell lineages. This is especially important for the iPSC-derived functional immune cells, as iPSC lines and their cellular derivatives are not expected to be inherently immunogenic, however, their cellular derivatives are at the same time expected to be fully functional, that in case of immune cell lineages is orchestration of their effector function profiles. In this context, lineage

Conclusions The field of regenerative medicine has witnessed several key breakthroughs in last two decades, including derivation of

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specific dedifferentiation and re-differentiation models could be useful. For example human DC-dedifferentiation and redifferentiation model has helped characterize the immunogenicity pathways in these iPSC lines using autologous DC as control, and it could also be useful in characterizing the functional profile of human iPSC-APC derived from these iPSC lines, without the underlying concerns associated with use of iPSC line from a different somatic cell source. These studies, together with findings from the ongoing hPSC-based clinical trials, could be very useful in devising best strategies to develop safe and effective CRT approaches against conditions that require administration of iPSC-derived cells at non-immune privileged sites. In summary, the progress in the PSC-based regenerative medicine field in last decade has been very encouraging, however, the foundation laid by these studies offer hope that the next decade is going to be even more exciting. Acknowledgements Arvind Chhabra conceived and wrote the manuscript. Arvind Chhabra made the figures, in part using the Servier-Medical Art Slides. Author offers heartfelt gratitude to authors of the manuscripts that are cited here and many more that could not be included because of the space limitations. Author also thanks Deepika Batra and Feny Rasania for help in the preparation of the manuscript. This work was supported by grants from the State of Connecticut Regenerative Medicine Program (10-SCA-23 and 13-SCB-05). Compliance with Ethical Standards Conflict of Interest The author has no financial or otherwise conflict of interest associated with this study to disclose.

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