Human somatic cell nuclear transfer and parthenogenesis: Preliminary Indian experience

The aim of the present study was to conduct preliminary experiments towards generation of autologous human embryonic stem cell lines by somatic cell nuclear transfer (SCNT) and parthenogenesis. A total of fifteen MII human oocytes were obtained from six female donors from a collaborating IVF clinic. Each woman having produced more than 20 eggs, donated 2-3 eggs for research with written informed consent. Nine oocytes were used for SCNT and six for parthenogenesis. For SCNT, the meiotic spindle of MII human oocytes was removed after visualization using a POLSCOPE, and the somatic cell was injected into the enucleated cytoplast, circumventing the need for electrofusion of the somatic cell and cytoplast. Six out of nine oocytes survived reconstruction during SCNT and four divided. Nuclear assessment of SCNT embryos by Hoechst 33342 staining confirmed nuclear division in some embryos post SCNT. Parthenogenetic activation was carried out by calcium ionomycin followed by 6-DMAP treatment. All the activated eggs showed presence of pronuclei after 16 hours, four out of six oocytes showed cleavage and one embryo developed into a compacted morula. It was placed on a feeder layer but neither attached nor showed any further growth. We believe the generation of autologous ES cell lines is essential to circumvent immune rejection if embryonic stem cells are to be used widely for therapy, however extensive standardization and optimization of technology is warranted.