Establish β- Thalassemia Embryonic Stem Cell Line - (a) Three Kinds Of Broth System To Establish Embryonic Stem Cell Lines To Compare

OBJECTIVE:①To generate embryonic stem cell lines in three different generating and culturing systems.②To discuss the relationship between the quality of the blastocyst and the efficiency of generating human embryonic stem cell lines.METHODS:We applied three different human embryonic stem cell generating and culturing systems in our research which respectively are the conventional generating and culturing system; new、defined human embryonic stem cell culture medium mTESRl system and the combination of the mTESR1medium and the5signal conduction pathway inhibitors system. The embryos we applied in generating embryonic stem cell lines were from the IVF-ET patients in the Hainan provincial reproductive medical center of the affiliated hospital of Hainan medical college, these embryos were considered improper to be transferred clinically or about to be discarded. Embryos were cultured in the embryo culture medium G2till blastocyst stage, then those blastocysts were transferred into little HEPES drop, we applied the whole-mechanical method to separate the ICM and the trophoblast in HEPES drops, then the ICMs were transferred into OC dished coated with feeder cell layer or Matrigel for culturing. All these OC dishes were placed in incubator. We observed the efficiency of outgrowths of ICMs and the efficiency of generating embryonic stem cell lines in3different systems and discuss the relationship between the quality of the blastocyst and the efficiency of generating human embryonic stem cell lines. Finally, we tested the sternness of the embryonic stem cell lines we generated.RESULTS:In our research, totally129embryos were distributed into three different group based on different generating and culturing system, there were33embryos in conventional culturing system;46embryos in mTESR1system and50embryos in mTESRl+5i system respectively. In conventional culturing system, there were7outgrowths were detected, the efficiency of the appearance of outgrowths was21%. There were5human embryonic stem cell lines were generated under conventional culturing system, the efficiency was11%. In mTESRl system, there were12outgrowths were detected, the efficiency of the appearance of outgrowths was26%. There were6human embryonic stem cell lines were generated under mTESRl system, the efficiency was11%.In mTESRl+5i system, there were19outgrowths were detected, the efficiency of the appearance of outgrowths was38%. There were no human embryonic stem cell lines were generated under this system. In our research we found that, the quality of blastocysts could effect the efficiency of generating human embryonic stem cell lines critically. Among all those relevant effecting factors, the development of ICM was the most important one, the develope stage of embryos was less important and there was no relationship between the trophoblast and the success of generating human embryonic stem cell lines. In the verification of stem cell lines,We chose stem cell line P096generated under mTESRl system. In the AP staining assay, P096were AP staining positive, colonies were stained purple while the feeder cells were not stained. In the immunofluorescent staining, the totipotent markers OCT-4、SSEA-4、TRA-1-60and TRA-1-81were detected in P096stem cell line. In RT-PCR assay, P096expressed totipotent genes OCT-4、NANOG and SOX-2. In genotype assay, P096remained normal genotype after long-term culture. In teratoma assay,P096formed teratoma subcutaneously in SCID mouses, and in pathological sections we observed different cells for three germ layers.CONCLUSION:①The quality of blastocyst was critical in generating human embryonic stem cell line. Among all those relevant effecting factors, the development of ICM was the most important one, the develope stage of embryos was less important and there was no relationship between the trophoblast and the success of generating human embryonic stem cell lines. Blastocysts better than3BC were more suitable for generating human embryonic stem cell lines.②Some of the ICMs(with outgrowths)could establish hESC lines at the original spot of seeding without seperating of the remaining trophoblast.③The defined culturing systems mTESRl shared the same efficiency of generating embryonic stem cell lines and subculture compared with the conventional culturing system.④In mTESRl+5i culturing system, the outgrowth of ICM could be detected, though the capability of generating human embryonic stem cell lines was insufficient obviously.