Isolation And Developmental Potential Of Hybrid Parthenogenetic Stem Cells

In the present study, we successfully isolated embryonic stem cells from hybrid parthenogenetic blastocyts which were derived from transfer of EGFP transgenic mouse MII spindle to Kumming Mouse MII oocytes. The embryonic stem cells were evaluated by a series of detections including morphological evaulation, karyotype analysis, alkaline phosphatase staining, staining of OCT-4 and SSEA-1, differentiation of embryoid bodies. In order to study the in vivo differentiation of HP-ES, Chimeras of injecting stem cells into blastocysts were produced and found the stem cells involved in germ-line formation.1. Reconstruction of hybrid oocytes and their parthenogenetic developmentTwo genetically different mouse oocytes were used for MII spindle transfer: the EGFP transgenic mouse oocytes were as spindle donors, and the in vivo matured Kunming mouse MII oocytes as recipients. Total of 114 reconstituted oocytes (71.25%) were obtained, and 103 (90.35%) were fused. Of the fused oocytes 69 (67.0%) expelled two polar bodies and formed two pronuclear. These embryos were then cultured in vitro and 12 of them (17.4%) developed to blastocysts.2. Isolation and evaluation of ES cells Twelve blastocysts were placed directly on treated feeder cells cultured in embryonic stem cells medium (ESM). After 5-6 days, 7 blastocysts developed into ICM-derived clump, 3 HP-ES cell lines were established.Analyses of the established stem cells showed that 1) morphologically, cells formed tightly packed colonies in the shape of nest. The edge of the cells was hard to define. ES cell clones showed a strong green fluorescence under fluorescence microscopy. 2) the ES cells had a normal diploid karyotype (2n=40) . 3) alkaline phosphatase . (AKP) staining positive. ES cell clones were stained in dark blue, showing alkaline phosphatase activity. 4) SSEA-1 and OCT-4 staining positive. 5) the resultant embryoid bodies from EGFP-ES cells showed good differentiation potential and had a strong green fluorescence.3. In-vivo differentiation of ES cells from hybrid chimeric blastocysrsTen to 15 ES cells were injected into Kunming mouse blastocyst cavity. After incubation of the injected blastocysts for 1 hr, 175 blastocysts were selected to transfer into 11 pseudo-pregnant uteruses. Total of 44 fetuses were delivered, and 6 of which were chimeric mice. After mating of these 6 chimeric mice with KM mice, one of them delivered offspings in black hair. This result suggested that the strain of ES cells had germ-line transmission ability. The flow cytometry analysis showed that the chimeric rates of HP-ES in heart, spleen, kidney and marrow were 27.88%,12.48%,1.8% and 6.93%, respectively.