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

Abstract

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 Potocki-Shaffer disease, 11p11.2-11p11.12 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.

Developmental potential of clinically discarded human embryos and associated chromosomal analysis

“…A major focus of these efforts has involved the study of abnormally fertilized zygotes, which are predominantly discarded 2 3 8 . Several groups have facilitated the derivation of diploid hESCs from 0PN (without any pronucleus) zygotes 3 , mononuclear (1PN) zygotes 9 and 3PN zygotes 3 8 10 . Chen et al reported that 130 3PN zygotes were collected from patients undergoing conventional in vitro fertilization (IVF).…”

“…In addition, Suss-Toby et al reported the derivation of normal karyotypical hESCs from 1PN zygotes 9 . In a recent study from our group, hESCs were derived from 3PN zygotes from patients with known diseases 10 .…”

Molecular Support for Heterogonesis Resulting in Sesquizygotic Twinning

“…Data on heterogonesis in humans are lacking, but indirect evidence that has been observed in vitro includes the facts that maternal and paternal spindle apparatuses can form independently of each other in the fertilized ovum 17 and that tripronuclear zygotes can develop into diploid embryos. 18 Fertilization of a single ovum by a diploidic spermatozoon is an alternative explanation, but because this would also result in the formation of a tripolar spindle apparatus, the heterogoneic model still holds true. Our results show that the twins share 78% of their paternal genome, which is 1.5 SD above the expected 50% allele sharing of independent paternal genomes.…”