Robert Morey

Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.

BackgroundThe pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play.ResultsWe present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant.ConclusionsThe vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.

Metabolic rescue in pluripotent cells from patients with mtDNA disease

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Proof of concept studies exploring the safety and functional activity of human parthenogenetic-derived neural stem cells for the treatment of Parkinson's disease.

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinsons disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

The epigenome in pluripotency and differentiation.

The ability to culture pluripotent stem cells and direct their differentiation into specific cell types in vitro provides a valuable experimental system for modeling pluripotency, development and cellular differentiation. High-throughput profiling of the transcriptomes and epigenomes of pluripotent stem cells and their differentiated derivatives has led to identification of patterns characteristic of each cell type, discovery of new regulatory features in the epigenome and early insights into the complexity of dynamic interactions among regulatory elements. This work has also revealed potential limitations of the use of pluripotent stem cells as in vitro models of developmental events, due to epigenetic variability among different pluripotent stem cell lines and epigenetic instability during derivation and culture, particularly at imprinted and X-inactivated loci. This review focuses on the two most well-studied epigenetic mechanisms, DNA methylation and histone modifications, within the context of pluripotency and differentiation.

Melanocytes Derived from Transgene-Free Human Induced Pluripotent Stem Cells

TO THE EDITOR Defects in melanocytes have been implicated in the etiology of a variety of human skin diseases and disorders (Lin and Fisher, 2007; Fistarol and Itin, 2010; Rees, 2011). There is long-standing interest in studying the development and dysfunction of human melanocytes, but there has not been a reliable and accessible system to study early events in human melanocyte differentiation. An in vitro system that reliably and efficiently produces normal human melanocytes from embryonic stage cells would allow us to better dissect the physiological and pathological development of melanocytes. Recent advances in stem cell biology have led to the establishment of human induced pluripotent stem cell (hiPSC) techniques that enable researchers to reprogram somatic cells to the pluripotent state (Takahashi et al., 2007). Differentiation of human and mouse pluripotent stem cells (PSCs) toward the melanocyte lineage has been reported (Yamane et al., 1999; Pla et al., 2005; Fang et al., 2006; Nissan et al., 2011; Ohta et al., 2011; Yang et al., 2011), but existing protocols have shortcomings that may limit their research and clinical applications. For example, the use of embryonic stem cells could lead to allogeneic immunoincompatibility of differentiated melanocytes and transplant recipients. In addition, the use of hiPSCs generated by integrative reprogramming strategies raises concerns about reactivation of retained transgenes, some of which are oncogenes. In addition, the current methods for melanocyte differentiation from hiPSCs require optimization in order to reproducibly generate high-purity melanocytes from multiple hiPSC lines. We have established a strategy to produce human melanocytes in vitro for use as a platform for pigment cell research and the development of cell-based therapies. We first derived transgene-free hiPSCs from two distinct types of skin cells: human primary melanocytes (HMs) and human dermal fibroblasts (HDF51) (Figure 1a and Supplementary Figure S1a online). We used a nonintegrative reprogramming approach mediated by Sendai virus–based vectors independently encoding POU5F1, SOX2, KLF4, and MYC (Fusaki et al., 2009; Macarthur et al., 2012). As shown in Figure 1b and Supplementary Figure S1b online, biomarkers of cellular pluripotency, including endogenous OCT4/POU5F1, NANOG, Tra-1-81, and UEA-I (Wang et al., 2011), were positive in HMi-506, HMi-503, and HDF51i-509 hiPSCs. Cells were also shown to be pluripotent using a gene expression diagnostic test (PluriTest; Muller et al., 2011), by differentiation into cells that express biomarkers relevant to all three germ layers in vitro (Figure 1c and Supplementary Figure S1c, S1d and S1e online) and by generation of teratomas (Supplementary Figure S1d online). Figure 1 Generation and differentiation of transgene-free human induced pluripotent stem cell (hiPSCs). (a) HMi-506 cells generated from human primary melanocyte (HM) cells using a Sendai virus–based reprogramming system were cocultured with mouse embryonic ... We newly developed two differentiation protocols based on previously reported methods. One protocol involves an aggregation-in-suspension step, whereas the other does not (Supplementary Figure S2 online). Both protocols generated cells displaying typical melanocyte morphology and pigmentation (Figure 1d) from hiPSCs after 30 days of directed differentiation, suggesting that the aggregation-in-suspension step is dispensable. The melanin granules that accumulated at the dendritic tips of differentiated cells were intensely stained by Fontana–Masson staining, indicating that the pigmentation of these cells was due to melanogenesis (Supplementary Figure S3 online). In addition, MITF (microphthalmia-associated transcription factor), a marker for melanocyte progenitors, was expressed in more than 90% of the differentiated derivatives after 30 days (Figure 1e and Supplementary Figure S4 online), which appears to be a higher differentiation efficiency than other reported protocols (Nissan et al., 2011; Ohta et al., 2011). As expected, MITF was not detected in the undifferentiated hiPSCs, and was present in the primary melanocytes (Figure 1e). Notably, our protocols resulted in similarly high levels of melanocyte differentiation for all four independent hiPSC lines examined, highlighting their reproducibility. Other melanocytic biomarkers including TYR (tyrosinase), MLANA (melan-A), TYRP1 (tyrosinase-related protein 1), PMEL (premelanosome protein), PAX3 (paired box 3), and SOX10 (SRY-box 10) were highly expressed in the differentiated derivatives (similar to primary melanocytes, Figure 2a and b). The melanin content and cell signaling involved in melanin production in the differentiated derivatives was increased by treatment with α-melanocyte-stimulating hormone (α-MSH) in a dose-dependent manner (Figure 2c and d and Supplementary Figure S5 online). These findings indicate that the differentiated derivatives possess molecular features of bona fide melanocytes and accurately mimic their ability to respond to α-MSH, which is the factor that activates melanogenesis and enhances skin pigmentation during the tanning response (Thody, 1999). Figure 2 Molecular and functional characterization of the melanocyte-like differentiated cells. (a) Heat map and dendrogram of melanocytic biomarkers showing that these transcripts were preferentially expressed in human primary melanocyte (HM) cells and HMi-506_Mel ... Genome-wide gene expression profiling and unsupervised hierarchical clustering revealed that the melanocytes (HMi-506_Mel Diff_1 and HMi-506_Mel Diff_2) differentiated from the HMi-506 cells were closely clustered with HMs and were distinct from all undifferentiated hiPSC samples (Figure 2e). As genetic abnormalities may occur in hiPSC genomes during the reprogramming and differentiation processes, we tested the genomic stability of the cells by comparing the differentiated derivatives with the parental primary melanocytes using high-resolution single-nucleotide polymorphism (SNP) genotyping and copy number variation analysis. As shown in Figure 2f, the HMi-506_Mel Diff derivatives and parental cells showed highly similar genotyping profiles, showing that the cellular genome remained stable during reprogramming and differentiation. Similar to human melanocytes in vivo, the differentiated derivatives in semiautologous skin reconstructs were located at the dermis–epidermis interface and interspersed with keratinocytes (Supplementary Figure S6a, S6b, S6c and S6d online), indicating their ability to integrate with the skin tissue of transplant recipients. Similar to the autologous dermal fibroblasts used for generating transgene-free hiPSCs, the differentiated derivatives stimulated limited proliferation of peripheral blood mononuclear cells that were isolated from the blood of the same individual in a mixed lymphocyte reaction assay (Supplementary Figure S6e online). These results attest to the clinical advantages of melanocytes differentiated from hiPSCs using the reprogramming and differentiation approaches described here. In this study, we have demonstrated that genetically stable melanocytes can be efficiently differentiated from transgene-free hiPSCs generated from two different types of cutaneous cells. This differentiation protocol takes less time than previously reported melanocytic differentiation protocols, and we showed that it is equally effective for multiple independent hiPSC lines. We performed a thorough investigation of the differentiated cells, including genome-wide gene expression analysis and SNP genotyping in addition to functional assays. Our approach can serve as an unlimited source of custom human melanocytes that can be used for novel approaches for modeling human skin disease (e.g., melanoma and vitiligo) and to provide material for transplantation.

Concise Review: Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer: A Horse in the Race?

Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT‐ESCs) and transcription factor‐mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT‐ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT‐ESCs—the ability to replace mutant mitochondrial DNA in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT‐ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from fertilized embryos. Thus, although NT‐ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT‐ESCs and bona fide ESCs may make NT‐ESCs superior for future applications in regenerative medicine. Stem Cells 2017;35:26–34

Spontaneous Single-Copy Loss of TP53 in Human Embryonic Stem Cells Markedly Increases Cell Proliferation and Survival.

Genomic aberrations have been identified in many human pluripotent stem cell (hPSC) cultures. Commonly observed duplications in portions of chromosomes 12p and 17q have been associated with increases in genetic instability and resistance to apoptosis, respectively. However, the phenotypic consequences related to sporadic mutations have not been evaluated to date. Here, we report on the effects of a single‐copy deletion of the chr17p13.1 region, a sporadic mutation that spontaneously arose independently in several subclones of a human embryonic stem cell culture. Compared to cells with two normal copies of chr17p13.1 (“wild‐type”), the cells with a single‐copy deletion of this region (“mutant”) displayed a selective advantage when exposed to stressful conditions, and retained a higher percentage of cells expressing the pluripotency marker POU5F1/OCT4 after 2 weeks of in vitro differentiation. Knockdown of TP53, which is a gene encompassed by the deleted region, in wild‐type cells mimicked the chr17p13.1 deletion phenotype. Thus, sporadic mutations in hPSCs can have phenotypic effects that may impact their utility for clinical applications. Stem Cells 2017;35:872–885