Clinically failed eggs as a source of normal human embryo stem cells

Abstract

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

Derivation of Man-1 and Man-2 research grade human embryonic stem cell lines

“…In our hands, as reported by other groups (Pickering et al 2005), no derivations have been achieved from grades C or D blastocysts. Man-2 is our second line derived from a chemically activated oocyte, following the first (RCM-1) described in De Sousa et al (2009). It is not possible to comment on the success rate of deriving lines from blastocysts obtained by this method as only two lines have originated, but in our hands, it does not seem to be obviously lower than from blastocysts obtained from supernumerary IVF embryos.…”

“…Cleavage stage embryos and failed to fertilise oocytes were donated by couples undergoing IVF treatment at St Mary's Hospital after informed consent with approval of the Central Manchester Research Ethics Committee and under licence R0171 from the Human Fertilisation and Embryology Authority. Failed to fertilise oocytes were chemically activated using 5 μM ionomycin (SigmaAldrich) in G1 medium (Vitrolife, Göteborg, Sweden) for 5 min at 37°C, followed by 1 mM 6-DMAP (Sigma) and 5 μg/ml cycloheximide for 3-6 h (De Sousa et al 2009). Cleaving embryos were cultured in ISM-1/2 sequential media (Medicult, Jyllinge, Denmark) until blastocyst formation.…”

Gene expression analysis of a new source of human oocytes and embryos for research and human embryonic stem cell derivation

“…We were reassured to find that levels of both genes and the ratio between them were consistent between individual blastocysts and similar in activated vs. control embryos, indicating that oocyte activation results in blastocysts with a well-defined ICM of high enough quality for stem cell derivation. In confirmation of the latter, one blastocyst resulted in a new hESC line, RCM-1 (15). This line has a normal diploid karyotype of 46XX, and the genome was demonstrated to be of biparental origin.…”

“…We demonstrate that these embryos are comparable to control IVF embryos, as assessed by developmental progression and gene expression profiling. Moreover, blastocysts generated by this method are capable of giving rise to normal hESC lines (15,16). …”

“…Upon receipt, derivation was attempted on inactivated neonatal human dermal fibroblasts using the methods detailed by De Sousa et al (15). One hESC line (RCMAN1) was successfully derived using this method.…”