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.
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.
Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines ...from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in vitro and in vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.
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► Human iPSCs were generated from pancreatic beta cells using lineage-tracing systems ► Beta cell-derived iPSCs maintained an epigenetic memory at key beta-cell genes ► Beta cell-derived iPSCs showed preferred differentiation into insulin-producing cells ► The skewed differentiation of beta-iPSCs may be advantageous for cell therapy
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.
Although haploidy has not been observed in vertebrates, its natural occurrence in various eukaryotic species that had diverged from diploid ancestors suggests that there is an innate capacity for an ...organism to regain haploidy and that haploidy may confer evolutionary benefits. Haploid embryonic stem cells have been experimentally generated from mouse, rat, monkey, and humans. Haploidy results in major differences in cell size and gene expression levels while also affecting parental imprinting, X chromosome inactivation, and mitochondrial metabolism genes. We discuss here haploidy in evolution and the barriers to haploidy, in particular in the human context.
Naturally occurring haploidy in eukaryotic species (albeit not in vertebrates) with diploid ancestors suggests evolutionary benefits for the state and an innate capacity for organisms to regain haploidy. Sagi and Benvenisty discuss haploidy in evolution and haploidy barriers in light of recent isolation of haploid embryonic stem cells.
Parental imprinting is a form of epigenetic regulation that results in parent-of-origin differential gene expression. To study Prader-Willi syndrome (PWS), a developmental imprinting disorder, we ...generated case-derived induced pluripotent stem cells (iPSCs) harboring distinct aberrations in the affected region on chromosome 15. In studying PWS-iPSCs and human parthenogenetic iPSCs, we unexpectedly found substantial upregulation of virtually all maternally expressed genes (MEGs) in the imprinted DLK1-DIO3 locus on chromosome 14. Subsequently, we determined that IPW, a long noncoding RNA in the critical region of the PWS locus, is a regulator of the DLK1-DIO3 region, as its overexpression in PWS and parthenogenetic iPSCs resulted in downregulation of MEGs in this locus. We further show that gene expression changes in the DLK1-DIO3 region coincide with chromatin modifications rather than DNA methylation levels. Our results suggest that a subset of PWS phenotypes may arise from dysregulation of an imprinted locus distinct from the PWS region.
Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated ...violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.
This Review provides a comprehensive overview of the mechanisms that modulate genomic imprinting and the roles this epigenetic process plays across mammalian physiology.
Human embryonic stem cells (HESCs) and induced pluripotent stem cells (HiPSCs) offer an immense potential as a source of cells for regenerative medicine. However, the ability of undifferentiated ...HESCs to produce tumors in vivo presents a major obstacle for the translation of this potential into clinical reality. Therefore, characterizing the nature of HESC-derived tumors, especially their malignant potential, is extremely important in order to evaluate the risk involved in their clinical use. Here we review recent observations on the tumorigenicity of human pluripotent stem cells. We argue that diploid, early passage, HESCs produce benign teratomas without undergoing genetic modifications. Conversely, HESCs that acquired genetic or epigenetic changes upon adaptation to in vitro culture can produce malignant teratocarcinomas. We discuss the molecular mechanisms of HESC tumorigenicity and suggest approaches to prevent tumor formation from these cells. We also discuss the differences in the tumorigenicity between mouse embryonic stem cells (MESCs) and HESCs, and suggest methodologies that may help to identify cellular markers for culture adapted HESCs.
In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, ...it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.
Males and females have a variety of sexually dimorphic traits, most of which result from hormonal differences. However, differences between male and female embryos initiate very early in development, ...before hormonal influence begins, suggesting the presence of genetically driven sexual dimorphisms. By comparing the gene expression profiles of male and X-inactivated female human pluripotent stem cells, we detected Y-chromosome-driven effects. We discovered that the sex-determining gene SRY is expressed in human male pluripotent stem cells and is induced by reprogramming. In addition, we detected more than 200 differentially expressed autosomal genes in male and female embryonic stem cells. Some of these genes are involved in steroid metabolism pathways and lead to sex-dependent differentiation in response to the estrogen precursor estrone. Thus, we propose that the presence of the Y chromosome and specifically SRY may drive sex-specific differences in the growth and differentiation of pluripotent stem cells.
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•The male-specific gene SRY is expressed in male human ESCs and iPSCs•SRY promoter can be activated in the pluripotent state regardless of sex•Male and female pluripotent cells differ in autosomal gene expression•SRY induces differential utilization of cholesterol to produce sex steroids
Sexually dimorphic traits are mostly hormonally driven and are established following gonadal development. By focusing on Y chromosome transcription in undifferentiated human embryonic stem cells, Ronen and Benvenisty now show that the male-determining gene SRY is expressed in these cells. This transcription factor, along with its target autosomal genes, may drive early differences in steroid metabolism and neuronal development between male and female embryos.
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