The recent finding that reprogrammed human pluripotent stem cells can be derived by nuclear transfer into human oocytes as well as by induced expression of defined factors has revitalized the debate ...on whether one approach might be advantageous over the other. Here we compare the genetic and epigenetic integrity of human nuclear-transfer embryonic stem cell (NT-ESC) lines and isogenic induced pluripotent stem cell (iPSC) lines, derived from the same somatic cell cultures of fetal, neonatal, and adult origin. The two cell types showed similar genome-wide gene expression and DNA methylation profiles. Importantly, NT-ESCs and iPSCs had comparable numbers of de novo coding mutations, but significantly more than parthenogenetic ESCs. As iPSCs, NT-ESCs displayed clone- and gene-specific aberrations in DNA methylation and allele-specific expression of imprinted genes. The occurrence of these genetic and epigenetic defects in both NT-ESCs and iPSCs suggests that they are inherent to reprogramming, regardless of derivation approach.
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•Isogenic human NT-ESCs and iPSCs were derived from the same somatic cell cultures•Human NT-ESCs and iPSCs show similar profiles of gene expression and DNA methylation•De novo coding mutations occur at the same rate in human NT-ESC and iPSC lines•Loss of imprinting occurs in both NT-ESC and iPSC lines at similar frequencies
Johannesson et al. compare human somatic cells reprogrammed to pluripotency via SCNT and as iPSCs. They find similar gene expression and DNA methylation profiles, as well as comparable levels of genomic aberrations such as coding mutations and imprinted gene expression defects. This suggests that neither reprogramming method is superior in this regard.
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.
Human pluripotent stem cells (hPSCs) are being increasingly utilized worldwide in investigating human development, and modeling and discovering therapies for a wide range of diseases as well as a ...source for cellular therapy. Yet, since the first isolation of human embryonic stem cells (hESCs) 20 years ago, followed by the successful reprogramming of human‐induced pluripotent stem cells (hiPSCs) 10 years later, various studies shed light on abnormalities that sometimes accumulate in these cells in vitro. Whereas genetic aberrations are well documented, epigenetic alterations are not as thoroughly discussed. In this review, we highlight frequent epigenetic aberrations found in hPSCs, including alterations in DNA methylation patterns, parental imprinting, and X chromosome inactivation. We discuss the potential origins of these abnormalities in hESCs and hiPSCs, survey the different methods for detecting them, and elaborate on their potential consequences for the different utilities of hPSCs.
This review summarises our current knowledge on the epigenetic abnormalities occurring in human pluripotent stem cells, their origins as well as consequences for modeling development and disease.
In mammals, X chromosome inactivation (XCI) is a process in which one of the two X chromosomes is silenced, following
XIST expression. Mouse female pluripotent stem cells do not express
Xist, and ...harbor two active X chromosomes. However, analysis of XCI in human embryonic stem cells (hESCs), mainly based on
XIST expression, was not conclusive. Here, we studied XCI in hESCs by meta-analysis of the expression of the entire set of genes on the X chromosome in 21 female hESC lines. Thus, we could divide the ES cell lines into three categories: lines with no XCI, lines with full XCI, and lines with partial XCI. The partial inactivation of the X chromosome always involved the middle of the chromosome, surrounding the
XIST transcription site. The status of XCI in some of the cell lines was validated by either allelic-specific expression or DNA methylation analysis. Interestingly, analysis of 10 female human-induced pluripotent stem cell (hiPSC) lines demonstrated similar heterogeneity in the inactivation of X chromosome and could also be classified into the same three categories detected in hESCs. Thus, we could show that in some hiPSC lines, the X chromosome was activated on reprogramming. Based on our analysis, we propose a model of the dynamics of XCI in pluripotent stem cells.
Human embryonic stem cells have excellent potential for being the ultimate source of transplantable cells for many different tissues. To enable their clinical use, differentiation protocols should be ...developed and safety standards must be met. The cells should improve symptoms without generating side effects and their immune rejection must be overcome. Profiling of the immune antigens expressed on the cells has revealed that upon differentiation the cells express molecules of the major histocompatibility complex. Here, we propose ways of overcoming the rejection of human embryonic stem cells after transplantation.
Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the ...field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.
Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.
Cell cycle regulation is a complex system consisting of growth-promoting and growth-restricting mechanisms, whose coordinated activity is vital for proper division and propagation. Alterations in ...this regulation may lead to uncontrolled proliferation and genomic instability, triggering carcinogenesis. Here, we conducted a comprehensive bioinformatic analysis of cell cycle-related genes using data from CRISPR/Cas9 loss-of-function screens performed in four cancer cell lines and in human embryonic stem cells (hESCs).
Cell cycle genes, and in particular S phase and checkpoint genes, are highly essential for the growth of cancer and pluripotent cells. However, checkpoint genes are also found to underlie the differences between the cell cycle features of these cell types. Interestingly, while growth-promoting cell cycle genes overlap considerably between cancer and stem cells, growth-restricting cell cycle genes are completely distinct. Moreover, growth-restricting genes are consistently less frequent in cancer cells than in hESCs. Here we show that most of these genes are regulated by the tumor suppressor gene
, which is mutated in most cancer cells. Therefore, the growth-restriction system in cancer cells lacks important factors and does not function properly. Intriguingly, M phase genes are specifically essential for the growth of hESCs and are highly abundant among hESC-enriched genes.
Our results highlight the differences in cell cycle regulation between cell types and emphasize the importance of conducting cell cycle studies in cells with intact genomes, in order to obtain an authentic representation of the genetic features of the cell cycle.
Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. ...Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb-3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.
Human embryonic stem cells (hESCs) provide a platform for studying human development and understanding mechanisms underlying diseases. Retinoblastoma-1 (RB1) is a key regulator of cell cycling, of ...which biallelic inactivation initiates retinoblastoma, the most common congenital intraocular malignancy. We developed a model to study the role of RB1 in early development and tumor formation by generating RB1-null hESCs using CRISPR/Cas9. RB1−/− hESCs initiated extremely large teratomas, with neural expansions similar to those of trilateral retinoblastoma tumors, in which retinoblastoma is accompanied by intracranial neural tumors. Teratoma analysis further revealed a role for the transcription factor ZEB1 in RB1-mediated ectoderm differentiation. Furthermore, RB1−/− cells displayed mitochondrial dysfunction similar to poorly differentiated retinoblastomas. Screening more than 100 chemotherapies revealed an RB1–/–-specific cell sensitivity to carboplatin, exploiting their mitochondrial dysfunction. Together, our work provides a human pluripotent cell model for retinoblastoma and sheds light on developmental and tumorigenic roles of RB1.
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•RB1-null hESCs were generated using CRISPR/Cas9•RB1−/− hESCs generate large, neural-enriched teratomas, possibly by ZEB1 activation•RB1 inactivation triggers aberrant mitochondrial abundance and function•Unbiased drug screening found that carboplatin specifically targets RB1-null cells
Retinoblastoma is the most common congenital intraocular malignancy, caused by retinoblastoma-1 (RB1) inactivation. In this paper, Benvenisty and colleagues used CRISPR/Cas9 to generate RB1−/− human embryonic stem cells, and utilized them to reveal cellular, developmental, and tumorigenic aspects of this mutation. Furthermore, they have used this model in a drug screening, identifying a chemotherapy specifically targeting these mutant cells.
Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The ...Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.
The International Stem Cell Initiative organized a conference to review current understanding of the detection, origins, and consequences of genetic and epigenetic variants that arise in cultures of human pluripotent stem cells. This report provides an overview of the discussions and a recommendation to form an advisory group to help reach an international consensus on risk assessment for clinical applications.
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