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 non-pluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to non-pluripotent 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, while 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.

SUMMARY Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting, and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's 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.

New small molecules that regulate the step-wise differentiation of human pluripotent stem cells into dopaminergic neurons have been identified. The steroid, guggulsterone, was found to be the most effective inducer of neural stem cells into dopaminergic neurons. These neurons are extensively characterized and shown to be functional. We believe this new approach offers a practical route to creating neurons of sufficient quality to be used to treat Parkinson's disease patients.

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.