Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biol

Journal of Fertilization: In Vitro - IVF-Worldwide, Reproductive Medicine, Genetics & Stem Cell Biol
Open Access

ISSN: 2375-4508

+44 1478 350008

Research Article - (2015) Volume 3, Issue 2

Genomic Instability in Embryonic Stem Cell: A Mechanism for Adaptation and Pluripotency Maintenance

Clara I Esteban-Pérez1,2*, Harold H Moreno-Ortiz1,2, Carolina Lucena2, Nancy A Reichert1 and Dwayne A Wise1
1Department of Biological Sciences, Mississippi State University, Mississippi State, MS, 39762, USA
2Reproductive Biomedicine, Colombian Center of Fertility and Sterility, CECOLFES, Bogota, Colombia, South America, USA
*Corresponding Author: Clara I Esteban-Pérez, Colombian Center of Fertility and Sterility, CECOLFES, Bogota, South America, Colombia, Tel: 571-7420505, Fax: 571-7422235 Email:

Abstract

Embryonic stem (ES) cells have the ability to maintain pluripotency and self-renewal during in vitro maintenance, which is a key to their clinical applications. ES cell quality has been widely evaluated through the determination of their specific genetic and epigenetic profiles. The hypothesis of this study is that genetic stability in repetitive sequences located near key genes involved in pluripotency, self-renewal, differentiation, chromatin assembly, and imprinting could be a signal for adaptation of the ES cell in vitro. Instability in specific repetitive sequences is present and increases during ES cell passages. ES cells displayed significant mean frequencies of instability in twelve markers out of 64 related to pluripotency (OCT4, D1S551), early differentiation (G60405, D18S63, and D1S468), chromatin assembly (D22S447, D6S2252, D10S529, and HISTB2), and imprinting (GRB10-promoter, D2S144, and IGF2- promoter). Interestingly, instability was different between H1 and H7 ES cell lines. In summary, these results suggest that instability in tandem repeat sequences located near early embryonic developmental genes is associated with failure of ES cell pluripotency and self-renewal maintenance over consecutive culture passages. These results suggest that instability determination is a potential indicator of gene deregulation and epigenetic modification that involves chromatin modification and imprint establishment during ES cell culture. Finally, instability in specific genes could be a signal that contributes to the adaptation of ES cells to in vitro culture or could be the switch that initiates early cell specialization in vitro.

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Keywords: Genomic instability; Embryonic stem cells;Genome repetitive sequences; Self-renewal biomarkers

Introduction

Since the first human embryonic stem (ES) cells were isolated two decades ago, this field of research has generated countless advances and knowledge about early embryonic development and cell fate differentiation [ 1-4]. Studies of ES cell pluripotency and self-renewal as the source of all cell types from the three embryonic germinal layers led to significant discoveries and clinical applications. However, continued maintenance in vitro leads to cellular, genetic, and epigenetic changes in the ES cells, which creates many questions about their real therapeutic potential. The accepted culture conditions used for ES cell maintenance around the world are limited due to different protocols between laboratories. Because of the wide range of variability in the maintenance of homogeneous and undifferentiated ES cells over time during culture passages, the clinical importance of ES cell research is sometimes doubted [5,6].

Several studies have reported changes in ES cell gene expression profiles that occur during long term culture [3,7,8]. Also, the presence of chromosomal abnormalities in late passage cultures of ES cells has been reported [9-13]. Furthermore, the signals or initial steps that lead to gene expression and epigenetic changes remain unknown. A simple screening method to select the best ES cells would be of great use in the field. This study focuses on determining the role of instability in repetitive DNA sequences as a signal of ES cell adaptation or differentiation, and the identification of possible biomarkers useful for screening and determining the quality of ES cells to be used for regenerative therapies.

Instability in flanking regions of developmental genes could affect enhancer or repressor elements that regulate transcriptional patterns of ES cells during in vitro maintenance. In order to understand how genomic instability affects pluripotency of ES cells, self-renewal, and differentiation, we have used a key characterization method to evaluate the effect of the ES cell. As a first step, we have investigated the instability effects of repetitive sequences on ES cells over time, and also we have determined the mean frequency of instability in different markers located close proximity to sequences of important genes responsible for ES cell pluripotency, self-renewal, cell differentiation, chromatin assembly, and imprinting. We have analyzed H1 and H7 ES cell lines during early, middle, and late passages to compare the genomic instability across passages. By determining the mean frequencies of instability for each marker, we identified sensitive repetitive markers that showed significant instability in ES cell cultures over time. In addition, specific genes that were identified as related to the unstable marker were evaluated. This study has established that instability in these specific regions could modulate gene expression and epigenetic signals that determine ES cell adaptation or differentiation stages.

Materials and Methods

Embryonic stem cell maintenance

Frozen aliquots of human ES cells H1-WA01 passage 27 and H7- WA07 passage 26 were purchased from the National Stem Cell Bank Wisconsin International Stem Cell Bank. H1 and H7 ES cells were seeded onto a mouse embryo fibroblast-CF1 (MEF) feeder layer previously inactivated with mitomycin C. The culture medium consisted of Dulbecco’s Modified Eagle Medium (DMEM) knockout medium (Invitrogen, Carlsbad, CA) supplemented with 20% knockout serum replacement (Invitrogen, Carlsbad, CA), 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA), 100 mM L-glutamine (Invitrogen, Carlsbad, CA) plus β-mercaptoethanol, 2 ug/ml basic fibroblast growth factor (b-FGF) (Invitrogen, Carlsbad, CA), and 1% non-essential amino acids (Invitrogen Carlsbad, CA). ES cells were maintained in a humidified atmosphere at 37°C in 5% CO2. Medium was changed daily.

Mouse embryo fibroblast CF1 feeder layer

The mouse embryo fibroblast (MEF-CF1) feeder layer cells were purchased from American Type Culture Collection (ATCC, Rockville, MD). MEF feeder layer cells were cultured in a T-25 flask (Falcon, Becton Dickinson Labware, NJ). The culture medium consisted of DMEM high glucose medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA). MEF cells were mitotically inactivated for 2 hours with 10 μg/ml mitomycin C (Sigma Aldrich, Saint Louis, MO), seeded at densities of 130,000 cells/ml in gelatin coated one-well dishes (Falcon, Becton Dickinson Labware, NJ) and cultured 24 to 48 hours before ES cells were seeded onto the feeder layer. These cells were maintained in a humidified atmosphere at 37°C in 5% CO2.

Embryonic stem cell passages

ES cell colonies with undifferentiated morphologies were mechanically dissected into small pieces under a stereomicroscope and seeded onto a fresh MEF feeder layer during 20 passages (5 months). Cells were passaged every 4-6 days (Supplementary Material Figure S1). Periodically, ES cells were tested for the presence of alkaline phosphatase activity, which is an indicator of the undifferentiated state. We used the alkaline phosphatase detection kit following the manufacturer’s recommended protocol (Millipore, Chemicon, Billeria, MA). Samples of ES cell colonies were dissected for isolation of DNA and RNA early in the culture time (passage 27-28) and during the middle of the culture time (passage 40-42) in both ES cell lines.

Immunohistochemical analysis

ES cell colonies were fixed with 4% paraformaldehyde (Sigma Aldrich, Saint Louis, MO) for 15 minutes at room temperature, washed in PBS, and immunostained. The primary antibodies used were rabbit anti-OCT4 polyclonal antibody, mouse anti-SOX2 monoclonal antibody, and mouse anti-SSEA-1 alexa fluor 488 (Chemicon/Millipore, Billerica, MA). Secondary antibodies included goat-anti-rabbit IgG rhodamine, and CY5-conjugated antibody (Chemicon/Millipore, Billerica, MA). Each antibody was diluted 1:200 in PBS, 0.1% Triton X-100, and 3% BSA. Nuclei were visualized with 4’-6-diamidino-2- phenylindole (DAPI) staining (Vysis Abbott Laboratories, Abbott Park, IL). Staining without primary antibody served as a negative control. Images were captured using a fluorescence microscope Axiovert 135 (Carl Zeiss International) with FITC and rhodamine filter set. Fluorescence intensities were measured with image software developed at the National Institute of Health (Bethesda, MD) downloaded from http://rsb.info.nih.gov/ij/index.html.

DNA isolation

ES cells colonies before isolation of DNA were passage into matrigel plates for eliminate MEFs contamination. DNA was prepared from each sample of ES cells in early passage (27-28) and middle passage (40-42). DNA from late passage (78-82) was provided by the Michigan Center for human ES Cell Research (Ann Arbor, MI) who followed the same protocol for maintenance and passages of cells like our research. DNA was isolated with the Purelink genomic DNA mini kit (Invitrogen Carlsbad, CA) following the manufacturer’s protocol. All DNA samples were quantified using a NanoDrop™ ND1000 spectrophotometer (Thermo Scientific, Wilmington, DE).

Single tandem repeat markers selection and standardization

Single tandem repeats (STRs) are located in or near promoter regions of specific genes responsible for embryonic stem cell pluripotency and self-renewal. We identified DNA sequences that were approximately 1000 bp upstream or downstream of the promoter using UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/) gene sorter and uni-STSNCBI database (http://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi). A total of 64 STR were selected and classified according to ES genetic network regulation database available at (http://www.wi.mit.edu/ young/hESregulation/). Eleven markers were related to pluripotency genes, 33 were related to differentiation genes, 12 were related to chromatin modification genes, and 8 were related to imprinting genes (Table 1). Each STR was optimized to obtain amplified products with robust signal intensity and balanced peak heights from ES cell samples in early passage (27-28), middle passage (40-42), and late passage (78- 82).

Pluripotency Differentiation Chromatin Structure Imprinting
OCT4 D16S3034 D4S1542 D7S488 GRB10PROM
D1S1656 D12S1719 DXS981 D6S1001 D20S821
D1S551 D4S2623 D14S588 HISTH4A IGF2R
D12S1682 D2S134 D3S2459 HISTHB2 DIRAS3PROM
D1S2630 D11S1331 D17S2180 D10S529 PEG10PROM
D6S2384 D4S1625 EGFR D22S447 SNURF10PROM
D6S416 D1S430 D16S3091 D8S11268 IGF2PROM
D2S2327 D2S290 D1S468 D22S941 IGF
kLF4-1 D3S1583 TNFa3 D7S638  
NANOG DXS458 D15S983 D6S2252  
D9S1840 D21S1909 DXS1208 D2S144  
  D6S1698 D5S426 DNMT3  
  D10S1653 D3S1541    
  D11S909 G60405    
  D5S2021 D3S1611    
  D18S63 D11S2179    

Table 1: List of single tandem repeat marker Eleven markers were related to pluripotency genes, thirty-three were related to differentiation genes, twelve were related to chromatin structure genes, and eight were related to imprinting genes

Samples were analyzed with differing amounts of genomic DNA: large DNA concentration (DNA concentration of 0.1 to 1 ng/μl) and single cell DNA concentration (single genome equivalent between DNA concentrations 12.5 to 50 pg/μl). The average for amplifiable DNA (λ) was calculated by Poisson distribution: λ= - ln (number of replicates with non-amplification / total number of replicates) [80]. A λ < 2 means that single genome equivalent of DNA was present in the amplification.

Each locus was standardized in separate PCR reactions to optimize and ensure specificity and sensitivity of the system. Labeled primers with either 6-FAM or HEX dye were used to allow automatic detection. Primers were tested at concentrations of 0.8-1.5 μM in standard PCR conditions and reagents.

Genomic instability determination by single cell PCR

Single cell PCR was performed on 64 STRs (Table 1). Less than a single diploid genome-equivalent of DNA (25-50 pg/μl), was used to perform single cell PCR analysis in 48 replicates for each marker. These concentrations of DNA ensure sensitivity of the PCR to detect wild type and mutated alleles at their appropriate frequency [14]. Total reaction volume of 10 μl containing 1X of buffer D (800 mM Tris HCL, 200 mM (NH4)2SO4, 0.2% w/v Tween 20) (US DNA, Fort Worth, TX), 2.5 mM of MgCl2 (US DNA, Fort Worth, TX), 1.25 U of Hot-MultiTaq DNA polymerase 5 U (US DNA, Fort Worth, TX), 4% of DMSO (Sigma Aldrich, Saint Louis, MO), 0.4 mg/ml of BSA (Thermo Scientific, Rockford, IL), 300 μM of dNTPs mix (Applied Biosystems, Foster City, CA), and 1X of Solution L 5X (enhancer solution for amplification of difficult templates) (US DNA, Fort Worth, TX). The primer volumes for each primer are shown on Supplementary Material Table S1.

PCR was performed on a PE 9600 thermocycler using a ramping protocol: 1 cycle of 95°C for 11 minutes; 1 cycle of 96°C for 1 minute; 10 cycles of [94°C for 30 seconds, ramp 68 seconds to 58°C (hold for 30 seconds), ramp 50 seconds to 70°C (hold for 60 seconds)]; 25 cycles of [90°C for 30 seconds, ramp 60 seconds to 58°C (hold for 30 seconds), ramp 50 seconds to 70°C (hold for 60 seconds)]; 1 cycle of 60°C for 30 minutes for final extension; and hold 4°C. Negative controls per run were included to check for contamination.

Amplified products were mixed with Hi-Di™ formamide and GeneScan™ 500 LIZ Size Standard (35-500 bp) (Applied Biosystems, Foster City, CA) and denatured for 3 min at 95°C to be separated and detected by fragment analysis on a Genetic Analyzer AB3130xl (Applied Biosystems, Foster City, CA). Data were analyzed with the software, GeneMapper version 4.0 (Applied Biosystems Foster City, CA). Quantification of the allele size in comparison with the internal lane size standard was scored in each single cell replicate. An average of 48 replicates per sample plus negative controls were amplified and scored for both ES cell lines.

STR makers are classified according to their repeat motif (number of nucleotides): mononucleotides (1 nucleotide motif), dinucleotide (2 nucleotide motif), trinucleotide (3 nucleotide motif), tetranucleotide (4 nucleotide motif), and pentanucleotide (5 nucleotide motif). Wild type alleles were determined for each microsatellite. Repeat motif shifts from the wild type allele size were considered a mutant allele. Mutant alleles for mononucleotides (e.g. GRB10-PROM, IGF2-PROM, and HISTBH2) were determined by a repeat shift greater than 3 repeats or less than 3 repeats. For dinucleotides (e.g. D18S63, D6S2252, and D10S529), mutants were determined by a repeat shift greater than 2 repeats or less than 3 repeats. For trinucleotides (e.g. D17S2180), tetranucleotides (e.g. OCT4, and D1S551) and pentanucleotides (e.g. DIRAS3-PROM), mutants were determined by a repeat shift greater than 1 repeat or less than 2 repeats (Supplementary Material Figure S2) [14-18].

Statistical analysis of genomic instability

Mutation frequencies (total number of wild type alleles related to the mutant alleles in each marker) were determined for each ES cell line and passage number by SP-PCR software version 2.0 (M.D. Anderson Cancer Center Houston, TX). Differences in mutation frequencies were calculated with a two tailed t-test using raw mutation frequencies using a package SAS/win 9.2 (SAS Institute, Cary, NC). Mutation frequencies of informative markers were considered statistically significant when a p-value was ≤0.05, and were considered marginally significant if the p value was ≤ 0.10

Results

Embryonic stem cell culture maintenance

ES cells were continuously cultured for 20 passages to explore the potential role of genomic instability during ES cell maintenance in vitro under standard conditions with MEFs and growth factors (b-FGF). ES cells from both cell lines (H1 and H7) retained their growth and morphological characteristics: homogenous round and compact colonies, a prominent nucleus and high nucleus: cytoplasm ratio, positive alkaline phosphatase activity and positive expression of the specific pluripotency marker OCT4 and negative expression of the differential marker SSEA-1.

Embryonic stem cells displayed morphological changes across passages

ES cell cultures, in general, could contain fewer than 20% of colonies with heterogeneous morphology corresponding to differentiation. These heterogeneous colonies were removed with a pipette using a stereomicroscope before the subsequent passage [19-21]. H7 ES cells were subcultured more than 20 times continuously for more than 5 months. During that time, they exhibited round and compact colony morphologies. In contrast, H1 ES cells were cultured under the same conditions and time, yet they exhibited an increased number of irregular shapes of colonies with some differentiated cells at the periphery (Figure 1). To explore the ES cell morphological characteristics over passages, we compared differences in the shape of the colonies between H1 and H7 ES cells; we quantified the number of regularly and irregularly shaped colonies from passages 28-42 in H1 ES cells and 27-42 in H7 ES cells. We found that H1 ES cells showed a significant increase in the colonies that exhibited signs of cell differentiation across passages in comparison to the H7 ES cell line (p=0.04). H1 ES cells in passage 40 showed a higher percentage (37%) of irregular colonies than did those from passage 27 (14%) (p=0.047) (Figure 2). The H7 cell line did not show any significant difference across passages. Taken together, these results indicate that H1 ES cells failed to promote complete self-renewal across passages.

fertilization-in-vitro-Morphologies

Figure 1: Morphologies of H1 ES cell colonies
(A) Phase contrast image shows heterogeneous colony morphology with differentiation at the periphery of colony. (B) Phase contrast image shows an undifferentiated homogeneous colony. Phase contrast photomicrographs have a magnification of 10X.

fertilization-in-vitro-colony-morphology

Figure 2: Percentage of H1 and H7 ES colony morphology changes vs culture passages
ES cells were subcultured/passaged approximately 20 times over 4 months by mechanical dissection of the colonies. Throughout, ES cells failed to retain their normal morphology. X axe values are the percentage of colonies with irregular morphology across passages. The differences in morphology for colonies of ES cell lines were statistically significant between H1 and H7 ES cell lines when compared to Y axe culture passages stages: early (passages 27-28) and middle (passages 40-42). p<0.05 (n=4).

Genomic instability in single tandem repeat markers was associated with embryonic stem cell culture adaptation

Because embryonic stem cells in culture maintain pluripotency and self-renewal via genetic rearrangements [3,9-13], we asked whether ES cell cultures are genetically stable in long term cultures. The efficiency of ES cells to maintain genomic stability was evaluated by analyzing single tandem repeat markers found close to specific genes involved in ES cell pluripotency and self-renewal (Table 1). Samples of DNA from H1 and H7 ES cells at three different times (early, middle, and late passages) were analyzed to determine genomic instability in specific markers. There was significant genomic instability in 21 out of 64 single tandem repeat markers evaluated. Both ES cell lines were unstable in these markers over cell passages. However, H1 ES cells became much more unstable than H7 ES cells. H1 ES cells showed significant instability differences between early to middle (p=0.002) and between early to late passages (p=0.025) but differences were not significant between middle to late passage. In contrast, H7 ES cells show a significant difference only between early to middle passage (p=0.057) (Figure 3). These results indicate genomic instability was present during long term ES cell cultures and suggest these could be a signal of cell adaptation.

fertilization-in-vitro-unstable-markers

Figure 3: Number of unstable markers across culture passages
H1 cells show statistically significant differences for frequencies of unstable markers across passages in comparison to H7 cells (p<0.05). Values represent the number of markers that show instability through the passages in each ES cell line. Early (passages 27-28), middle (passage 42), and late (passages 78-82).

Genomic instability could be a signal of embryonic stem cell pluripotency and self-renewal loss during long term cell culture

Increasing evidence suggests that culture passages of ES cells lead to significant changes in gene expression [3,7,8,22]. Our results have shown that during long term culture and subsequent passages, ES cells accumulated instability in single tandem repeats. These markers are located near important genes involved in pluripotency and differentiation. H1 ES cells were unstable in three markers related to pluripotency genes (OCT4, D1S551, and D1S2630) that were completely stable in H7 ES cells over passages. In addition, H1 ES cell showed instability in eight markers related to genes expressed during early differentiation (D2S134, D3S1583, G60405, D11S909, D18S63, DXS981, D17S2180, and DXS1208). In contrast, H7 ES cells showed instability in three different markers related to differentiation (D16S3091, D1S468, and D12S1682). Both ES cell lines showed instability in the differentiation marker DXS1208, but the difference did not reach significance. Statistically significant differences were observed in two pluripotency related markers (OCT4 and D1S551) and three differentiation related markers (G60405, D18S63, and D1S468). D1S551, D18S63, and D1S468 markers showed higher mean values of mutation frequencies at a significant level (p<0.05) compared with the other unstable markers analyzed (Figure 4 and Table 2). We suggest that the presence of genomic instability in markers located near to these specific pluripotency or differentiation genes could be a signal of gene expression changes that induce adaptation or differentiation of the ES cell during long term cultures and multiple passages.

fertilization-in-vitro-pluripotency

Figure 4: Unstable markers related to genes involved in pluripotency and early differentiation
Differences in the number of unstable markers and mean mutation frequencies were observed between H1 and H7 ES cell lines. (A) Shows the number of unstable markers per ES cell line, and the cellular status of either pluripotency or differentiation. (B) Mean values of mutation frequencies of unstable markers related to pluripotency genes. (C) Mean values of mutation frequencies of unstable markers related to differentiation genes. Values represent the mean value of mutation frequency of sample replicates (n=48) per marker that was calculated with SP-PCR software (MD Anderson Cancer Houston, TX). Statistically significance differences *p≤0.05, marginally significance **p≤0.10

ES cells Passage   Oct4     D1S551     G60405     D18S63     D1S468  
  Number n m f n m f n m f n m f n m f
  28 46 2 0.031 54 0 0 33 0 0 37 0 0 45 0 0
H1 42 37 0 0 56 2 0.024 48 2 0.029 37 2 0.028 29 0 0
  82 35 1 0.016 63 0 0 32 0 0 41 0 0 33 0 0
  27 33 0 0 48 0 0 33 0 0 63 0 0 55 0 0
H7 42 27 0 0 74 0 0 25 0 0 34 0 0 64 3 0.028
  78 75 0 0 74 0 0 35 0 0 42 0 0 61 0 0
  p-value     0.06     0.046     0.077     0.036     0.05

Table 2: Mutation frequencies of five single tandem repeat markers located near genes related to pluripotency and differentiation Number of normal alleles (n), number of mutated alleles (m), and mean value of mutation frequency (f) calculated by SP-PCR software (MD Anderson Cancer Houston, TX). p-values ≤0.05 are in bold, p-value ≤0.10 in italic.

Epigenetic changes that occur during embryonic stem cell in vitro culture could result from genomic instability

Imprinting, chromatin assembly, and methylation are essential epigenetic mechanisms that modulate ES cell maintenance [23-25]. We found significant differences in ES cell genomic instability following passages. H1 ES cells showed instability in three markers (D22S447, D6S2252, and D10S529) and H7 ES cells in two markers (D10S529 and HISTHB2) that were related to chromatin assembly (Figure 5 and Table 3). All four chromatin assembly markers were significantly unstable. D22S447 and D6S2252 showed higher mean values of mutation frequencies at significant levels (p<0.05). Instability of the HISTHB2 marker was highly statistically significant in the H7 ES cells (p<0.001). H1 and H7 ES cells showed significant instability differences in the D10S529 marker (p<0.03) (Figure 5). Additionally, unstable markers for imprinting genes were determined. A single tandem repeat in the promoter of GRB10 imprinting gene was found to be unstable in both H1 and H7 ES cells, with a significant difference between them (p=0.026) (Figure 6) (Table 4). H7 ES cells also showed high instability in two additional markers (D2S144 and IGF2-PROM), whereas H1 ES cells were stable for these markers. D2S144 was significantly unstable compared with the IGF2-promoter marker that showed less significance (p=0.04 and p=0.08 respectively) (Figure 6 and Table 4). These findings showing instability of markers located near genes that participate in epigenetic modifications support the idea that genomic instability could be essential to generating epigenetic modifications during ES cell maintenance in vitro.

fertilization-in-vitro-chromatin-assembly

Figure 5: Unstable markers related to chromatin assembly genes
Differences in the number of unstable markers and mean mutation frequencies were observed between H1 and H7 ES cell lines. (A) Number of unstable markers per ES cell line. (B) Mean values for mutation frequencies of unstable markers related to chromatin assembly genes. Values represent the mean mutation frequency of sample replicates (n=48) per marker calculated with SPPCR software (MD Anderson Cancer Houston, TX). HISTHB2 shows highly significant differences in mean mutation frequencies of H7 ES cells (p<0.001). D10S529 shows instability in both ES cells lines, but H7 ES cells show a significantly higher mutation frequency compared to H1 ES cells (p=0.03). Statistically significance *p<0.05.

ES cells PassageNumber   D6S2252     H1STHB2     D10S529     D22S447  
  n m f n m f n m f n m f
  28 67 0 0 47 0 0 47 0 0 46 0 0
H1 42 41 3 0.054 38 0 0 39 0 0 37 2 0.028
  82 49 0 0 40 0 0 40 1 0.012 45 0 0
  27 33 0 0 35 7 0.102 40 3 0.034 53 0 0
H7 42 42 0 0 34 0 0 40 0 0 38 0 0
  78 63 0 0 37 0 0 36 0 0 68 0 0
  p-value     0.018     <0.001     0.03     0.05

Table 3: Mutation frequencies of three single tandem repeat markers located near genes related to chromatin assembly
Number of normal alleles (n), number of mutated alleles (m), and mean value of mutation frequency (f) calculated by SP-PCR software (MD Anderson Cancer Houston, TX). p-value ≤ 0.05 are in bold

fertilization-in-vitro-imprinting-genes

Figure 6: Unstable markers related to imprinting genes
Differences in the number of unstable markers and mean mutation frequencies were observed between H1 and H7 ES cell lines. (A) Number of unstable markers per ES cell line. (B) Mean values for mutation frequencies of unstable markers related to imprinting genes. Values represent the mean mutation frequency of sample replicates (n=48) per marker calculated with SP-PCR software (MD Anderson Cancer Houston, TX). GRB10-PROM shows instability for both ES cell lines, but H1 ES cells show a significantly higher mutation frequency compared to H7 ES cells (p = 0.026). Statistically significance *p<0.05. Marginally significant differences **p <0.10

ES cells PassageNumber   GRB10-PROM     D2S144     IGF2-PROM
  n m f n m f n m f
  28 44 0 0 45 0 0 66 0 0
H1 42 45 1 0.016 28 0 0 54 0 0
  82 40 0 0 34 0 0 65 0 0
  27 35 0 0 37 0 0 53 0 0
H7 42 71 1 0.009 41 1 0.02 58 2 0.022
  78 63 0 0 64 0 0 45 0 0
  p-value     0.026     0.04     0.08

Table 4: Mutation frequencies of three single tandem repeat markers located near genes related to imprinting genes. Number of normal alleles (n), number of mutated alleles (m), and mean value of mutation frequency (f) calculated with SP-PCR software (MD Anderson Cancer Houston, TX). p-values ≤ 0.05 are in bold, p-value ≤ 0.10 in italic.

Discussion

Embryonic stem cells have the capacity for unlimited stem cell proliferation and the ability to differentiate into all the cell lineages derived from the three germinal layers. Questions about the molecular signals of pluripotency and self-renewal maintenance in vitro are still unanswered and are the key to clinical ES cell applications. We evaluated microsatellite marker instability in sequences located near genes known to be responsible for pluripotency and cell differentiation characteristics of ES cells.

Accumulation of DNA damage is observed during cellular stress responses. ES cells in long term cultures have shown genomic instability in response to environmental changes in the form of chromosomal abnormalities after more than 100 passages during in vitro maintenance [9-12]. Genomic instability in single tandem repeats create frame-shift mutations, and enhancer, or repressor modifications that could effect gene expression changes affecting cellular processes. This has been explored widely in tumorigenesis studies [26-30].

ES cells and tumors have common molecular pathways that maintain their cellular characteristics and functions [31-35]. Instability of a single tandem repeat located downstream or upstream of specific pluripotency and self-renewal genes is a reliable tool to characterize genomic stability during ES cell culture in vitro. It can be a potential biomarker to predict and evaluate pluripotency loss and uncontrolled cell differentiation processes during ES cell maintenance.

Our data suggest that instability in pluripotency and differentiation markers is a signal of balance between culture adaptation of ES cells and the differentiation process that is observed as morphological characteristics and genetic stability. H1 colonies became more irregular than did H7 colonies through culture passages. Colony irregularities are morphological signs of differentiation during cell culture and could be related to the DNA instability found in specific markers located near essential genes responsible for optimal ES cell functions. ES cells show low instability during early passages when compared to the mean frequencies of instability during middle and late passages. Several reports suggest that late passages significantly increase the frequency of chromosomal instability due to environmental signals from the in vitro system used to maintain ES cell lines in culture [9-12]. Our results support the idea that ES cell lines exhibit different adaptation processes, seen as genomic instability in early and middle passages as a part of cell adaptation in vitro. However, during later passages, chromosomal instability that enables maintenance of ES cell pluripotency occurs in some stem cell lines. Some studies report that the H1 ES cell line showed trisomy in chromosomes 12 and 17 at 144 passages [10,12,35]. In contrast, H7 ES cell line showed trisomy in chromosome 20 and translocation between chromosome 6 and 17 at passage 209 [10,12,35]. Apparently, chromosomal instability and single tandem repeat instability occur by independent processes that happen during longterm ES cell culture. H1 and H7 ES cell lines showed high rates of single tandem repeat instability during passages 27-28 and 42, but instability frequencies decreased at late passages (78-82 respectively) (Figure 3).

The key findings that emerged from this data included the failure of ES Cells to maintain pluripotency, tendency to differentiate, and epigenetic changes over passages. We identified twelve unstable markers localized near pluripotency, differentiation, chromatin assembly, and imprinting genes that play important roles during early embryogenesis. These genes are involved in specific cell signals that determine genetic and epigenetic modifications relevant to the ES cell: DNA transcription, cell cycle, cell differentiation, tissue specification, apoptosis, and DNA repair.

ES cell genes for pluripotency and self-renewal are actively expressed and are responsible for maintaining all characteristics of the ES cell. When genomic instability occurs around these specific genes, it could lead to loss of pluripotency and self-renewal in the ES cells. We found two unstable pluripotency markers in H1 ES cells; OCT4 and D1S551. OCT4 (POU class 5 homeobox 1) is a transcription factor that plays a role in embryonic development and has been identified as an important gene for ES cell pluripotency [36-38]. OCT4 is part of the ES cell gene network that regulates pluripotency by transcription regulation. OCT4, NANOG, and SOX2 are transcription factors that regulate themselves and bind common target developmental genes important for ES cell maintenance and embryonic development [37,39- 41]. D1S551 is located near a regulator of a G- protein signaling gene. G-proteins are involved in many cell signaling pathways [42-45]. In mouse ES cells, G- protein signaling is present during early neurogenesis and provides control of neuronal differentiation. Studies in mice and rats demonstrated that G- protein is a modulator of calcium channels, and gamma-aminobutyric acid (GABA), and opioid receptors [42,46].

Several reports have shown how gene expression changes occur during ES cell culture passages, but the exact mechanism is not clear [7,22]. Accumulation of DNA damage creates changes in gene expression that induce decline in cell function and loss of the cell’s integrity over time [3,7,8,22]. Long term culture and passages generate oxidative stress that is a source of DNA damage, apoptosis, and cell cycle defects [47-50]. For example, mouse ES cells, after exposure to ionizing radiation, show DNA damage that induces fibroblast cell differentiation [51,52]. Our results, show genomic instability could be a signal of gene expression deregulation. Early embryonic differentiation genes showed genomic instability in H1 ES cells over multiple passages. H1 ES cells cannot completely maintain pluripotency, whereas H7 ES cells can. Differentiation markers that showed instability in H1 ES cells were D2S134, D11S909, D18S63, and DXS981. Interestingly, these are specific markers located near to genes expressed during early embryonic neuroectoderm specialization [53-59]. D17S2180 and DXS1208 are related to endoderm and mesoderm specialization genes, respectively [60-64] (Table 5). In comparison to H7 ES cell unstable markers, D16S3091 is related to early mesoderm gene differentiation, D1S468 is a gene that promotes apoptosis, and D12S1682 is both an endoderm and mesoderm differentiation gene [65-67] (Table 5).

Name Marker Gene Symbol Gene Name Gene Function References
Oct4 POU2F1 POU class 2 homeobox 1 Developmental transcription factor [36,72]
D1S551 RGS4 Regulator of G-protein signalling 4 Signal transduction regulator [42,43]
D1S2630 POU2F1 POU class 2 homeobox 1 Developmental transcription factor [38,40,41]
D2S134 ME1S1 Meishomeobox 1 Embryo development [57,59]
D3S1583 RARB Retinoic acid receptor, bet Embryo development  
G60405 ERCC6 Excision repair cross-complementing group 6 DNA repair  
D11S909 ST5 suppression of tumorigenicity 5 Tumor suppressor  
D18S63 TG1F1 TGFB-induced factor homeobox 1 growth factor activity [54,56]
DXS981 SPG16 spastic paraplegia Developmental gene [53]
D17S2180 HOXB5 homeobox B5 Developmental transcription factor [60,62]
DXS1208 HSPB1 Heat shock thermic protein Developmental transcription factor [64]
D16S3091 CDF13 cadherin 13, H-cadherin (heart) Growth factor activity  

Table 5: List of unstable markers Summary of characteristics of genes located in close proximity to unstable markers involved in embryonic development. Twelve markers showed statistically significant instability frequencies

Genomic instability is a multistep process that involves genetic and epigenetic modifications that induce opposite effects on the status of ES cell pluripotency. Epigenetic changes such as chromatin assembly, imprinting, and methylation are responsible for determining transcriptional patterns dependent upon the cell stage. Imprinting is a switch for gene transcription that ensures cell proliferation, development, and tissue specific functions [50,66,68- 71]. Developmental genes for ES cells have a specific pattern of histone modifications that determine the status of activation of specific genes involved in embryonic development and cell fate during differentiation by de novo methylation. For example, the OCT4 gene is unmethylated during pluripotency by bivalent histone modifications to ensure cell proliferation and development. However, OCT4 is completely repressed when cell differentiation occurs [72-74]. ES cell lines in vitro fail to maintain a specific epigenetic pattern, inducing changes in the cellular status that leads to loss of ES cell pluripotency over time [23,25,50,75]. In our study, H1 and H7 ES cells showed significant differences of instability in markers that were located next to chromatin assembly and imprinting genes across time. Genomic instability was observed in markers such as D22447, D6S2252, HISTHB2, and D10 S529, all of which were located near to genes that code for histone proteins. The D6S2252 marker is located next to HIST1H2AH (linker histone H1), which interacts with the DNA between nucleosomes and is responsible for chromatin compaction [33,76,77]. D10S529 is a marker for a variant histone, H2AFY2 that contributes to the inactivation of the X chromosome [78-80]. In zebra fish embryos, it has been observed that H2AFY2 is involved in the activation of neuronal differentiation genes such as the homeobox A1 gene (HOXA), which encodes a DNA-binding transcription factor to control gene expression during embryonic development and cell differentiation [79]. D22S447 is a histone cell cycle regulator A (HIRA) that is a homolog of Saccharomyces cerevisiae histone. HIRA is responsible for controlling cell growth by regulation of cell cycle genes [81]. Taken together, our results suggest that instability in these markers could be the signals that induce X chromosome inactivation, ES cell growth, and differentiation through changes in expression of developmental and differentiation genes over multiple passages. Additionally, imprinting markers showed instability and are involved in the embryonic methylation process. D2S144 is a marker for the DNA (cytosine-5-)-methyltransferase 3 alpha gene (DNMT3A) that is responsible for epigenetic modification of de novo DNA methylation important for embryonic development, differentiation, imprinting, and X-chromosome inactivation [82,83]. Other unstable markers are located next to the promoter region of imprinting genes, such as GRB10 and IGF2, which are imprinted in a tissue specific manner. These results confirm that H1 and H7 ES cells have a constant and actively regulated process across passages that control genetic and epigenetic outcomes to ensure ES cell growth, maintenance of cell feature characteristics or cell differentiation in vitro.

In conclusion, our findings indicate that maintenance of ES cell genetic and epigenetic characteristics is compromised by the loss of DNA integrity in tandem repeat sequences that flank specific genes that are responsible for the pluripotency and self- renewal of ES cell maintenance, cell fate during differentiation, chromatin assembly, imprinting, and methylation. From our data, we can support the idea that genomic instability could be responsible for genetic and epigenetic imbalances originating in long term ES cell cultures. The exact signals that coordinate this process are complex and not completely known. Even so, our data support our hypothesis that instability in repetitive sequences located close to specific genes could be the signal for adaptation or differentiation of ES cells in culture passages over time. Future studies could be focus in evaluate the level of expression and methylation of these candidate proximal genes.

Furthermore, our results identify biomarkers that could be part of an ES cell characterization process that evaluates genomic integrity through in vitro maintenance procedures. Understanding the role of genomic instability in ES cell maintenance could lead to the origin of an accurate approach for the safety and reliability needed in regenerative medical applications of human ES cells.

Acknowledgements

We would like to thank the National Stem Cell Bank for providing the H1 (WA01) and H7 (WA07) human embryonic stem cell lines. Also, we extend our thanks to Dr. K. Sue O’Shea and Crystal Pacut at the Consortium for Stem Cell Therapies at the University of Michigan for providing the DNA samples from late passage of H1 and H7 ES cell lines. We would like to thank Dr. Karen Coats, Dr. Janet Donaldson, and Kortney Wilkinson for manuscript editing. This research was funded by the Department of Biological Sciences; Office of Institutional Research; Office of the Graduate School, and the College of Art and Sciences at Mississippi State University.

References

  1. Evans, MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154-156.
  2. Thomson JA,Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. (1998) Embryonic stem cell lines derived from human blastocysts. 282: 1145-1147.
  3. Brimble SN, Zeng X, Weiler,DA, Luo Y, Liu Y, et al. (2004) Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev 13:585-597.
  4. Enver T, Soneji S, Joshi C, Brown J, Iborra F, et al. (2005) Cellular differentiation hierarchies in normal and culture adapted human embryonic stem cells. HumMolGenet 14: 3129-3140.
  5. Toyooka Y,Shimosato D, Murakami K, Takahashi K, Niwa H (2008) Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135: 909-918.
  6. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, et al. (2008) The ground state of embryonic stem cell self-renewal. Nature 453: 519-523.
  7. Abeyta MJ, Clark AT, Rodriguez RT, Bodnar MS, Pera RA, et al. (2004) Unique gene expression signatures of independently-derived human embryonic stem cell lines. Hum Mol Genet 13: 601-608.
  8. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, et al. (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2: 185-190.
  9. Amit M, Carpenter MK, Inokuma,MS, Chiu CP, Harris CP, et al. (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. DevBiol 227: 271-278.
  10. Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, et al. (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22: 53-54.
  11. Inzunza J, Sahlén S, Holmberg K, Strömberg AM, Teerijoki H,et al. (2004) Comparative genomic hybridization and karyotyping of human embryonic stem cells reveals the occurrence of an isodicentric X chromosome after long-term cultivation. Mol HumReprod10: 461-466.
  12. Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, et al. (2005) Genomic alterations in cultured human embryonic stem cells. Nat Genet 37: 1099-1103.
  13. Ogawa K,Nishinakamura R, Iwamatsu Y, Shimosato D, Niwa H (2006) Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. BiochemBiophys Res Commun 343: 159-166.
  14. Coolbaugh-Murphy M,Maleki A, Ramagli L, Frazier M, Lichtiger B, et al. (2004) Estimating mutant microsatellite allele frequencies in somatic cells by small-pool PCR. Genomics 84: 419-430.
  15. Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, et al. (1998) National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58: 5248-5257.
  16. Suraweera N, Duval A, Reperant M, Vaury C, Furlan D, et al. (2002) Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroent123: 1804-1811.
  17. Coolbaugh-Murphy MI, Xu J, Ramagli LS, Brown BW, Siciliano MJ (2005) Microsatellite instability (MSI) increases with age in normal somatic cells. Mech Ageing Dev 126: 1051-1059.
  18. Goel A,Nagasaka T, Hamelin R, Boland CR (2010) An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers. PLoS One 5: e9393.
  19. International Stem Cell Initiative,Adewumi O, Aflatoonian B, Ahrlund-Richter L, Amit M, et al. (2007) Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25: 803-816.
  20. Veraitch FS, Scott R, Wong JW, Lye GJ, Mason C (2008) The impact of manual processing on the expansion and directed differentiation of embryonic stem cells. BiotechnolBioeng99: 1216-1229.
  21. Kent L (2009) Culture and maintenance of human embryonic stem cells. J Vis Exp .
  22. Gu B, Zhang J, Wang W, Mo L, Zhou Y, et al. (2010) Global expression of cell surface proteins in embryonic stem cells. PLoS One 5: e15795.
  23. Bibikova M,Chudin E, Wu B, Zhou L, Garcia EW, et al. (2006) Human embryonic stem cells have a unique epigenetic signature. Genome Res 16: 1075-1083.
  24. Collas P1 (2009) Epigenetic states in stem cells. BiochimBiophysActa 1790: 900-905.
  25. Ahmed K,Dehghani H, Rugg-Gunn P, Fussner E, Rossant J, et al. (2010) Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo. PLoS One 5: e10531.
  26. Cahill DP,Lengauer C, Yu J, Riggins GJ, Willson JK, et al. (1998) Mutations of mitotic checkpoint genes in human cancers. Nature 392: 300-303.
  27. Roelofs H,Mostert MC, Pompe K, Zafarana G, van Oorschot M, et al. (2000) Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis. Am J Pathol 157: 1155-1166.
  28. Smiraglia DJ,Plass C (2002) The study of aberrant methylation in cancer via restriction landmark genomic scanning. Oncogene 21: 5414-5426.
  29. Kremenskoy M,Kremenska Y, Ohgane J, Hattori N, Tanaka S, et al. (2003) Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses. BiochemBiophys Res Commun 311: 884-890.
  30. Gorringe KL, Chin SF, Pharoah P, Staines JM, Oliveira C, et al. (2005) Evidence that both genetic instability and selection contribute to the accumulation of chromosome alterations in cancer. Carcinogen 26: 923-930.
  31. Summersgill BM, Jafer O, Wang R, Goker H, Niculescu-Duvaz I, et al. (2001) Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses. Cancer Genet Cytogenet128: 120-129.
  32. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, et al. (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. ProcNatlAcadSci U S A 100: 13350-13355.
  33. Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, et al. (2004) Role of histone H2A ubiquitination in Polycomb silencing. Nature 431: 873-878.
  34. Andrews PW, Martin MM, Bahrami AR, Damjanov I, Gokhale P, et al. (2006) Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: Opposite sides of the same coin. BiochemSoc T 33: 1526-1530.
  35. Baker DE, Harrison NJ, Maltby E, Smith K, Moore HD, et al. (2007) Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25: 207-215.
  36. Ying QL, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115: 281-292.
  37. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, et al. (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122: 947-956.
  38. Masui S,Nakatake Y, Toyooka Y, Shimosato D, Yagi R, et al. (2007) Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol 9: 625-635.
  39. Babaie Y,Herwig R, Greber B, Brink TC, Wruck W, et al. (2007) Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 25: 500-510.
  40. Chen X,Xu H, Yuan P, Fang F, Huss M, et al. (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133: 1106-1117.
  41. Fernandez-Tresguerres B, Cañon S, Rayon T, Pernaute B, Crespo M, et al. (2010) Evolution of the mammalian embryonic pluripotency gene regulatory network. PNAS 107: 19955-19960.
  42. Strübing C,Rohwedel J, Ahnert-Hilger G, Wiedenmann B, Hescheler J, et al. (1997) Development of G protein-mediated Ca2+ channel regulation in mouse embryonic stem cell-derived neurons. Eur J Neurosci 9: 824-832.
  43. Charlesworth P, Komiyama N, Grant S (2006) Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: Normal electrophysiological and morphological properties in vitro. BMC Neuroscience 7: 47-58.
  44. Ebert PJ, Campbell DB, Levitt P (2006) Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of g protein signaling 4 during development. I. Cerebral cortex. Neurosci 142: 1145-1161.
  45. Rusin KI, Moises HC (1998) Mu-opioid and GABA(B) receptors modulate different types of Ca2+ currents in rat nodose ganglion neurons. Neurosci 85: 939-956.
  46. Wu X, Noh SJ, Zhou G, Dixon JE, Guan KL (1996) Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells. J BiolChem 271: 3265-3271.
  47. Lengauer C,Kinzler KW, Vogelstein B (1997) Genetic instability in colorectal cancers. Nature 386: 623-627.
  48. Eden A,Gaudet F, Waghmare A, Jaenisch R (2003) Chromosomal instability and tumors promoted by DNA hypomethylation. 300: 455.
  49. Allegrucci C, Wu YZ, Thurston A, Denning CN, Priddle H, et al. (2007)Restriction landmark genome scanning identifies culture induced DNA methylation instability in the human embryonic stem cell epigenome. HumMol Genet 16: 1253-1268.
  50. Saretzki G, Armstrong L, Leake A, Lako M, von Zglinicki T (2004) Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells. Stem Cells 22: 962-971.
  51. Maynard S,Swistowska AM, Lee JW, Liu Y, Liu ST, et al. (2008) Human embryonic stem cells have enhanced repair of multiple forms of DNA damage. Stem Cells 26: 2266-2274.
  52. Tamagaki A,Shima M, Tomita R, Okumura M, Shibata M, et al. (2000) Segregation of a pure form of spastic paraplegia and NOR insertion into Xq11.2. Am J Med Genet 94: 5-8.
  53. Pazmany T, Tomasi TB (2006) The major histocompatibility complex class II transactivator is differentially regulated by interferon-gamma and transforming growth factor-beta in microglial cells. J Neuroimmunol 172: 18-26.
  54. Wang Y, Wang L, Wang Z (2008) Transgenic analyses of TGIF family proteins in Drosophila imply their role in cell growth. J Genet Genomics 35: 457-465.
  55. Hamid R, Brandt SJ (2009) Transforming growth-interacting factor (TGIF) regulates proliferation and differentiation of human myeloid leukemia cells. MolOncol 3: 451-463.
  56. Mojsin M, Stevanovic M (2009) PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region. BiochemJ 425: 107-116.
  57. Göhring I,Tagariello A, Endele S, Stolt CC, Ghassibé M, et al. (2010) Disruption of ST5 is associated with mental retardation and multiple congenital anomalies. J Med Genet 47: 91-98.
  58. Xiang P, Lo C, Argiropoulos B, Lai CB, Rouhi A, et al. (2010) Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1. ExpHematol 38: 798-798, 808.
  59. Fu M,Lui VC, Sham MH, Cheung AN, Tam PK (2003) HOXB5 expression is spatially and temporarily regulated in human embryonic gut during neural crest cell colonization and differentiation of enteric neuroblasts. DevDyn 228: 1-10.
  60. Wu Q, Lothe RA, Ahlquist T, Silins I, et al. (2007) DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB, SCGB3A, and CRABP1 as novel targets. MolCancer 10: 45.
  61. Lui VCH, Cheng WWC, Leon TYY, Lau DKC, Garcia-Bareclo MM, et al. (2008) Perturbation of Hoxb5 signaling in vagal neural crests down-regulates ret leading to intestinal hypoganglionosis in mice. Gastroenterol 134: 1104-1115.
  62. Kumarapeli A, Horak K, Wang, X (2010) Protein quality control in protection against systolic overload cardiomyopathy: The long term role of small heat shock proteins. Am JTransl Res 2: 390-401.
  63. Schwarz L, Vollmer G, Richter-Landsberg C (2010) The small heat shock protein HSP25/27 (HSPB1) is abundant in cultured astrocytes and associated with astrocytic pathology in progressive supranuclear palsy and corticobasal degeneration. Int J Cell Bio 717520: 1-10.
  64. Wechsler J, Choi YH, Krall J, Ahmad F, Manganiello VC, et al. (2002) Isoforms of cyclic nucleotide phosphodiesterase PDE3A in cardiac myocytes. J BiolChem 277: 38072-38078.
  65. Kim KP, Thurston A, Mummery C, Ward-van Oostwaard D, Priddle H, et al. (2007) Gene-specific vulnerability to imprinting variability in human embryonic stem cell lines. Genome Res 17: 1731-1742.
  66. Sayan BS, Yang AL, Conforti F, Tucci P, Piro MC, et al. (2010) Differential control of TAp73 and DeltaNp73 protein stability by the ring finger ubiquitin ligase PIR2. ProcNatlAcadSci U S A 107: 12877-12882.
  67. Kamakaka RT, Thomas JO (1990) Chromatin structure of transcriptionally competent and repressed genes. EMBO J 9: 3997-4006.
  68. Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33 Suppl: 245-254.
  69. Dhara SK,Benvenisty N (2004) Gene trap as a tool for genome annotation and analysis of X chromosome inactivation in human embryonic stem cells. Nucleic Acids Res 32: 3995-4002.
  70. Shen Y, Matsuno Y, Fouse SD, Rao N, Root S, et al. (2008) X-inactivation in female human embryonic stem cells is in a nonrandom pattern and prone to epigenetic alterations. PNAS 105: 4709-4714.
  71. Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, et al. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet 38: 431-440.
  72. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, et al. (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448: 553-560.
  73. Chamberlain SJ, Yee D, Magnuson T (2008) Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency. Stem Cells 26: 1496-1505.
  74. Yu J,Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. 318: 1917-1920.
  75. Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, et al. (2005) Formation of macroH2A-containing senescence associated heterochromatin foci and senescence driven by asf1a and hira. DevCell 8: 19-30.
  76. Petty EL, Collette KS, Cohen AJ, Snyder MJ, Csankovszki G (2009) Restricting dosage compensation complex binding to the X chromosomes by H2A.Z/HTZ-1. PLoS Genet 5: e1000699.
  77. Chadwick BP, Willard HF (2001) Histone H2A variants and the inactive X chromosome: identification of a second macroH2A variant. Hum Mol Genet 10: 1101-1113.
  78. Buschbeck M, Uribesalgo I, Wibowo I, Rue P, Martin D, et al. (2009) The histone variant macroH2A is an epigenetic regulator of key developmental genes. Nature StructMol Bio 16: 1074-1079.
  79. Gamble MJ,Frizzell KM, Yang C, Krishnakumar R, Kraus WL (2010) The histone variant macroH2A1 marks repressed autosomal chromatin, but protects a subset of its target genes from silencing. Genes Dev 24: 21-32.
  80. Ahmad A, Kikuchi H, Takami Y, Nakayama T (2005) Different roles of n-terminal and c-terminal halves of hira in transcription regulation of cell cycle related genes that contribute to control of vertebrate cell growth. J BiolChem 280: 32090-32100.
  81. Chen T, Ueda Y, Xie S, Li E (2002) A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J BiolChem 277: 38746-38754.
  82. Wienholz BL, Kareta MS, Moarefi AH, Gordon CA, Ginno PA, et al. (2010) DNMT3L modulates significant and distinct flanking sequence preference for DNA methylation by DNMT3A and DNMT3B in vivo. PLoS Genet 6: e1001106.
Citation: Esteban-Pérez CI, Moreno-Ortiz HH, Lucena C, Reichert NA, Wise DA(2015) Genomic Instability in Embryonic Stem Cell: A Mechanism for Adaptation and Pluripotency Maintenance. JFIV Reprod Med Genet 3:142.

Copyright: © 2015 Esteban-Pérez CI, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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