Article

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: 7.7). 07/2008; 26(6):1484-9. DOI: 10.1634/stemcells.2007-0993
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
135 Views
  • Source
    [Show abstract] [Hide abstract]
    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; DOI:10.1093/molehr/gat077 · 3.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Genomic instability is recognized as one of the most important hurdles in the expanding field of stem cell-based therapies. In the recent years, an accumulating body of evidence has shown that human stem cells undergo a diverse program of biological changes upon ex vivo cultivation that include numerical and structural chromosomal abnormalities, point mutations, variation of telomere length and epigenetic instability. As the field moves forward, the growing awareness of the risk factors associated with human genome plasticity strongly advocates for the use of extensive genetic screening as part of a quality control platform to attest to the safety of stem cell-based products. Here we present a timely and comprehensive review that addresses the current status and emerging trends of the field, ultimately underscoring the need to implement new regulatory standards that are able to streamline the route to therapeutic applications. Stem Cells 2014
    Stem Cells 11/2014; 32(11). DOI:10.1002/stem.1796 · 7.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However, optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs, including being karyotypically normal and their ability to generate teratomas containing all 3 germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20, they remain highly useful. In particular, CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus, 10(8) CA1S cells can be generated within one week from 10(6) cells to seed 10(6) wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm, a finding that was confirmed with H1 hESCs, although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines, these cells should ease the development of optimized hESC growth and differentiation protocols. In particular, they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 10/2013; 110(10). DOI:10.1002/bit.24936 · 4.16 Impact Factor

Full-text (3 Sources)

Download
39 Downloads
Available from
May 28, 2014