Factors Affecting Human Blastocyst Formation In Vitro And The Basic Research For Establishment Of Human Embryonic Stem Cells Line

Traditional IVF-ET (in vitro fertilization-embryo transfer) procedure usuallytransfer D3 embryos, but the blastocyst transfer is adapted to physiological procedure, decrease the transfer number and avoid multiple pregnancy and complication, wehave gotten a number of blastocysts through sequential-culture for D3 discarded embryos, studied the correlative factors of blastocyst formation. Then, we used these blastocysts to do some basic research for establishment of human embryonic stem cells (hESCs) line cultivated on the human embryonic pulmonary fibroblasts (HEPFs) feeder layer.There are three parts in this study which includes factors affecting human blastocyst formation in vitro, preparation of feeder layer and the basic research for establishment of hESCs line.Through sequential culturing the D3 discarded embryos to D5.7, we observedthe blastocyst formation and detect the relationship between the D3 embryo cells number, quality grade and the blastosysts quality, the formation of the high-qualityblastocysts, providing the academic basis for the blastocyst transplantation. 203 D3 discarded embryos entered this experiment. Cultured these embryos with G2.3,observed theirs changes daily till D5.7. 51 blastocysts were formed including 14 high-quality blastocysts using the blastocyst grade standard from Gardener. The blastulation rate was 25.12%. Among 32 patients who have blastocysts, 16 patients pregnancied, the pregnancy rate is higher than whose without blastocysts(50%Vs28.79%, PO.05). The blastultions rate is positively correlative with the D3 embryo cells numbers. The blastulation rate of the embryos with I - II+ degree is high than those with III-IV degree.hESCs hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies.Traditionally, hESCs have been cultured on mouse embryonic fibroblasts (MEFs) feeder layers, which permit continuous growth in an undifferentiated stage. To use these cells in human therapy, animal free culture system should be used which will prevent expose to retroviruses, such as human feeder layer, matrix media, serum-free and feeder-free system, and so on. In this study, we used HEPFs as feeder layer and compared the MEFs to HEPFs in many aspects including characterization, passage method, and advantages and disadvantages. Mouse embryos were harvest at 12.5-14.5 days post-coition, made theprimary MEFs and expanded to P3.5 , P7 HEPFs were expanded to P25. As a feeder layer for hESCs, MEFs can be used within P5, while the HEPFs can be used withinP25> the optimum treatment time of mitomycin C is 3.25-3.5 hrs. HEPFs canmaintain and proliferate hESCs and propagate hESCs in an undifferentiated state effectively.Finally, we did some basic research for establishment of hESCs line, which could proliferate and maintain undifferentiated state with normal karyotype. Thoseblastocysts cultured from D3 discarded embryos were treated by immunosurgery and inner cell mass (ICM) were isolated and plated on the mitomycin C treated HEPFs. After 10-14 days, ICM will be ready to subcultivate. Subcultivation is accomplished by mechanical dissection of the growing colony into approximately 0.5mm2 pieces on fresh organ culture dishes pretreated with gelatin and containing feeder cells. Subculture may then be carried out every 5-7 days. We established hESCs-1121 lines which had been propagated to 17 passages with a high ratio of nucleus to cytoplasm, prominent nucleoli, compact colony morphology, normal karyotype and positive alkaline phosphatase (AKP).