Derivation Of Neural Progenitor Cells From Teratomas Of Human Embryonic Stem Cells And Effects Of Knockdown Of Dnmt3a And Dnmt3b On Human Embryonic Stem Cells

Human embryonic stem cells(hESCs) were derived from human early blastocysts,were able to be expanded long-term in vitro with stable karyotype,and were able to self-renewal and differentiate into all cells types of all three germ layers.The hESCs provided great promise for regenerative medicine and provided valuable tools for researching early human embryonic development and differentiation,gene functions and for evaluating of pharmacologic action.Based on introducing hESCs stain-HSF6,following studies were carried out.Chapter 1 Study on culturing system of hESCs-HSF6.Aims:to study and establish culturing system of hESCs-HSF6.Methods:MEF cells were derivated from embryos of 13.5-days-pregency ICR mice.The MEF were treated with 10μg/mL mitomycin C or 3 000 Radγ-ray.The hESCs were maintained on the irradiated MEF in KO-DMEM with daily medium changes.The hESCs were passaged using two methods:enzymatical method with 1mg/mL collagenaseⅣand lmg/mL dispase and machanical method with Pasteur glass pipets.The characterizations of hESCs were carried out using immunostaining and differentiating.Results:the passage 3 to 5 MEF did not proliferate after being treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray.The enzyme-treated expansion rapidly produced greater amounts of hESCs within a limited time frame.However,the cell clumps varied in size,and there was a probability that both the differentiated and undifferentiated cells would be transferred.The mechanical method was laborious and time-consuming,but the technique permitted efficient transfer of undifferentiated hESCs and results in similar clump sizes.In cases in which there were differentiated colonies,the combination of two methods allows mass production of hESCs by excluding differentiated colonies from passage by manual selection before enzyme treatment.Results of immunostaining and differentiaon showed that the hESCs could self-renewal and maintain undifferetiation in the culture system.Conclusions:①ICR-MEF were derivated successfully from embryos of 13.5-days-pregency ICR mice;it was efficient to make feeder for hESCs-HSF6 with passage 3 to 5 ICR-MEF treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray MEF;②Both mechanical and enzymatic transfer methods for hESCs depent on experimental purpose. The mechanical transfer method was good for maintenance of hESC lines.Chapter 2 Derivation of NPC from teratomas of hESCs.Aims:to differentiate hESCs into teratomas in vivo and to derivate neural progenitor cells(NPC) from the teratomas.Methods:hESCs clumps were harvested and were injected into SCID-beige mice.The mice were sacrificed and teratomas were got and identified.NPC were derivated from teratomas through plating cells onto polyornithine-(PO) and fibronectin(FN)-coated dishes in serum-free medium DMEM/F12 supplemented with B27 supplement,and 10ng/ml bFGF.The NPC were fixed for immunostaining with mouse monoclonal anti-nestin.The NPC were induced to differentiate by withdrawing bFGF and the differenctiated cells were fixed for immunostaining with mouse monoclonal anti-TuJⅠand mouse monoclonal anti-GFAP.Results:The hESCs grafted into SCID mice consistently developed into teratomas after 5- to 8-week.They were analyzed histologically and contained representative tissues of the three germ layers.NPCs were derived successfully from the teratomas of hESCs through consecutive passages and culture in PO/FN-coated plate.Using antibodies against neural stem cell marker nestin,the cells were detected nestin positive.The NPC could differentiate into neurons and astrocytes.Using antibodies against neuronal markerβⅢtubulin(TuJⅠantigen) and astroglial marker glial fibrillary acidic protein(GFAP),it was found that TuJⅠ-positive or GFAP-positive cells were presented in these differentiated teratoma-derived NPCs.Conclusions:①NPCs were derived successfully from the teratomas of hESCs through culture in PO/FN-coated plate;②the model of "hESCs-SCID mice-teratoma" provided a new approach or tool for cell-based therapy and reseach of differentiation or development.Chapter 3 Effects of knockdown of Dnmt3a and Dnmt3b on hESCs.Aims:to study on effects of knockdown of DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b(Dnmt3b) on hESCs.Methods:The morphous and growth of hESCs were observed after the Dnmt3a and Dnmt3b were knockdown.Immunostaining with makers of hESCs were used to detect their characterizations.Reverse transcription PCR(RT-PCR) was used to detect Oct3/4,Nanog and Sox2 that were correlated to hESCs self-renewal.Potentials of differentiation of hESCs after being knockdown Dnmt3a and Dnmt3b were examined:in vitro be induced into embryoid body(EB) and in vivo be induced into teratoma.Results:The hESCs could maintain self-renewal and undifferentiation after being knockdown Dnmt3a and Dnmt3b,and they grow colonly on MEF similar to wild type hESCs.Results of immunostaining showed that they still were SSEA-4-,TRA-1-60-,TRA-1-81- and OCT3/4-positive. They still expressed Oct3/4,Nanog and Sox2.The AKP staining of EB showd that they were difficult to differentiate into embryo bodies(EB) in vitro and knockdown of Dnmt3b could cause neuroectoderm emerge ahead of schedule in EBs.Both hESCs of knockdown of Dnmt3a and Dnmt3b could not differentiate into teratoma in SCID mice.Conclusions:①Dnmt3a and Dnmt3b may be not essential for hESC maintaining self-renewal and undifferentiation;②Dnmt3a and Dnmt3b may be essential for hESC differentiation normally.