Assisted Oocyte Activation Technology And Isolation,Culture Of Embryonic Stem Cells

Human embryonic stem cells (ES cells) are important both for their potential in regenerative medicine and as a window on early human embryology. However, research on human ES cells raises profound ethical concerns, because extracting the stem cells destroys the embryos, and thus destroys its potential for life. In addition, the scarcity of human embryos is critical for human ES cells. How to minimize the ethical concerns surrounding human ES cells research and solve the scarcity of human embryos? In present study, we attempted to activate mouse or human oocytes and unfertilized oocytes after ICSI for deriving ES cells. In addition, we isolated mouse embryonic stem cell from normal embryos.Part I PARTHENOGENETIC ACTIVATION OF MOUSE OOCYTES WITH CHEMICAL STIMULUS AND ISOLATION OFPARTHENOGENETIC STEM CELLSObjective: To investigate the optimal protocol for parthenogenetic activation on mouse oocytes and whether the parthenogenetic stem cells could be derived from parthenogenetic blastocysts. Methods: The mouse oocytes were activated by different chemical stimulus. First, the parthenogenetic activation of ethanol on different age mouse oocytes was investigated according to the repetitive three factors three levels orthogonal experiment design. Then, the ideal age mouse oocytes were exposed to ethanol and subsequently were treated with 2mM 6-DMAP /10μg/ml puromycin for 4 hours. Third, mouse oocytes were exposed to 5μM A23187 for 5 minutes or subsequently were treated with 6-DMAP/puromycin. The activation rate, proportion of oocytes that showed pronucleus formation and second polar body extrusion werecalculated at 6 hours after activation. The parthenogenetic embryos were cultured for 3-4 days and their developmental potential were examined. Culture the parthenogenetic blastocysts on feeder cells and observe the embryos daily to monitor hatching, attachment, inner cell mass (ICM) appearance and ES cell colonies formation. Results: When the mouse oocytes at 9-hour age were treated with 7% ethanol for 5 minutes, the highest activation rate and the best development potential were obtained. However, the disruption rate of oocytes increased with age of oocytes and dose of ethanol. The better development potential of parthengenetic embryos were achieved by the combination of ethanol with 6-DMAP/puromycin. Calcium ionophore A23187 could effectively activate the mouse oocytes, whereas the combination of A23187 with 6-DMAP/puromycin improved the developmental potential of parthenogenetic embryos. Some parthenogenetic embryos activated by A23187 and 6-DMAP could develop to blastocyst stage. The parthenogenetic embryos could attach on the feeder cell and ICM appeared as normal embryos, but ES cell colonies formation was low. Conclusion: Ethanol and A23187 could effectively activate the mouse oocytes, but the developmental potential of parthenogenetic embryos was restricted. The combination of them with 6-DMAP/puromycin could effectively improve developmental potential of parthenogenetic embryos. The parthenogenetic stem cells could be isolated, but they tended to differentiate.Part II PARTHENOGENETIC ACTIVATION OF CALCIUM IONOPHORE A23187 AND 6-DMAP ON HUMAN OOCYTESMATURED IN VITROObjective: To investigate the parthenogenetic activation of calcium ionophore A23187 and 6-DMAP on the human oocytes matured in vitro. Methods: Immature oocytes at germinal vesicle stage or metaphase of first meiosis from immature oocyte aspiration were harvested and cultured in vitro. Following in vitro maturation, 102mature oocytes were assigned to three groups according to the time of maturation in vitro: 24-hour group, 48-hour group, 72-hour group. The oocytes were exposed to 5uM A23187 for 5 minutes or subsequently were treated with 2mM 6-DMAP for 4 hours. After incubation, the oocytes were cultured in vitro for 3-5 days. The activation rate, proportion of oocytes that showed pronucleus formation and second polar body extrusion were calculated at 16-18 hours after activation. Sex chromosomal analysis of the parthenotes derived from two pronuclei (2PN) was performed by dual color fluorescence in situ hybridization (FISH). Results: The activation rate in 48-hour group after treatment was 38.9% (7/18) in the A23187 group and 80.8% (21/26) in the combination of A23187 and 6-DMAP. Cleavage rate and the developmental potential of parthenotes in combinative group were significantly higher than in the A23187 and control groups, respectively. Furthermore, the activation rate in 24-hour group was similar to the 48-hour group's, which was significantly higher than 72-hour group's. FISH analysis showed 9 embryos with XX in 10 parthenotes. Conclusions: The combination of A23187 with 6-DMAP proved to be an effective method of producing diploid parthenotes.Part m EFFECT OF ACTIVATION WITH CALCIUM IONOPHORE A23187 AND PUROMYCIN ON HUMAN OOCYTES THAT FAILED TO FERTILIZE AFTER ICSIObjective: To investigate the effect of oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that failed to fertilize after ICSI. Methods: All 180 discarded oocyes that showed no evidence of fertilization at 16-18 hours after conventional intracytoplasmic sperm injection cycles (ICSI) and in vitro maturation/ICSI cycles (IVM-ICSI) were assigned to six groups according to the time after ICSI: ICSI 22-hour group, ICSI 44-hour group, ICSI 68-hour group, IVM-ICSI22-hour group, IVM-ICSI 44-hour group, and control. All unfertilized oocytes were exposed to 5\iM A23187 for 5 minutes and subsequently were incubated with lOjig/ml puromycin for 4 hours. After incubation, the oocytes were cultured in vitro for 3~5 days. Sex chromosomal analysis was performed on 16 embryos that displayed two pronuclei and a second polar body (2PN2PB). The expression of IGF- II on the activated embryos, normal embryos, and parthenotes was examined. Results: The combination A23187 with puromycin could activate the unfertilized oocytes 22~68 hours after ICSI. Best results were achieved in ICSI 22-hour group, which elicited 91.2% (31/34) of activation rate, 64.7% (22/34) of cleavage rate and 44.1% (15/34) of high-quality embryos. Some embryos in the group could develop to blastocyst stage. The activation rates of unfertilized oocytes and developmental potential of activated embryos decreased with the cultured time after ICSI. Sex chromosome analysis indicated 9 male and 4 female embryos. The expression of IGF- II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes. Conclusions: The combination of A23187 with puromycin could effectively activate unfertilized oocytes 22~68 hours after ICSI. Moreover, the activation efficiency and developmental potential of activated embryos inversely correlated with culture time after ICSI and the combination of ICSI with assisted oocyte activation should perform within 22 hours after ICSI. The 2PN2PB zygotes activated by A23187/puromycin could develop normally. It is suggested that the activated embryos might be great resource for deriving human ES cells.Part IV ISOLATION AND CULTURE OF KUNMING MOUSEEMBRYONIC STEM CELLSObjective: To investigate the optimal culture system for islolation and culture of Kunming mouse ES cells, and establish the Kunming mouse ES cells. Methods: The Kunming mouse blastocysts cultured on SNL cells, primary mouse embryo fibroblasts(PMEF) and geletin-coated plastic without feeder cells using different media. Observe the embryos daily to monitor hatching, attachment and ICM formation. When the ICM appeared and extended above the flat trophoblasts and feeders, divide ICM into several clumps and transfer them to fresh feeder layer. The mouse ES cells were subjected to characterize for their morphology, and alkaline phosphatase (AKP), stage special embryonic antigen (SSEAs), Oct4 expression. They also identified for karyotype analysis. They were cultured in suspension to form embryoid bodies and differentiated in vitro. Mouse ES cells were injected in nude mouse to investigate the differentiation in vivo. Results: The attachment of mouse embryos, ICM and ES colonies formation on SNL cells were similar to that of PMEF, which were higher than that of plastic. The media with LIF improved ES cell colonies formation. One ES cell line was established from Kunming mouse blastocyst, which displayed typical ES cell morphology. ES cell colonies showed strong positive immunostaining for AKP, SSEA-1 cell surface markers. Most ES colonies retained normal karyotypes and Oct4 expression in prolonged subculture. During in vitro differentiation of ES cells, three embryonic germ layer cells appeared. It formed teratomas containing cell derivatives from all three embryonic germ layer in nude mouse. However, microscopic observations revealed the presence of immature tissue and high frequency of mitotic figures, indicating their malignant nature. After further passaged to another nude mouse, another teratomas was formed that also contained three mature embryonic germ layer, but the malignant nature disappeared. Conclusions: The feeder and LIF could improve the attachment of blastocyst, ICM and ES cell colonies formation. Kunming mouse ES cells could establish on SNL cells. However, the teratomas derived from ES cells contained immature tissue, which could differentiate to benign tissue after further passage. All these data indicated that more care must be taken before using ES cell transplantation as a therapeutic option for patients with degenerative disease.