Copy Number Variant Analysis of Human Embryonic Stem Cells

Department of Psychiatry and Biobehavioral Sciences, University of California Los Angeles, Los Angeles, California 90095-1732, USA.
Stem Cells (Impact Factor: 6.52). 07/2008; 26(6):1484-9. DOI: 10.1634/stemcells.2007-0993
Source: PubMed


Differences between individual DNA sequences provide the basis for human genetic variability. Forms of genetic variation include single-nucleotide polymorphisms, insertions/duplications, deletions, and inversions/translocations. The genome of human embryonic stem cells (hESCs) has been characterized mainly by karyotyping and comparative genomic hybridization (CGH), techniques whose relatively low resolution at 2-10 megabases (Mb) cannot accurately determine most copy number variability, which is estimated to involve 10%-20% of the genome. In this brief technical study, we examined HSF1 and HSF6 hESCs using array-comparative genomic hybridization (aCGH) to determine copy number variants (CNVs) as a higher-resolution method for characterizing hESCs. Our approach used five samples for each hESC line and showed four consistent CNVs for HSF1 and five consistent CNVs for HSF6. These consistent CNVs included amplifications and deletions that ranged in size from 20 kilobases to 1.48 megabases, involved seven different chromosomes, were both shared and unique between hESCs, and were maintained during neuronal stem/progenitor cell differentiation or drug selection. Thirty HSF1 and 40 HSF6 less consistently scored but still highly significant candidate CNVs were also identified. Overall, aCGH provides a promising approach for uniquely identifying hESCs and their derivatives and highlights a potential genomic source for distinct differentiation and functional potentials that lower-resolution karyotype and CGH techniques could miss. Disclosure of potential conflicts of interest is found at the end of this article.

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Available from: Kshama Mehta, Oct 06, 2015
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    • "Since the introduction of high-resolution molecular cytogenetic methods for the screening for genetic changes in human embryonic stem cells (hESC), numerous laboratories worldwide have reported the gain of 20q11.21 as a recurrent mutation (Lefort et al., 2008; Spits et al., 2008; Wu et al., 2008; Elliott et al., 2010; Närvä et al., 2010; Laurent et al., 2011; reviewed in Lund et al., 2012 and Nguyen et al., 2012). Recently, in a large joint study, the International Stem Cell Initiative (ISCI) screened 125 hESC lines worldwide and found this aberration in over 20% of the lines (Amps et al., 2011). "
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    ABSTRACT: Gain of 20q11.21 is a chromosomal abnormality that is recurrently found in human pluripotent stem cells and cancers, strongly suggesting that this mutation confers a proliferative or survival advantage to these cells. In this work we studied three human embryonic stem cell (hESC) lines that acquired a gain of 20q11.21 during in vitro culture. The study of the mRNA gene expression levels of the loci located in the common region of duplication showed that HM13, ID1, BCL2L1, KIF3B and the immature form of the micro RNA miR-1825 were up-regulated in mutant cells. ID1 and BCL2L1 were further studied as potential drivers of the phenotype of hESC with a 20q11.21 gain. We found no increase in the protein levels of ID1, nor the downstream effects expected from over-expression of this gene. On the other hand, hESC with a gain of 20q11.21 had on average a three-fold increase of Bcl-xL (the anti-apoptotic isoform of BCL2L1) protein levels. The mutant hESC underwent two- to three-fold less apoptosis upon loss of cell-to-cell contact and were approximately two-fold more efficient in forming colonies from a single cell. The key role of BCL2L1 in this mutation was further confirmed by transgenic over-expression of BCL2L1 in the wild type cells, leading to apoptosis-resistant cells, and BCL2L1-knock-down in the mutant hESC, resulting in a restoration of the wild type phenotype. This resistance to apoptosis supposes a significant advantage for the mutant cells, explaining the high frequency of gains of 20q11.21 in human pluripotent stem cells.
    Molecular Human Reproduction 11/2013; 20(2). DOI:10.1093/molehr/gat077 · 3.75 Impact Factor
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    • "EBs were generated from hESC (HSF1, H9) [2], [33] and hiPSC (hiPSC2, XFiPSC2) [6], [34] lines (with or without feeders) under standard culture conditions. EBs were embedded and cryo-sectioned before immunofluoresence staining. "
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    ABSTRACT: Epithelial to mesenchymal transitions (EMTs) are thought to be essential to generate diversity of tissues during early fetal development, but these events are essentially impossible to study at the molecular level in vivo in humans. The first EMT event that has been described morphologically in human development occurs just prior to generation of the primitive streak. Because human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are thought to most closely resemble cells found in epiblast-stage embryos prior to formation of the primitive streak, we sought to determine whether this first human EMT could be modeled in vitro with pluripotent stem cells. The data presented here suggest that generating embryoid bodies from hESCs or hiPSCs drives a procession of EMT events that can be observed within 24-48 hours after EB generation. These structures possess the typical hallmarks of developmental EMTs, and portions also display evidence of primitive streak and mesendoderm. We identify PTK7 as a novel marker of this EMT population, which can also be used to purify these cells for subsequent analyses and identification of novel markers of human development. Gene expression analysis indicated an upregulation of EMT markers and ECM proteins in the PTK7+ population. We also find that cells that undergo this developmental EMT retain developmental plasticity as sorting, dissociation and re-plating reestablishes an epithelial phenotype.
    PLoS ONE 11/2012; 7(11):e50432. DOI:10.1371/journal.pone.0050432 · 3.23 Impact Factor
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    • "Aberration Detection Method 2 (ADM2) algorithm (threshold 5.0) was used to identify DNA copy number aberrations. We applied a filtering option of a minimum of 3 aberrant consecutive probes [61] and a minimum absolute average log 2 ratio of 0.30. UCSC human genome assembly hg18 was used as a reference and CNVs were identified with a database integrated into the Agilent Genomic Workbench analytic software. "
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    ABSTRACT: The importance of the genetic factor in the aetiology of premature ovarian failure (POF) is emphasized by the high percentage of familial cases and X chromosome abnormalities account for 10% of chromosomal aberrations. In this study, we report the detailed analysis of 4 chromosomal abnormalities involving the X chromosome and associated with POF that were detected during a screening of 269 affected women. Conventional and molecular cytogenetics were valuable tools for locating the breakpoint regions and thus the following karyotypes were defined: 46,X,der(X)t(X;19)(p21.1;q13.42)mat, 46,X,t(X;2)(q21.33;q14.3)dn, 46,X,der(X)t(X;Y)(q26.2;q11.223)mat and 46,X,t(X;13)(q13.3;q31)dn. A bioinformatic analysis of the breakpoint regions identified putative candidate genes for ovarian failure near the breakpoint regions on the X chromosome or on autosomes that were involved in the translocation event. HS6ST1, HS6ST2 and MATER genes were identified and their functions and a literature review revealed an interesting connection to the POF phenotype. Moreover, the 19q13.32 locus is associated with the age of onset of the natural menopause. These results support the position effect of the breakpoint on flanking genes, and cytogenetic techniques, in combination with bioinformatic analysis, may help to improve what is known about this puzzling disorder and its diagnostic potential.
    Molecular Cytogenetics 07/2012; 5(1):32. DOI:10.1186/1755-8166-5-32 · 2.14 Impact Factor
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