Regenerative capacity of mammals is limited and can rarely regenerate a specific organ or tissue fully. Due to these limitations, regenerative medicine seeks efficient and safe cell sources for regeneration of damaged tissues and organs or treatment for incurable diseases. Human embryonic stemcells (HESCs) hold two important properties called self renewal and pluripotency. However, the use of embryonic pluripotentstemcells in cell therapy faces two major obstacles. First, immunological incompatibility of ES cells with the recipient, and the second, ethical concerns about the destruction of human embryos during the ES cells. Thus, induction of somatic cells of individuals can be a proper way to overcome these problems. So far, several methods have been utilized to induce Pluripotency in Somatic cells. One of these methods is the technology of inducedpluripotentstemcells (iPS) in contribution with Pluripotency factors. Yet, the use of these cells in the clinic, owing to application of viral vectors to transfer Pluripotency inducing factors, is quite limited. Therefore, recognition of a combination of small molecules to be replaced with exogenous factors is the ultimate goal of the study for the purpose of generating iPS cells. Recent progresses in development of iPS cells will be discussed here.
Certain growth factors, Wnt-3a being a notable example, are well known to act both stimulatory and inhibitory to cardiomy- ocyte differentiation depending on the application time during the differentiation process . We found that FGF-10 only enhanced cardiomyocyte differentiation when applied at day 2 or day 4 (Figure 2A, and 2B), which coincided with the expression pattern of the FGF-10 gene (Figure 1C). Also, the stimulatory effect of FGF-10 was concentration-dependent, and immunoblotting confirmed the increased expression of three structural proteins cardiac tropomyosin (Tpm), cardiac troponin-T (cTnT), and cardiac troponin-I (cTnI) (Figure 2B, 2C and 2D). EGFP-positive cells were observed to have more synchronized beating in the FGF-10 treatment group through real-time imaging of the contractile embryoid bodies (Video S1, S2, S3, S4) as well as a more obvious sarcomeric structure through confocal imaging using antibodies against cTnI, cTnT, and Tpm (cTnI data shown in Figure 2G, other data not shown). Our data showed that the
Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage, which are restricted to form only sperm and eggs following their specification from pluripotent epiblast cells. Evidence suggests that Blimp1 is the key determinant of germ cell specification as it initiates the germ cell-specification program in pluripotent epiblast cells at embryonic day (E) E6.5–7.5 . Furthermore, the Blimp1/Prmt5 complex plays a decisive role not only during specification of founder population of PGCs, but also thereafter in maintenance of early germ cells as they migrate into the developing gonads between embryonic day (E) 8.5–11.5 . It is precisely during this interval that PGCs can be induced to dedifferentiate into pluripotent embryonic germ (EG) cells in vitro when exposed to exogenous signaling molecules, LIF, FGF-2 and SCF [3–5]. While PGCs show expression of some key pluripo- tency-specific genes, they differ significantly from EG cells in being monopotential, and unlike EG cells, they are incapable of participating in chimeras when introduced into blastocysts . The process of dedifferentiation of PGCs into EG cells signifies the reversal of their developmental program, which may provide insights into how a phenotypically and developmentally restricted group of cells can undergo dedifferentiation into self-renewing pluripotentstemcells.
Disease modeling with iPSc has several distinct advantages. First, iPSc lines have virtually no expansion limit and therefore provide an inexhaustible source of cells for study. Second, the mutation causing the disease is naturally present in the original cells reprogrammed; this precludes the need for complex procedures to create the genetic defect and avoids potential secondary effects of the genetic engineering approach chosen. Third, under the right conditions, iPSc can differentiate into virtually any cell type in vitro, providing a source of disease relevant cells. In the case of GD, a number of protocols have been developed that allow differentiation to monocytes/macrophages and several subtypes of neurons [95,98,105]. Finally, the cells are of human origin and species specific differences are avoided. The combination of these four advantages makes iPSc disease modeling a powerful preclinical model for study of basic pathogenic mechanisms and development of potential therapeutic, and an important complement to animal model systems . In recent years, the GD experimental paradigm has shifted somewhat from mouse models to iPSc models. This shift has allowed new insights into a number of aspects of GD molecular and cellular pathology, including, as described throughout this review, electrophysiology, calcium signaling, inflammatory response, autophagic flux, lysosomal defects and, importantly, α-synuclein accumulation and the mechanistic relationship between GD and the Parkinson’s related synucleinopathies. In particular, iPSc modeling may prove useful to finally answer the question of how much of the GD phenotype is cause by loss of function vs. gain of function mechanisms.
It is widely assumed that autologous iPSCs and their derivatives should be immunologically tolerated by the recipient. However, this dogma was challenged by a study showing T-cell-dependent immune rejection of syngeneic mouse iPSCs (miPSCs) following transplantation , in which miPSCs derived via the episomal approach were less prone to immune-mediated attack than those generated using viral vectors. Another study showed that short-term immuno- suppression by inhibiting leukocyte co-stimulatory molecules promoted engraft- ment of embryonic andinducedpluripotentstemcells . Recently, it was reported that low immunogenicity of less immunogenic cells could be retained after cell reprogramming and further differentiation . Nevertheless, another finding has demonstrated limited or no immune response including T cell infiltration in tissues derived from autologous iPSCs or allogenic ES cells . Interestingly, it was shown that hiPSCs-derived CD34 + hematopoietic progenitor
Figure S1 Characterization of human induced pluripo- tent stemcells. a, Primary human keratinocytes (PHK) derived from skin biopsy were reprogrammed to iPSC with a modified version of our ‘hit and run’ vector and subsequently infected with Adeno-Cre to remove the vector. PCR primers specific to lentiviral DNA were used to determine whether polycistronic reprogramming factor sequences in individual hiPSC clones were successfully deleted. PCR primers for endogenous genomic DNA were used as controls. b, hiPSCs growing in the plates were tested for alkaline phosphatase expression. c, Primary human keratino- cytes (PHK), hESC (H1), and reprogramming factor-free hiPSC lines(hiPSC-19 and hiPSC-21) were examined for expression of pluripotent markers (KLF4, NANOG, OCT4, SALL4, SOX2, ZFP42, and UTF1) by nCounter analysis (NanoString Technol- ogies). Expression levels of each gene in H1 cells were set to 1, and gene expression levels in PHK and hiPSCs were compared to H1. d, Teratomas were formed by injecting hiPSC-19 and hiPSC-21 cells into the dorsal flanks of NSG mice. The tumors were removed after 8 to 12 weeks and histological sections demonstrat- ed tissues derived from all three germ layers.
Cells in the hypoblast (endoderm) at the cephalic margin of the disc form the anterior visceral endoderm that expresses head-forming genes, including Orthodenticle Homeobox 2 (OTX2), LIM homeobox 1 (LIM1), and HESX Homeobox 1 (HESX1) and the secreted factor Cerberus. These genes establish the cranial end of the embryo before gastrulation. In initial somithogenesis, at the posterior notochord end a structure, called node, the loss of the bilateral symmetry in the left to right direction (L>R) begins. (13,14) The information for the LR, generated in the node begins to propagate to the mesodermal tissue at the periphery of the embryo, the lateral plate mesoderm (LPM), which will trigger an asymmetric cascade of LR gene expression. Nodal, a member of the TGF-β family of genes, is then activated and initiates and maintains the integrity of the node and streak. Once the streak is formed, a number of genes regulate formation of dorsal and ventral mesoderm, head and tail structures. Bone morphogenetic protein 4 (BMP-4) in the presence of fibroblast growth factor (FGF), ventralizes mesoderm during gastrulation and so that it forms intermediate LPM. Chordin, noggin, and follistatin antagonize BMP-4 activity and dorsalize mesoderm to form the notochord and somitomeres in the head region. These structures formation in more caudal regions is regulated by the Brachyury (T) gene. (13–17)
The ROCK inhibitor pretreatment on the FBS+DMSO formulation showed promising results. An improvement of 100% (CM monolayers) and 44% (cardiospheres) on metabolic activity recovery was observed when ROCK inhibitor pretreatment was performed using this medium formulation. Previous work has stated that ROCK inhibitor enhances cell –cell adhesion and cell aggregation by modulating gap junctions, thereby blocking the pathway to apoptosis (reviewed in ). In addition, the improvement observed with ROCK inhibitor pretreatment may also be related to the ability to avoid anoikis, which is a subtype of apoptosis induced by the loss of cell adhesion, e.g. the loss of anchorage to the extracellular matrix, as adhesion to the extracellular matrix been shown to prevent caspase activity, thus preventing apoptosis . Despite the improvement observed in cell recovery yields and viability with ROCK inhibitor pretreatment, some difference weas observed when comparing the results obtained from 2D and 3D cryopreservation using FBS+DMSO w/o ROCKi, as the 2D approach presented lower metabolic activity recoveries. The presence of cell –cell and cell –surface interactions on monolayers has been shown to render cells more susceptible to freezing injury [161, 162]. These interactions are likely sites for monolayer damage by the osmotic stresses and phase changes in cryopreservation, and have been associated with enhanced susceptibility to intracellular ice formation . The cells extended morphology may also create conditions for cryopreservation-induced damage to the cells structure (cytoskeleton or gap junctions) due to mechanical forces, such as extracellular ice . DMSO was the only compound added to prevent cryopreservation damage unlike CS10, which contains numerous high quality cryo-protecting components. These features may explain the difference observed between cryopreservation of monolayers and cardiospheres using this solution. Despite this fact, from an economical perspective, the FBS+DMSO formulation is less costly and easy to prepare in any stem cell lab, as most of its components exist for common lab procedures.
shown). Thus our data emphasize the importance of using serum- free medium for consistent results with riPS cell derivation. During the time course of our studies another group reported a riPS cell derivation protocol that used both REFs and rat neural precursors as starting cell types and relied on serum-containing medium, non- excisable moloney murine leukemia virus (MMLV)-based delivery (in combination with ES cell extracts), and mitotically inactivated REFs as feeder cell layer . A direct comparison of this and our method is not feasible. However, our approach seems to offer several advantages: (1) well-known superior reprogramming capacity of HIV-based, compared to MMLV-based retroviral vectors; (2) a true hit-and-run strategy involving lentivirus excision from the rat genome (without any obvious chromosomal rearrangements) and, thus, the elimination of the risks associated with reprogramming factor re-activation and, as a subsequence, altered differentiation properties of riPS cells; (3) the approach as it has been discussed above, eliminates the dependence on serum batch variation. Recently proposed virus-free iPS derivation systems [31,32,33,34,35] remain to be tested in the rat, however, for the time being, the hit-and-run protocol offered in this paper appears to be the most efficient and consistent.
Demyelinating diseases such as multiple sclerosis (MS) are characterized by damage to the myelin sheath surrounding neurons, causing impaired nerve impulses that lead to a constellation of neurological symptoms. Recent research on cell transplantation has yielded new insights into the novel possibilities of using stem cell-derived oligodendrocytes in graft-based remyelination therapy to restore action potential conduction. However, to date, an efficient and reliable cell source has not been introduced (for review, see . The recent groundbreaking developments regarding inducedpluripotentstemcells (iPSCs) generated from easily accessible somatic cells  appear to offer a nearly inexhaustible source of transplantable, autologous neural stemcells (for review, see [3,4]. Many studies have demonstrated that mouse and human iPSCs are highly morphologically, molecularly and phenotypically similar to their respective embryo-derived embryonic stem cell (ESC) counterparts [5,6]. The use of iPSCs also circumvents the ethical issue related to using ES cellsand producing human disease models in vitro (for review, see .
OCT-4/3 and NANOG have been used as epigenetic markers for iPSCs [8–10,26,27]. We previously showed candidate epigenetic markers by analyzing 6 iPS lines . Here we identified 8 novel epigenetic markers more closely by defining 9 genes with the hypo- methylated stem cell-specific DMRs and significantly higher expression, and 17 genes with the hyper-methylated stem cell- specific DMRs and significantly lower expression in iPSCs/ESCs from 22 iPS lines. DNA methylation and expression of these genes, especially the 8 genes, SALL4, EPHA1, PTPN6, RAB25, GBP4, LYST, SP100 and UBE1L, can now be used as epigenetic markers for pluripotentstemcells. Among these 8 genes, SALL4 has been used as an expression marker, and is revealed for the first time as an epigenetic marker. These epigenetic changes during reprogram- ming can be detected by 3 different methods (Illumina assay, COBRA and bisulfite sequencing), and is evident, i.e. CpG sites are methylated or unmethylated in an all-or-none fashion. The identification of these novel epigenetic markers can be another tool for the validation of pluripotentstemcells that are iPSCs and ESCs. The hypo-methylated stem cell-required DMRs may have an important role for reprogramming as do the stem cell-specific DMRs, because reprogramming is dependent on the type of parent cells. In fact, genes associated with the hypo-methylated stem cell-required DMRs include a large number of transcription factors that are involved in pluripotency. Establishment of the stem cell-required DMRs database in iPSCs derived from different types of parent cells can help to generate human iPSCs in a fast and easy manner. Hypo-methylated stem cell-specific regions have been reported to be abundant in CpG islands [28–30]. In this study, the hypo-methylated stem cell-specific DMRs were significantly biased towards CpG islands, whereas the hyper- methylated stem cell-specific DMRs were biased to non-CpG islands, suggesting that genes with CpG islands have a propensity to be demethylated during reprogramming towards pluripotentstemcells. The higher number of the hyper-methylated stem cell- specific DMRs in iPSCs indicates that the Yamanaka factors activate only limited numbers of stem cell-specific/associated genes through demethylation of the specific DMRs shown in this study on the genome in parallel with methylating most genes associated with tissue-specific function during reprogramming.
ABSTRACT: The pluripotentstemcells can potentially be used to counter a wide range of diseases, from diabetes to spinal cord injury, to childhood leukemia, to heart disease. Human iPS cells are similar to human embryonic stem (ES) cells in terms of proliferation and differentiation ability, and can be generated from adult somatic cells. Now we can easily generate i PS cells from patient’s somatic cellsand those iPS cells have all genomic information of the patient genome. Many human diseases are caused by genomic mutation. Disease modeling using human iPS cells is newly emerged research field to analyze genetic human diseases. Actually, there are many fatal genetic diseases without effective therapy. To develop newly effective therapies for those diseases, first of all we have to generate disease models. In the past, there had been solely animal models of human genetic disease, such as specific gene knockout mice, transgenic mice and autochthonous diseased animals. Although those models gave us many valuable information regarding the mechanisms of human genetic diseases, most crucial problem is that those models are not human. So it is often difficult to model human diseases using experimental animals. One of the important points is, among humans, each individual shows highly rich in genomic diversity in terms of racial differences and single nucleotide polymorphisms (SNPs). So it has been highly expected to generate not only disease- specific models, but also disease-specific and patient-specific disease models. To generate patient- specific disease models, now we can use iPS cells. 1
in turn activates Osx expression. Because epigenetic conditions during embryonic development are quite different from those during ESC/iPSC differentiation, the transcription factors re- quired for TNAP expression may be different. In embryos, Runx2 is required for the differentiation of prechondrogenic mesenchy- mal cells into osteoblasts, whereas Osx is believed to induce subsequent maturation of osteoblasts and inhibit chondrogenic differentiation. In Osx-null embryos, cartilage forms normally but the embryos completely lack bone [17–19]. OSX, which is specifically and exclusively expressed in all osteoblasts, showed markedly high expression in TNAP-positive cells, although TNAP- positive and -negative cells expressed almost similar levels of RUNX2 (Fig. 3b). These findings indicated that iPSCs may not require the prechondrogenic process and may induce Osx without a Runx2 surge. Several pathways have been reported to increase Osx expression. Mitogen-activated protein kinases, particularly p38, Erk1/2, and protein kinase D, activate Osx expression accompanied by TNAP activation. Ascorbate-dependent prolyl hydroxylase domain protein induces Osx expression. Endoplasmic reticulum stress also increases Osx induction . These cascades may play an important role in the Osx surge and the increase in TNAP in iPSCs. We found that continuous culture of these TNAP-positive cells in OBM eventually led to increased expres- sion of RUNX2, TNAP, COL1A1, and OSX as well as other osteogenic markers, such as BSP and OCN. These results indicated that TNAP-positive cells derived from hiPSCs are OSX-positive osteoprogenitors, not chondrogenic cells. Furthermore, TNAP- positive cells are capable not only of differentiating into osteogenic cells but also of responding to active vitamin D treatment. Vitamin D treatment effectively upregulated OCN and downregulated TNAP, indicating that these cells could differentiate into cells in the late phase of osteogenesis and may be able to differentiate into terminally differentiated osteocytes.
[17,18]. Therefore, we sought to reprogram human UCs through episomal system without using serum, feeders and c-MYC. To test the episomal system in UCs, we transfected an episomal vector encoding EGFP into these cells through electroporation. We showed that EGFP expression efficiency was remarkable, about 35% cells were positive for EGFP expression (Fig. 1C). Next, we found that UCs could survive and grow in serum-free media mTeSR1 and E8 which were used to maintains human ES or iPS cells [21,22], albeit slower than in their optimal medium (Fig. 1D). These data suggested that it was possible to use episomal system and defined serum-free medium for iPS generation with UCs as illustrated in Figure 1E. Because involvement of oncogene c-MYC during reprogramming might increase the risk of genomic toxicity , we tried to omit it by using OCT4, SOX2, SV40T, KLF4 (OSTK, encoded by pEP4EO2SET2K). However, we failed to obtain stable iPS colonies from UCs or skin fibroblasts (Fig. 1F), suggesting that the OSTK four factor were insufficient for non- integrating iPS cell generation under serum-free conditions. We and several other groups had shown that miR-302-367 cluster could greatly enhance somatic reprogramming efficiency [24,25,26]. In addition, we found that mice chimeras with genome integration of miR-302-367 cluster and their offspring are tumors- free for over 2 years. Thus, miR-302-367 cluster might be less genomically toxic and even suppress tumorigenecity of human pluripotentstemcells  and be a better choice for iPS cells generation than c-MYC. We then constructed an episomal vector expressing human miR-302-367 cluster (named pCEP4-miR302- 367 cluster. Fig. S2A and S2B) and simultaneously transfected it into UCs with OSTK through electroporation. The transfected UCs were then cultured on matrigel-coated plate and in totally defined and serum free medium (mTesR1 or E8) for reprogram- ming. Around 20 days after electroporation, we could observe
The laboratory rat (Rattus norvegicus) was the first mammalian species to be used for scientific research, and has been widely applied as an animal model for studies in physiology, pharmacol- ogy, toxicology, nutrition, behavior, immunology, and neoplasia . The availability of many kinds of spontaneous models for diseases such as hypertension and diabetes has made the rat the preferred choice for scientific investigations. Furthermore, rats are profitable tools for transplantation studies and motor functional analysis because of their body size and ease in handling and care. In 2006, Yamanaka et al. reported the generation of pluripotentstemcells from mouse somatic cells by transduction of four transcription factors (Oct3/4, Sox2, Klf4, and Myc) . These cells are referred to as inducedpluripotentstemcells (iPSCs). The discovery of iPSCs has contributed a great step forward in stem cell research, because iPSCs generated from patients are a great resource for novel therapeutic strategies. Moreover, iPSCs can be extremely valuable research tools, especially for rats and other species for which embryonic stemcells (ESCs) are not available or are difficult to isolate. The generation of iPSCs from disease model rats could help to clarify the pathogenesis of various disorders.
Advancing pluripotentstem cell research to clinical applications requires adapting laboratory-scale cultivation methods to less conventional and current Good Manufacturing Practices- (cGMP- ) compliant platforms [1,2]. As a first step, we set out to improve and simplify conventional methods used to subculture hPSCs. Current practices include the propagation of adherent colony cultures, as multicellular aggregates, using one or a combination of methods that include manual scraping, manual microdissection, enzymatic and non-enzymatic procedures to detach the cells from their matrix. Manual microdissection and scraping portions of selected colonies is labor intensive, and highly dependent on the proficiency of skilled technical personnel. Enzymatic and existing non-enzymatic methods are time-critical. Over-treatment of the cells with the detachment solution often results in the increased production of single cells . Unfortunately, the continuous cultivation of adherent hPSCs from single cells  may play a role in promoting chromosomal abnormalities and genetic alterations in a hPSC population over time [5,6]. Moreover, single cells produced during the subculture of hPSC colonies are susceptible to dissociation-induced apoptosis .
Normal or iPSC-derived keratinocytes were seeded onto gelatin-coated four-well chamber slides at a cell density of 2 × 10 4 cells/well in CnT-07 media. Cultures were allowed to expand until they were about 70% confluent. Cells were incubated with freshly sonicated fluorescent microspheres 0.5μm (red) in diameter (Life Technologies) (previously demonstrated to be an appropriate model for melanosome uptake in epidermal keratinocytes ). Microspheres were pre-incubated for 1 hour at 37°C in CnT-07 media (containing 10% FBS) before being incubated with cells for different time points (0, 2, 4 or 6 hour) at a final concentration of 288,000 particles/ml in CnT-07 media. Media was removed andcells were washed 3 times in fresh cold media followed by a final wash in cold PBS to remove non-ingested microspheres. Cells were then fixed in ice-cold methanol (Fisher Scientific) for 10 minutes at RT, rinsed with PBS, coverslipped using Vectashield mounting media containing DAPI and examined using a Zeiss LSM 5 Exciter confocal laser scanning microscope. Quantitative analysis of the beads was performed by counting the number of internalized beads in 30 cells for each time point, ran- domly taken from 3 microscopic fields (10 cells per microscopic field) in 3 different experi- ments (resulting in 90 cells analyzed per condition), and values are expressed as the mean value ± SEM. Statistical analysis was performed using the Student’s t test and significance level has been identified as p<0.05. In order to determine that only internalized beads were counted in the quantitative analysis we performed parallel phase contrast and fluorescence microscopy.
The efficient commitment of a specialized cell type from inducedpluripotentstemcells (iPSCs) without contamination from unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone marrow mesenchymal stemcells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal, hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P,0.05). After culturing with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7–9 days, CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or from the spontaneous differentiation groups (P,0.05), as assessed by flow cytometry analysis. These inducedcells expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of the mechanisms of hematopoiesis and drug screening.
Unsupervised hierarchical clustering of upregulated genes in hiPSCs and donor cells with respect to hESCs further confirmed the proximity of hiPSCs to their corresponding cell of origin (Figure 5) as compared to other donor cell types. For each set of iPS-donor cell types, IPA analysis was performed for functional annotation of the set of upregulated genes (Supplementary Table S1-A to S1-D). We clarified the role of these genes in various basic processes (cellular growth and proliferation, tissue development, cellular function, lipid metabolism, connective tissue development, DNA repair, cellular maintenance, etc). Next, we examined the expression of fibroblast , fat [33,34,35,36], and keratinocyte  specific genes within the upregulated gene sets. We found significant residual gene expression of fibroblast (Figure 6A), adipocyte (Figure 6B), and keratinocyte genes (Figure 6C) within their corresponding hiPSCs. Specifically, fibroblast genes in Figure 6A such as PLAT and PLAU [32,38] play important roles in remodeling the extracellular matrix and other functions in the coagulation system. Other fibroblast genes include CXCL1, which is involved in cell migration , and FOXF1 and FOXP1, which are forkhead family transcription factors expressed in fibroblasts Figure 2. Distance between hiPSC, hESC and donor cells. (A) Relative distances of the hiPSC states from the corresponding somatic states (donor cells), and from the hESC state. (B) Global clustering among hESC, hiPSC, and donor cells.
RE14, RE17 and RE19 from S7) carried fewer mutations, of which a subset was shared between them as well as with EPCs from the same individual. None of the shared SNVs were detected in the corresponding fibroblasts or whole blood, indicating that these SNVs were somatically acquired by the EPCs in vivo (Fig 1D and 1E). In addition, private SNVs were detected which were unique to each monoclonal-derived iPSC line and these were not found in EPCs or the individual’s reference genome. Deep sequencing of the donor EPC genome revealed that some of the mutations detected in the iPSCs were in fact present in the EPCs but at very low frequen- cies (Fig 1D and 1E, orange boxes; S7 and S8 Tables), suggesting that these mutations were acquired by the EPCs during the in vitro expansion process, prior to reprogramming. Notably no known driver mutations (using COSMIC database), which could confer a selective advan- tage, were identified in any of the iPSC lines. These results demonstrate that iPSCs derived from monoclonal somatic cells can be used to identify in vivo acquired somatic mutations. The mutational burden of iPSCs reflects mutations accumulated in vivo in the ancestral somatic cell lineages and mutations acquired during in vitro cell culture and subsequent repro- gramming. The iPSCs from heterogeneous somatic cells usually do not share any mutations but the exome sequencing data demonstrated that by using monoclonal cell sources it is possi- ble to resolve mutations acquired in vivo from those arising during in vitro cell culture. Fur- thermore, identifying shared mutations in somatic cell lineages could be used to construct a cellular phylogenetic tree. We therefore performed whole genome sequencing on the