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 The inherited neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by GAA•TTC triplet repeat hyper-expansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA•TTC repeats causes heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA•TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells, occupies FXN intron 1, and shRNA silencing of MSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.

Objective To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients. Methods We used a human FRDA neuronal cell model, derived from patient induced pluripotent stem cells, to determine the efficacy of a 2-aminobenzamide HDACi (109) as a modulator of FXN gene expression and chromatin histone modifications. FRDA patients were dosed in 4 cohorts, ranging from 30mg/day to 240mg/day of the formulated drug product of HDACi 109, RG2833. Patients were monitored for adverse effects as well as for increases in FXN mRNA, frataxin protein, and chromatin modification in blood cells. Results In the neuronal cell model, HDACi 109/RG2833 increases FXN mRNA levels and frataxin protein, with concomitant changes in the epigenetic state of the gene. Chromatin signatures indicate that histone H3 lysine 9 is a key residue for gene silencing through methylation and reactivation through acetylation, mediated by the HDACi. Drug treatment in FRDA patients demonstrated increased FXN mRNA and H3 lysine 9 acetylation in peripheral blood mononuclear cells. No safety issues were encountered. Interpretation Drug exposure inducing epigenetic changes in neurons in vitro is comparable to the exposure required in patients to see epigenetic changes in circulating lymphoid cells and increases in gene expression. These findings provide a proof of concept for the development of an epigenetic therapy for this fatal neurological disease.

Background: Friedreich ataxia is caused by a GAA⅐TTC triplet-repeat expansion in the first intron of the FXN gene. Results: Expansion of the repeats is observed in induced pluripotent stem cells (iPSCs) and can be blocked with either shRNAs to mismatch repair enzymes or small molecules targeting the repeats. Conclusion: MutS␣ and MutS␤ are involved in repeat expansion. Significance: iPSCs provide a model system for studying triplet-repeat expansion.

Summary During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single cell transcriptomic data identified 41 distinct populations of progenitors, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/beta-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders.