RESEARCH ARTICLE Molecular Reproduction & Development 82:344–355 (2015)

Derivation of Normal Diploid Human Embryonic Stem Cells From Tripronuclear Zygotes With Analysis of Their Copy Number Variation and Loss of Heterozygosity XUEMEI CHEN,1,2 WENBIN NIU,1 FANG WANG,1 WENZHU YU,1 SHANJUN DAI,1 HUIJUAN KONG,1 YIMIN SHU3, YINGPU SUN1*

AND

1

Reproductive Medical Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China Department of Human Anatomy, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China 3 Department of Obstetrics and Gynecology, Stanford University Medical Center, Palo Alto, California 2

SUMMARY This study sought to establish archives of genetic copy number variation (CNV) in human embryonic stem cell (hESC) lines that are associated with known diseases. We collected patients’ fresh, discarded zygotes from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) protocols. A total of 208 fresh, tripronuclear, discarded zygotes were also collected in this study from patients on the third day of their treatment cycle, prior to transfer. The blastula-formation rates were 13.51% (26/ 192) and 26.7% (4/15) while the high-quality blastocyst formation rates were 5.8% (11/ 192) and 20% (3/15) in the IVF and ICSI groups, respectively. The inner cell mass (ICM) from each embryo was mechanically separated, and then grown on feeder layers consisting of mouse embryonic fibroblasts and human foreskin fibroblasts (a 1:1 mixture). The hESC karyotype was determined by traditional G-banding; analysis of the results for the Zh19P25 and Zh20P24 cell lines showed that both were 46 XY. CNV and loss-of-heterozygosity analysis of hESC gDNA was performed to assess the genetic characteristics associated with molecular diseases using the high-resolution Infinium High-Density HumanCytoSNP-12 DNA chip. Seven CNVs in Zh19P25 and Zh20P24 were deletions, and a region that corresponds to PotockiShaffer disease, 11p11.211p11.12 in Zh20P24, showed a 2.98-Mb loss. These data together suggest that single-nucleotide polymorphism (SNP) microarray analysis for molecular cytogenetic features can help to distinguish hESC lines with a normal karyotype from tripronuclear zygotes with known, disease-related characteristics. Mol. Reprod. Dev. 82: 344355, 2015. ß 2015 Wiley Periodicals, Inc. Received 6 June 2014; Accepted 3 March 2015

INTRODUCTION Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the blastocyst (Thomson et al., 1998), which are most commonly obtainedpursuant to law from discarded embryos from assisted reproduction treatments and/or frozen-recovered embryos that were

ß 2015 WILEY PERIODICALS, INC.



Corresponding author: Reproductive Medical Center The First Affiliated Hospital of Zhengzhou University Number one, Constructive East Rd Zhengzhou City 450052, China. E-mail: [email protected]; [email protected]

Grant sponsor: National Natural Science Foundation of China; Grant number: 31271605

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22485

stored but no longer needed. When blastocysts are developed in vitro from discarded fertilized eggs, 0-, 1-, and Abbreviations: CNV, copy number variation; hESCs, human embryonic stem cells; ICM, inner cell mass; ICSI, intra-cytoplasmic sperm injection; IVF, in vitro fertilization; PN, pronucleus; SNP, single nucleotide polymorphism

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3-pronucleus (PN)-containing zygotes have been reported to give rise to a karyotypically normal hESC line (Suss-Toby et al., 2004; Lavon et al., 2008; Strom et al., 2010). The efficiency to establishing hESCs is approximately 310%, independent of the source of the blastocyst (Hanson and Caisander, 2005). hESCs have self-renewal and unlimited-proliferation capabilities in vitro, and maintain their potential to differentiate into various cells of the human body. Indeed, scientists have developed methods to specifically differentiate hESCs into cells of several tissues and organs. These approaches provide potential new treatment options in the field of regenerative medicine, but also create great opportunities and challenges; one of the biggest hurdles is to ensure the safety of hESCs for clinical treatment. For example, hESCs retain their indefinite proliferation potential and pluripotency characteristics, which may be hazardous for clinical applications. Moreover, the stability of the genetic content of hESCs is of great importance since the unstable genomes can accelerate the onset of diseases including cancer. Therefore, the genetic stability of these cells must be re-evaluated before they are used extensively in the clinic. Established hESCs should have a normal genetic background, but may acquire chromosome deletions, mutations, or undergo methylation during routine culturing and passaging in vitro, which can lead to changes in the genetic background or epigenetic characteristics of the lines (Yu et al., 2012). For example, an aneuploid karyotype can appear spontaneously in hESCs after long-term subculturing (Buzzard et al., 2004; Maitra et al., 2005). But how often do smaller changes occur? The resolution of conventional karyotyping using G-banding, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) is low; for example, G-banding can resolve only 320 Mb (Narva et al., 2010). New DNA array-based methods, such as single nucleotide polymorphism (SNP) detection, have increased resolution from the Mb to the single-base level. Indeed, SNP arrays have been used to study copy number variations (CNVs) and loss of heterozygosity (Standfuss et al., 2012).

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With the inherent movement toward hESC-based clinical therapies in mind, this study aimed to identify the pluripotency and genetic stability of hESCs derived from 3PN embryos. The molecular cytogenetics HumanCytoSNP-12 chip was used to detect DNA duplications, deletions, amplifications, copy-neutral loss-of-heterozygosity, and mosaicism. The average SNP call rate and reproducibility were greater than 99% in this study, including dense coverage of approximately 250 genomic regions and targeted coverage in approximately 400 additional diseaserelated genes. Therefore, incorporating HumanCytoSNP12 chip analysis to clarify the molecular genetic backgrounds of hESCs related to diseases may provide the profiles of hESC lines needed to determine their appropriateness for clinical applications.

RESULTS Derivation of hESC Lines From Fresh, Discarded Zygotes A total of 208 3PN fresh, discarded human zygotes were collected on the third day from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with embryo transfer treatment cycles. These zygotes were then cultured in vitro until the blastocyst stage for establishing hESC lines. Thirty blastocysts formed (Fig. 1 and Table 1), representing a blastocyst-formation rate of 13.51% from the IVF group and 26.67% from the ICSI group. Highquality blastocysts (representing at least a grade of 4BB) were less abundant, forming at a rate of 5.80% from the IVF group and 20.00% from the ICSI group (Table 2). There were no significant differences in the high-quality blastocyst formation rate between the IVF and ICSI (P > 0.05) groups (Table 3).

Characterization of hESC Lines Morphology and alkaline phosphatase activity. The 14 high-quality blastocysts with a grade greater than 3BB were dissected, and their ICMs were transferred to a

Figure 1. Fresh, discarded 3PN zygotes, and their resultant blastocyst formation. (A) A 3PN zygote. (B) An 8-cell embryo. (C) A hatched blastocyst. Scale bar, 100 mm.

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TABLE 1. Blastulation and Derived hESCs Cell Lines Embryos

ART

Embryos source

Cells

D3 grade*

Blastocyst grade*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

IVF IVF IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF ICSI IVF IVF ICSI IVF ICSI IVF IVF IVF IVF IVF IVF IVF IVF IVF IVF IVF IVF

3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN 3PN

10 12 8 8 4 4 8 5 5 8 5 8 4 6 4 10 5 4 5 8 Fragmented 5 Fragmented 4 12 8 8 8 6 8

IV III II II IV II II III III III III III III II III IV III IV III II IV III IV II II II III II II II

5CC 1CC 5CC 5BB 6BB 4CC 6BB 6BB 6CB 4BB 6AA 4BC 5BB 5BB 5CB 4BB 6AA 5CC 4CB 5AA 4BC 4CB 3BC 4BB 4BB 4BC 6BA 5BC 5CC 5CB

hESCs cell lines

ZZU-hES-20

ZZU-hES-19

*D3 and blastocyst grades are defined in the Methods section.

negative for SSEA-1. The feeder layer cells were negative for all markers (Fig. 3).

mitomycin C-treated mixed feeder layer. Nine founder clones formed, and two hESCs cell lines were derived (ZZU-hES-19 and -20, referred to herein as Zh19 and Zh20). These clones exhibited a circular-nest colony growth habit, with very clear boundaries in the surrounding layer and closely packed cells within the colony. The cells were small and contained relatively large nuclei, a high nuclear-to-cytoplasmic ratio, and prominent nucleoli. In addition, the hESCs showed strong alkaline phosphatase activity, whereas the feeder layer cells did not show any (Fig. 2).

Differentiation of hESCs. Both of the hESC lines formed embryoid bodies in vitro. In each case, positive staining was observed for ectoderm neuronal (nestin), mesoderm (myocardial troponin), and endoderm (alpha fetal protein, AFP) markers (Fig. 4).

Expression of stem cell-lineage genes. Pluripotency-specific gene expression was assessed by reverse-transcriptase PCR in Zh19 and Zh20 lines. The H9P25 hESC line was used as a positive control, and mouse embryonic fibroblasts were used as a negative control. POU5F1 (191 bp) and NANOG (169 bp), both transcription factors associated with stem-cell pluripotency and self-renewal, were transcribed (Fig. 5). Semi-

Expression of hESC-specific antigens. Like the previously derived H9 embryonic stem cell lines, the two hESC lines derived herein stained positive for typical primate embryonic stem-cell surface markers (Thomson et al., 1998; Suss-Toby et al., 2004; Strom et al., 2010), such as SSEA-4, TRA-1-60, and TRA-1-81, but were

TABLE 2. Blastocyst-Formation Rate of Fresh, Discarded 3PN Embryos (%) Pronucleus 3PN

Blastocyst class

IVF

ICSI

P

Blastocyst High-quality

13.51 (26/192) 5.8% (11/192)

26.67 (4/15) 20% (3/15)

0.16 0.11

Confidence intervals were set at a ¼ 0.05 and P < 0.05 was considered significant.

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TABLE 3. Blastocyst Formation of Fresh, Discarded 3PN Embryos IVF ICSI

Non-blastocyst

Blastocyst

Total

Non-blastocyst Blastocyst

10 2

3 0

13 2

P ¼ 0.6286. (Fisher’s exact test). Confidence intervals were set at a ¼ 0.05, and P < 0.05 was considered significant.

quantitative analysis revealed no statistical difference in POU5F1 and NANOG transcript abundance between Zh19, Zh20, and H9 lines (Table 4).

Genetic Analysis of hESCs G-banding. When analyzed by conventional karyotype G-banding, Zh19 and Zh20 hESC lines showed normal 46 XY karyotypes (Fig. 6).

CNV analysis for Zh19P25 and Zh20P24. Using Kyrostudio software to analyze Zh19P25 and Zh20P24, we found losses at chr19q11 of 0.52 and 0.53 Mb in length for the two respective lines. The value of CNV for this region is 1, implying that a single copy of the locus was lost. In both cell lines, gene LOC148189 was affected. As LOC148189 was a single-copy gene, we used it as a reference to evaluate other losses within the cell lines using quantitative real-time PCR (qPCR). Within Zh20P24, we observed a loss at the pericentromeric region 11p11.2p11.12 that is associated with PotockiShaffer syndrome and contains FOLH1, LOC440040, OR4C13, OR4C12, LOC441601, LOC646813, OR4A5, and OR4C46 (Table 5, Figs. 7 and 8).

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DISCUSSION Genetic variation in hESCs has been reported to occur during long-term subculture (Narva et al., 2010). Indeed, hESCs possessing a normal karyotype may even have small or partial mutations that are not detected. As discarded human embryos are currently the mainand limitingsource for generating hESC cell lines, understanding the genomic stability of each line is important for their clinical application. Conventional karyotyping techniques including G-banding, FISH, and CGHare of low resolution, cannot effectively detect chromosome number, and lack the ability to identify small changes in DNA content (Narva et al., 2010; Slovak et al., 2010). The SNP gene-chip approach overcomes these limitations, allowing for the detection of chromosome genetic variations at the single-base level. We, therefore, used SNP technology in this study to detect genetic features of hESCs, define the genetic characteristics of the stem cell, and established archives for the molecular genetics of hESCs. Together, these data could provide more detailed information regarding stem-cell libraries as well as experimental data for the clinical application of hESCs. Of the embryos lacking a pronucleus (0PN), 57% had normal diploid chromosomes, 30% had polyploid chromosomes or chimera, and 13% had chromosomal aneuploidy (Manor et al., 1996). The 1PN embryos had approximately 48.7% normal diploid chromosomes (Yan et al., 2010), whereas the aneuploidy incidence in 3PN zygotes was approximately 7080% (Staessen and Van Steirteghem, 1997). Despite these aneuploidy frequencies, hESCs derived from 0-, 1-, and 3PN zygotes can possess a normal karyotype and express typical stem-cell surface markers (Heins et al., 2004; Suss-Toby et al., 2004; Lavon et al., 2008; Chen et al., 2012a; Jiang et al., 2013). Indeed, our hESCs Zh19 and Zh20 carried normal karyotypes even though they were derived from 3PN zygotes, which are not usually regarded as suitable for continued culture in assisted-reproduction systems; how these 3PN zygotes

Figure 2. hESC morphology and alkaline phosphatase staining. hESCs clones were examined under an inverted phase-contrast microscope (A), and then hESCs were fixed and stained with an alkaline phosphatase ES Cell Characterization Kit (B). Scale bar, 200 mm.

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Figure 3. Expression of pluripotency markers in cultured hESCs. hESCs were fixed onto the plate with 4% buffered formalin phosphate, and then stained for SSEA-1 (green), SSEA-4 (green), TRA-1-60 (red), and TRA-1-81 (red) using antibodies in an ES Cell Characterization Kit. Nuclei were counterstained with DAPI (blue). Scale bars, 50, 100, or 200 mm.

generated hESC lines with normal karyotypes remains unclear. Several reasons were proposed for this unexpected ‘‘rescue’’ observation (Lavon et al., 2008; Strom et al., 2010): (a) Aneuploid cells may contribute to the trophectoderm and therefore be excluded from the ICM. Indeed, mosaic embryos have been reported at all stages of development (Harper et al., 2004; Gianaroli et al., 2001). (b) Aneuploid cells contribute to the ICM at a low level, and may be eliminated or selected against during hESC culture since euploid cells divide faster than aneuploid cells (Lavon et al., 2008). Finally, (c) The third pronucleus is a consequence of second-polar-body retention in the zygote (Richard et al., 2003). Thus, the extra sets of chromosomes would be excluded from metaphase or discarded by the developing embryo through normal cell-checkpoint processes (Strom et al., 2010). Given that our hESC, derived from discarded 3PN zygotes, do not exhibit extensive aneuploidy, such samples may be an important resource for the isolation of new lines in the future, without significant ethical issues. Indeed, many abandoned, clinically abnormal zygotes develop into blastocysts, whose ICMs can be used to establish

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hESCs representing various chromosome disease models. Therefore, using discarded embryos is still the most effective, ethical approach for generating new hESCs. Abnormal karyotypes have been reported for different hESC lines, including H1, H7, H14, HUES5, HUES6, HUES7, HUES10, HUES13, HUES14, HUES17, Shef1, Shef4, and Shef5. The most-frequent karyotypic disruption observed was a gain in chromosomes 12, 17, and X (Baker et al., 2007). In contrast, the Zh19 and Zh20 cell lines derived herein showed a normal karyotype (46 XY) by G-banding; this stability could be a result of the mechanical methods used to separate the ICM from the trophectoderm, as well as to passage these hESCs. The accumulation of SNP, CNV, and loss-of-heterozygosity mutations during the hESC culturing process hav also been reported (Mosher et al., 2010; Narva et al., 2010; Amps et al., 2011). Duplications are common for chromosomes 1, 12, 17, 20, and X whereas chromosomal losses occur in 10p and 18q. Both of our hESC lines carried the same 0.52 Mb chromosomal deletion at locus 19 q11. Additional analysis of CNV-related diseases and genes revealed a 2.93 Mb loss of chromosome 11p11.2 p11.12

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Figure 4. Differentiation of hESCs in embryoid bodies. Embryoid bodies formed during in vitro differentiation were fixed onto the plate with 4% buffered formalin phosphate, and then stained for (A) nestin (green; ectoderm/neural), (B) myocardial troponin (red; mesoderm), or (C) alpha-fetal protein (red; endoderm) using antibodies in a Human Embryonic Germ Layer Characterization Kit. Nuclei were counterstained with DAPI (blue). Scale bars, 20 mm (B) or 50 mm (A, C).

in Zh20P24; this region is linked to PottsSchafer syndrome (at the proximal short arm of chromosome 11) as well as Wilms’ tumor (Bremond-Gignac et al., 2005). Genes within this locus include FOLH1, which encodes an M28 peptidase family II transmembrane glycoprotein that acts as a glutamate carboxypeptidase; its substrates include folic acid and neuropeptides. FOHL1 is expressed in the prostate, kidneys, and the central and peripheral nervous

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systems. Mutation s in FOHL1 can affect the ability of the small intestine to absorb folic acid, leading to decreased folic acid levels in the blood and, consequently, high bloodhomocysteine levels. FOHL1 levels in the brain are linked to the pathological changes of glutamate excitatory poisoning, and an increase in FOHL1 expression is indicative of prostate cancer (Collin et al., 2009; McKay et al., 2012). Interestingly, CNV analysis of Zh20P24 revealed an

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Figure 5. The expression of multipotency genes for hESCs. The PCR products were assessed by gel electrophoresis on 2% agarose ethidium bromide-stained gels and pictures were taken using gel imaging system. lane1: 2000 bp DNA Ladder Marker; lane24,(H9 hESC) GAPDH, POU5F1 and NANOG; lane5-7,(human embryonic fibroblasts) GAPDH, POU5F1 and NANOG; lane810:,(Zh19 hESC) GAPDH, POU5F1 and NANOG; lane1113: (Zh20 hESC) GAPDH, POU5F1 and NANOG.

association with renal and prostate cancer, whereas Zh19P25 had no clear correlation with a disease; these results point to the power of the single-nucleotide-level genomic analysis to pick up subtle differences among hESC lines.

CONCLUSION We used 3PN embryos to generate high-quality blastocysts, and established hESC lines carrying normal karyotypes. Analysis of both Zh19P25 and Zh20P24 cell lines on a SNP chip for genetic variation and molecular disease characteristics revealed clear genetic changes within these stem cells that can be used to better characterize them. This research provides a basis for the discrimination of hESC lines prior to their future clinical application.

MATERIALS AND METHODS Fresh, Discarded Embryos, and Their Culture The Ethics Committee and Reproductive Medical Ethics Committee of Zhengzhou University approved the derivation, characterization, and early differentiation of hESC lines from the donated supernumerary embryos in this study. All participating partners signed an informed-

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consent form after receiving verbal and written information. Specifically, those embryos that could not be used for infertility or assisted reproductive treatment were redirected to stem cell derivation and research. The 3PN zygotes used in this study were obtained from the Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University between June 2010 and December 2010. These zygotes were subsequently cultured in vitro to the blastocyst stage. Blastocysts were graded according to the Peter’s rated criteria (D3): Grade I, even-sized, symmetrical blastomeres with no obvious fragmentation; medium refractivity and complete zona pellucidae. Grade II, uneven-sized blastomeres, or a total cytoplasmic mass containing 50% cytoplasmic fragmentation. A three-part scoring system, based on blastocyst expansion and ICM and trophectoderm development, was also used. Blastocysts were given a numerical score from 1 to 6 on the basis of their degree of expansion, as follows: (i) a blastocyst with blastocoel cavity less than half of the volume of the embryo; (ii) a blastocyst with blastocoel cavity more than half the volume of the embryo; (iii) a full blastocyst with a blastocoel cavity completely filling the embryo; (iv) an expanded blastocyst with a blastocoel volume larger than that of the early embryo, with thinning of the shell; (v) a hatching blastocyst, coming out of the shell; or (vi) a hatched blastocyst. Development of the inner cell mass was assessed as follows: (i) tightly packed, many cells; (ii) loosely grouped, several cells; or (iii) very few cells. The trophectoderm was assessed as follows: (i) many cells forming a cohesive epithelium; (ii) few cells forming a loose epithelium; and (iii) very few large cells. (see Brinsden, 1999; Gardner et al., 2000).

The Establishment and Culture of hESCs Trophectoderm cells around the ICM were removed by mechanical separation with a syringe needle under a dissecting microscope; damage to the ICM was minimized or avoided. The ICM was seeded onto a mixed feeder layer that was previously inactivated with mitomycin C (Chen et al., 2012b). Primary mouse embryonic fibroblasts (MEF) and primary human foreskin fibroblasts (HFF), between passages 10 and 20, were treated with mitomycin C, and then mixed at a ratio of 1:1 of MEF:HFF to prepare the mixed feeder layer. The cultures were passaged every 37 days for the primary clone, then each cell line was passaged every 47 days by mechanical methods. The hESC cuture media

TABLE 4. Semi-Quantitative Analysis of the Expression POU5F1 and NANOG by Reverse-Transcriptase-PCR in hESC Lines

H9 Zh19 Zh20

POU5F1

t

P

NANOG

t

P

1.03  0.07 0.97  0.04 0.95  0.05

t ¼ 1.320 t ¼ 1.589

P ¼ 0.257 P ¼ 0.187

0.95  0.03 0.92  0.03 0.88  0.03

t ¼ 1.203 t ¼ 2.289

P ¼ 0.295 P ¼ 0.084

Semi-quantitative analysis of H9, Zh19, and Zh20 cell lines; H9 was used as a positive control. Zh19 and Zh20 were compared with H9 separately. Confidence intervals were set at a ¼ 0.05, and P < 0.05 was considered significant.

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Figure 6. Normal G-banding karyotypes of Zh19 and Zh20 hESCs.

consisted of 80% KnockoutDMEM, 20% knockout serum replacement (KSR), 1% non-essential amino acids (NEAA), 2 mM L -glutamine, 0.1 mM mercaptoethanol, and 8 ng/ml of basic fibroblast growth factor (Gibco, Grand Island, NY).

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In Vitro Differentiation of hESCs Simple and cyst-shaped embryoid bodies could be observed during in vitro differentiation. Embryoid bodies were collected for molecular characterization.

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TABLE 5. The Genome CNV of Zh19 and Zh20 hESCs

Chr

Length (Mb)

Zh19P25 Zh20P24

19 11

0.52 2.98

Loss Loss

1 1

19

0.53

Loss

1

CNV Value

Cytobands

Related genes

q11 p11.2 p11.12 q11

LOC148189 FOLH1,LOC440040,OR4C13,OR4C12,LOC441601,LOC646813,OR4A5, OR4C46 LOC148189

Alkaline Phosphatase Staining hESCs clones were examined under an inverted phasecontrast microscope. hESCs grown in a 60  15 mm dish (353037; BD, Becton, Dickinson and Company Becton Drive Franklin Lakes, New Jersey) were fixed and labeled with the alkaline phosphatase ES Cell Characterization Kit (Millipore: Massachusetts, Boston, USA).

Immunostaining hESCs or EBsboth washed once with PBS, then fixed to a plate with 4% buffered formalin phosphate (SigmaAldrich, St. Louis, MO, USA)were first incubated with primary antibodies that identified pluripotency markers (mouse anti-human SSEA-1, SSEA-4 [Santa Cruz Biotechnology, Inc., Santa Cruz, CA] http://www.scbt.com), TRA1-60, and TRA-1-81 antibodies in the ES Cell Characterization Kit (Millipore)) or germ layer markers (mouse antihuman nestin, -myocardial troponin, and -AFP antibodies in the Human Embryonic Germ Layer Characterization Kit (Millipore)). Primary antibody was incubated with the cells

overnight at 48C. The next morning, the cells were washed three times with phosphate-buffered saline (PBS), and were then incubated with the secondary antibody Alexa Fluor 488 (for SSEA-1 and SSEA-4) and Cy3 (for TRA-1-60 and TRA-1-81) donkey anti-mouse IgG (Jackson ImmunoResearch Inc, Pennsylvania, Amish country) for an hour at room temperature. The cells were then washed three times with PBS, and nuclear staining was performed by incubation with 1 mg/ml DAPI (SigmaAldrich) for 2 min. ProLong1 Gold (Invitrogen molecular probe Carlsbad, California USA.) was used to mount the coverglass, and then the samples were observed and imaged by a LSM700 laser confocal microscopy (Zeiss, German).

Reverse Transcriptase-PCR RNA extraction from mechanically-isolated, undifferentiated hESC clones was performed with All Prep DNA/ RNA/Protein Mini Kit 80004 Kit according to the manufacturer’s protocol (Qiagen, Germany). The quality and concentration of RNA were detected using a Nanodrop

Figure 7. Genome CNV of Zh20 is related to the PotockiShaffer syndrome-associated pericentromeric 11p region. A 2.98 Mb genomic loss was found between chromosomal 11p11.2p11.12. This CNV locus contains the genes FOLH1, LOC440040, OR4C13, OR4C12, LOC441601, LOC646813, OR4A5, and OR4C46.

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Dna Extraction, Purification, and Chip Analysis

Figure 8. Genomic-copy-number states differ between Zh19P25 and Zh20P24 hESCs. LOC148189 is one site determined to be lost at the affected chromosomal 11p11 region; SPATA7 is the control site used as a CNV control. There were significant gene-copy-number differences in the Zh19P25 and Zh20P24 samples.

spectrophotometer (Thermo Fisher Scientific, Massachusetts, Waltham, USA) and 1% agarose gel electrophoresis. Total RNA (1 mg) was reverse-transcribed using a PrimeScript1RT-PCR Kit (Takara, DRR014A, Japan). Complementary DNA samples were subjected to PCR amplification with human-specific primers (Table 6). The PCR products were assessed by gel electrophoresis on 2% agarose ethidium bromide-stained gels and pictures were taken using a gel imaging system.

Karyotype Analysis Karyotype analyses of hESCs were performed by G-banding. Samples of hESCs were treated with colchicine at a final concentration of 0.2 mg/ml (Gibco) for 3 hr, and then washed twice with PBS. The cells were digested with 0.05% trypsin, and separated into single cells before stopping the digestion. Individual cells were then treated with a hypotonic solution (0.075 M KCl), and fixed in a 3:1 solution of methanol and acetic acid (v/v). Metaphase spreads were G-banded by brief exposure to trypsin, stained for 10 min, and washed with Giemsa solution (SigmaAldrich). After air drying, these sections were observed and analyzed under an oil immersion microscope (Wang et al., 2012).

TABLE 6. List of Primers Gene

Primer sequence

50 -GCACCGTCAAGGCTGAGAAC 50 -TGGTGAAGACGCCAGTGGA POU5F1 50 -GTGCCGTGAAGCTGGAGAA 50 -TGGTCGTTTGGCTGAATACCTT NANOG 50 -CAACATCCTGAACCTCAGCTACAA 50 -GGCATCCCTGGTGGTAGGAA LOC148189 50 -ATGTCGCAGTAAAATTGCTCCTC 50 -AAGCCACCTTTTGTCTATCTCTGG SPATA7 50 - TTTTCTAGCCAGTAAACCTTG 50 - GCTGTACATATTCTATTTACTG GAPDH

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Product length 138 bp 191 bp 169 bp

Whole-genome DNA was extracted and purified with the All Prep DNA/RNA/Protein Mini Kit, according to the manufacturer’s protocol (Qiagen). The concentration and quality of the samples were determined using a Nanodrop spectrophotometer (Thermo Scientific) and 1% agarose gel electrophoresis with a reference DNA. Two hundred nanograms of DNA were used for each amplification. HumanCytoSNP-12 Bead Chip (Illumina) was performed using two samples (Zh19P25 and Zh20P24) followed by scanning on a BeadArray Reader (Illumina, Inc.). The gene-call threshold was set at 0.15, and the call rates were 0.9909876 and 0.9944094. The CNVs were compared using existing databases in the world DGV database (http://projects.tcag.ca/variation). The phenotypic data Library DECIPHER (http://decipher.sanger.ac.uk/), PUBMED (www.ncbi.nlm.nih.gov/pubmed/), and OMIM (www.ncbi.nlm.nih.gov/omim) databases were queried to determine if there were known pathogenic CNVs in Zh19P25 and Zh20P24 hESCs lines.

Quantitative Real-Time Polymerase Chain Reaction We separately used genomic DNA from Zh19P25 and Zh20P24 as templates to validate genomic CNV status by quantitative real-time PCR (qPCR). The concentration of the samples was measured with a Nanodrop spectrophotometer (Thermo Scientific). The primers for LOC148189 (detected loss site) and SPATA7 (normal CNV control site) were designed and synthetized by Takara company (Table 6). qPCR was performed with SYBR Green Supermix (Takara, RR820A, Dalian, China) with 2 ml of the template in a 20 ml total reaction volume. Gene expression levels were measured using the 7500 Fast Real-Time PCR System (AB Applied Biosystems, Lifetech, Life Technologies Corporation, New York, USA). The fold change between LOC148189 and SPATA7 was calculated based on the 2DCT method to determine gene copy number differences between LOC148189 and SPATA7 in the same sample.

Statistical Analysis Statistical analyses were performed with the SPSS 12.0 (SPSS, Inc., Chicago, IL) software. The rate comparisons were performed using the x2 test (Fisher’s exact test). Semi-quantitative analysis was used on the expression of POU5F1 and NANOG by reverse-transcriptase-PCR in H9, Zh19, and Zh20 cell lines; H9 was used as a positive control. Zh19 and Zh20 were compared with H9 separately. The experiment was repeated three times, and the analysis of the gray value was used in for the statistics. The gDNA of the Zh19P25 and Zh20P24 samples were used as templates to validate genomic copy number (CNV) states by qPCR. The independent-samples t-test was used to evaluate significance. Confidence intervals were set at a ¼ 0.05, and P < 0.05 was considered to be statistically significant.

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ACKNOWLEDGMENTS We are grateful to everyone who has contributed to this work. We especially acknowledge Professor Guangli Xu from the Maternal and Child Healthcare Hospital of Zhengzhou for their valuable advice regarding G-band karyotyping.

AUTHOR’S CONTRIBUTIONS Yingpu Sun and Xuemei Chen designed the experiments; Xuemei Chen was responsible for the coordination of the project and microarray experiments, data analysis, integration, and statistical analysis. Wenbin Niu was responsible for the microarray experiments. Shanjun Dai was responsible for embryo culture. Xuemei Chen, Fang Wang, Huijuan Kong, Wenzhu Yu, and Yimin Shu were responsible for cell culture and DNA extractions. Xuemei Chen contributed to writing the paper.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article.

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Derivation of normal diploid human embryonic stem cells from tripronuclear zygotes with analysis of their copy number variation and loss of heterozygosity.

This study sought to establish archives of genetic copy number variation (CNV) in human embryonic stem cell (hESC) lines that are associated with know...
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