A human embryonic stem cell line derived from a single blastomere of a 4-cell stage embryo

Introduction: Recently we have shown that blastomeres of a 4-cell stage human embryo were able to develop into blastocysts with an inner cell mass (ICM) and trophectoderm (TE). We therefore hypothesized that blastomeres are potentially totipotent and may not yet be allocated to either ICM or TE at this early stage of preimplantation development. To provide further evidence that blastomeres are pluripotent at this stage, we derived human embryonic stem cells (hESC) from single blastomeres of 4-cell stage embryos.
Material & methods: Mature oocytes were donated for research by couples treated at our IVF centre who had given their written informed consent. Embryos were obtained after ICSI using spermatozoa of a consenting donor. Three embryos showing less than 10% fragmentation and regular sized blastomeres underwent zona hatching using laser pulses and the blastomeres were removed by aspiration using a pipette with an inner diameter of 50 _m. All blastomeres were individually cultured in sequential medium as used in our IVF programme. On day 4, the blastomere-derived morulas were plated on inactivated mouse embryonic fibroblasts (MEFs) and further cultured in the same serum-free medium as for the hESC derivation and culture.
Results: Eleven out of twelve morulas were plated and eight attached to the MEFs. Eventually, one blastomere-derived morula without microscopically visible signs of cavitation produced an outgrowth. When the outgrowth showed a compact colony-like structure with cells of stem-cell-like morphology (day 22 post splitting), it was mechanically passaged onto fresh MEFs. Routine passaging was performed as described for blastocyst-derived hESC. The putative hESC line was shown to express the typical markers of stemness by immunocytochemistry and/or RT-PCR, i.e. POU5F1, NANOG, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, DNMT3B, GDF3, LIN 28, NPM 1, REX-1, SOX-2 and Alkaline Phosphatase. The morphology and growth rate of the colonies was similar to blastocyst-derived hESC. At passage 10, cells were harvested for molecular karyotyping. We observed a 46,XX karyotype, however, with a deletion of chromosome 18q23 and a duplication of chromosome 7q33. In order to demonstrate the pluripotency of this putative hESC line, cells were injected into NOD-SCID mice. After 9 weeks, a teratoma was formed containing cells from the three germ layers including squamous epithelium (ectoderm), cartilage (mesoderm) and gastro-intestinal epithelium (endoderm). The hESC line is currently at passage 45 and was successfully thawed and passaged after cryopreservation.
Conclusions: ESC have already been derived from single blastomeres of both mouse and human embryos at the 8-cell stage, but with a low success rate (4% and 2% respectively), indicating that some of these blastomeres are pluripotent at this stage. However, this derivation required the use of a co-culture system involving aggregation or merging microdrops with established ESC lines respectively. Here we describe hESC derivation starting from blastomere-derived morulas that simply attached to MEFs. One out of twelve single blastomeres developed into a hESC line (8% derivation rate). This does not prove that the four cells are pluripotent. Knowing the low derivation rate in general, it will be difficult/impossible to derive hESC from four single blastomeres. Interestingly, the formation of ICM cells has not been essential for the derivation of this particular hESC line. Further research is required to determine whether this blastomere-derived hESC line is functionally different from blastocyst-derived hESC lines.