Uri Ben-David

Identification and Classification of Chromosomal Aberrations in Human Induced Pluripotent Stem Cells

Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.

The tumorigenicity of human embryonic and induced pluripotent stem cells.

The unique abilities of human pluripotent stem cells to self-renew and to differentiate into cells of the three germ layers make them an invaluable tool for the future of regenerative medicine. However, the same properties also make them tumorigenic, and therefore hinder their clinical application. Hence, the tumorigenicity of human embryonic stem cells (HESCs) has been extensively studied. Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity.

Selective elimination of human pluripotent stem cells by an oleate synthesis inhibitor discovered in a high-throughput screen.

The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.

Large-Scale Analysis Reveals Acquisition of Lineage-Specific Chromosomal Aberrations in Human Adult Stem Cells

In this study, we assessed the genetic integrity of over 400 samples of human multipotent stem cells using gene expression data sets. Our analysis reveals that neural and mesenchymal stem cells acquire characteristic large chromosomal aberrations at a similar, or somewhat lower, frequency to that seen in pluripotent stem cells, sometimes within a few passages in culture. Some of the identified chromosomal abnormalities can also be detected in human tumors of the respective tissues.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting

UNLABELLED The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. SIGNIFICANCE We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803.

Immunologic and chemical targeting of the tight-junction protein Claudin-6 eliminates tumorigenic human pluripotent stem cells

The tumorigenicity of human pluripotent stem cells is a major safety concern for their application in regenerative medicine. Here we identify the tight-junction protein Claudin-6 as a cell-surface-specific marker of human pluripotent stem cells that can be used to selectively remove Claudin-6-positive cells from mixed cultures. We show that Claudin-6 is absent in adult tissues but highly expressed in undifferentiated cells, where it is dispensable for human pluripotent stem cell survival and self-renewal. We use three different strategies to remove Claudin-6-positive cells from mixed cell populations: an antibody against Claudin-6; a cytotoxin-conjugated antibody that selectively targets undifferentiated cells; and Clostridium perfringens enterotoxin, a toxin that binds several Claudins, including Claudin-6, and efficiently kills undifferentiated cells, thus eliminating the tumorigenic potential of human pluripotent stem cell-containing cultures. This work provides a proof of concept for the use of Claudin-6 to eliminate residual undifferentiated human pluripotent stem cells from culture, highlighting a strategy that may increase the safety of human pluripotent stem cell-based cell therapies.

Patient-derived xenografts undergo mouse-specific tumor evolution

Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of preexisting minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Notably, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have major implications for PDX-based modeling of human cancer.

Genome maintenance in pluripotent stem cells

Pluripotent stem cells (PSCs) must maintain their proper genomic content in order to preserve appropriate self-renewal and differentiation capacities. However, their prolonged in vitro propagation, as well as the environmental culture conditions, present serious challenges to genome maintenance. Recent work has been focused on potential means to alleviate the genomic insults experienced by PSCs, and to detect them as soon as they arise, in order to prevent the detrimental consequences of these genomic aberrations on PSC application in basic research and regenerative medicine.

Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs, inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation, differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs, mainly as a consequence of increased replication. Furthermore, trisomy 12 increases the tumorigenicity of hPSCs in vivo, inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last, a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together, these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications.

High Prevalence of Evolutionarily Conserved and Species-Specific Genomic Aberrations in Mouse Pluripotent Stem Cells

Mouse pluripotent stem cells (PSCs) are the best studied pluripotent system and are regarded as the “gold standard” to which human PSCs are compared. However, while the genomic integrity of human PSCs has recently drawn much attention, mouse PSCs have not been systematically evaluated in this regard. The genomic stability of PSCs is a matter of profound significance, as it affects their pluripotency, differentiation, and tumorigenicity. We thus performed a thorough analysis of the genomic integrity of 325 samples of mouse PSCs, including 127 induced pluripotent stem cell (iPSC) samples. We found that genomic aberrations occur frequently in mouse embryonic stem cells of various mouse strains, add in mouse iPSCs of various cell origins and derivation techniques. Four hotspots of chromosomal aberrations were detected: full trisomy 11 (with a minimally recurrent gain in 11qE2), full trisomy 8, and deletions in chromosomes 10qB and 14qC‐14qE. The most recurrent aberration in mouse PSCs, gain 11qE2, turned out to be fully syntenic to the common aberration 17q25 in human PSCs, while other recurrent aberrations were found to be species specific. Analysis of chromosomal aberrations in 74 samples of rhesus macaque PSCs revealed a gain in chromosome 16q, syntenic to the hotspot in human 17q. Importantly, these common aberrations jeopardize the interpretation of published comparisons of PSCs, which were unintentionally conducted between normal and aberrant cells. Therefore, this work emphasizes the need to carefully monitor genomic integrity of PSCs from all species, for their proper use in biomedical research. STEM CELLS 2012; 30:612–622