Authors:
H. Van de Velde, M. Geens, I. Mateizel, C. Spits, K. Sermon,
M. De Rycke, J. Van der Elst, P. Devroey, H. Tournaye, and I. Liebaers
Summary:
Introduction - Embryonic stem cell (ESC) lines have been derived from single blastomeres of mouse and human embryos at the 8-cell stage, indicating pluripotency at this stage. Success rates were low and co-culture with established ESC lines was necessary. Our research programme focuses on totipotency during preimplantation development. Recently we have shown that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with an inner cell mass (ICM) and trophectoderm (TE). We therefore hypothesize
that 4-cell stage blastomeres are potentially totipotent and may not yet be allocated to either ICM or TE. Totipotency at the early stage of development can be further demonstrated by deriving pluripotent human embryonic stem cells (hESC) from single blastomeres of 4-cell stage embryos.
Materials and 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 on day 2. Blastomeres were removed by aspiration using a pipette with an inner diameter of 50 mm. All blastomeres were individually cultured in sequential medium series 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 obtained from three embryos 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 embryo splitting), it was mechanically passaged onto fresh MEFs. Further routine passaging was done as described for blastocyst-derived hESC. The morphology and growth rate of the passaged colonies was similar to 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, LIN28, NPM1, REX-1, SOX-2 and Alkaline Phosphatase. At passage 10, cells were harvested for molecular karyotyping. A 46,XX karyotype was observed, however, with a deletion of chromosome 18q23qter and a duplication of chromosome 7q33qter. 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, named VUB26_QUATRO, is currently at passage 45 and has been successfully thawed and passaged after cryopreservation.
Conclusions - We report the first-time successful derivation of a hESC line from a single blastomere of a 4-cell stage human embryo. This result adds evidence to the hypothesis that the blastomeres are totipotent at the 4-cell stage. The derivation started from single blastomere-derived morulas that simply attached to MEFs without need for mixing with established ESC lines. Also, the formation of ICM cells has not been essential for the derivation of this particular
hESC line. Current research aims to determine whether the 4-cell stage single blastomere-derived hESC line VUB26_QUATRO is functionally different from blastocyst-derived hESC lines.
Source:
The 24th Annual Meeting of the ESHRE; Barcelona, Spain, O-274 Oral (07/09/08)