Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells

To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.     

Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue

AIM To investigate genotype variation among induced pluripotent stem cell(iPSC) lines that were clonally generated from heterogeneous colon cancer tissues using next-generation sequencing. METHODS Human iPSC lines were clonally established by selecting independent single colonies expanded from heterogeneous primary cells of S-shaped colon cancer tissues by retroviral gene transfer(OCT3/4, SOX2, and KLF4). The ten iPSC lines, their starting cancer tissues, and the matched adjacent non-cancerous tissues were analyzed using nextgeneration sequencing and bioinformatics analysis using the human reference genome hg19. Non-synonymous single-nucleotide variants(SNVs)(missense, nonsense,and read-through) were identified within the target region of 612 genes related to cancer and the human kinome. All SNVs were annotated using dbS NP135, CCDS, RefSeq, GENCODE, and 1000 Genomes. The SNVs of the iPSC lines were compared with the genotypes of the cancerous and non-cancerous tissues. The putative genotypes were validated using allelic depth and genotype quality. For final confirmation, mutated genotypes were manually curated using the Integrative Genomics Viewer. RESULTS In eight of the ten iPSC lines, one or two non-synonymous SNVs in EIF2AK2, TTN, ULK4, TSSK1 B, FLT4, STK19, STK31, TRRAP, WNK1, PLK1 or PIK3R5 were identified as novel SNVs and were not identical to the genotypes found in the cancer and non-cancerous tissues. This result suggests that the SNVs were de novo or pre-existing mutations that originated from minor populations, such as multifocal pre-cancer(stem) cells or pre-metastatic cancer cells from multiple, different clonal evolutions, present within the heterogeneous cancer tissue. The genotypes of all ten iPSC lines were different from the mutated ERBB2 and MKNK2 genotypes of the cancer tissues and were identical to those of the noncancerous tissues and that found in the human reference genome hg19. Furthermore, two of the ten iPSC lines did not have any confirmed mutated genotypes, despite being derived from cancerous tissue. These results suggest that the traceability and preference of the starting single cells being derived from pre-cancer(stem) cells, stroma cells such as cancer-associated fibroblasts, and immune cells that co-existed in the tissues along with the mature cancer cells.CONCLUSION The genotypes of iPSC lines derived from heterogeneous cancer tissues can provide information on the type of starting cell that the iPSC line was generated from.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells

It remains controversial whether the abnormal epigenetic modifications accumulated in the induced pluripotent stem cells (iPSCs) can ultimately affect iPSC pluripotency. To probe this question, iPSC lines with the same genetic background and proviral integration sites were established, and the pluripotency state of each iPSC line was characterized using tetraploid (4N) complementation assay. Subsequently, gene expression and global epigenetic modifications of “4N-ON” and the corresponding “4N-OFF” iPSC lines were compared through deep sequencing analyses of mRNA expression, small RNA profile, histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation. We found that methylation of an imprinted gene, Zrsr1, was consistently disrupted in the iPSC lines with reduced pluripotency. Furthermore, the disrupted methylation could not be rescued by improving culture conditions or subcloning of iPSCs. Moreover, the relationship between hypomethylation of Zrsr1 and pluripotency state of iPSCs was further validated in independent iPSC lines derived from other reprogramming systems.     

Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells

Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.     

Few Single Nucleotide Variations in Exomes of Human Cord Blood Induced Pluripotent Stem Cells

The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34⁺ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2–14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.     

New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.     

The tumourigenicity of iPS cells and their differentiated derivates

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

iPS cells: a game changer for future medicine

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC‐based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non‐responders by analyzing the efficacy of the drug on iPSC‐derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC‐derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro‐ and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.     

Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs)

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.     

Genetic Heterogeneity of Induced Pluripotent Stem Cells: Results from 24 Clones Derived from a Single C57BL/6 Mouse

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs “captures” the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically “fit” for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0–45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40–60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.     

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)¹. While improvements in several gene delivery methods have been described^2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor¹¹, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.     

DNA methylation dynamics in human induced pluripotent stem cells over time

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the "convergence" of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs.     

Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature

Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs), have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC) system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCL cultures from six unrelated donors to iPSCs on the ensuing gene expression patterns within and between individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures, increasing the number of genes with a detectable donor effect by an order of magnitude. The proportion of variation in gene expression statistically attributed to donor increases from 6.9% in LCLs to 24.5% in iPSCs (P < 10^-15). Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in iPSCs than in LCLs. Our findings indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation.     

Differential coupling of self-renewal signaling pathways in murine induced pluripotent stem cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors.     

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones,not Cell Type of Origin

Background

For regenerative therapy using induced pluripotent stem cell (iPSC) technology, cell type of origin to be reprogrammed should be chosen based on accessibility and reprogramming efficiency. Some studies report that iPSCs exhibited a preference for differentiation into their original cell lineages, while others did not. Therefore, the type of cell which is most appropriate as a source for iPSCs needs to be clarified.

Methodology/Principal Findings

Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.

Conclusions/Significance

The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation. Qualified and genetically matched human iPSC clone sets established in this study are valuable resources for further basic study of clonal differences.