| Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of early blastrocyts, which possess the ability of unlimitted proliferation and maintain normal dipoid keptypes under differentiation-inhibited culture in vitro, they will differentiate into multiple lineage cell types or occur commentent and differentiation without differentiation-inhibited factors or hi presence of certain induced-factors.. Based on the review of previous researches of ES cells, we conducted to isolate mouse ES cells from the ICM of early blasteocyts, to proliferate ES cell D3 line and to examine their ability of spontaneous differentiation in vitro, as well as to induce the ES cell D3 line into osteoblasts by conditioned-medium, ascorbic acid, β -glycerophosphate and/or dexame-thasone.1. Isolation and growth curve of MEFThe mouse embryonic fibroblasts (MEF) proliferated slowly that the time of doubling (Td) was 47.13h or 47.96h, respectively, and their growth state is poor when seeded into each well of 24-well plates at 5.0±103 cells with replacing fresh medium at 2- or 4-day intervals, whereas MEF flourished at 2.0±104 cells per well with replacing fresh medium at 4-day intervals (Td-35.38h) and it was appropriate to passage after 3 to 5 days of culture, and there was no benefits to the growth of the cells as replacing fresh medium at 2-day intervals (Td=41.56). The cell proliferation become rapidly when plated at 5.0±104 cells per well with replacing fresh medium at 2-day intervals (Td=53.12h), and needed to be subcultured after the culture of 2 to 3 days, if not, the cultured cells were easily expire, and on the contrary, the speed of the cell proliferation decreased as replacing fresh medium at 4-day intervals (Td=57.80h).2. Effects of mitomycin C on MEFThe MEF were inactivated by mitomycin C for 30, 90, 180min, respectively, at 24h after seeded into each well of 24-well culture plate at 3±104 cells. The low number of proliferating cells in all experimetial groups was significantly different from that of the control group (P<0.01) at the 3th, 5th, 7th, 9th, llth day of culture, respectively. Incontrast, there was not significantly different among the three treated groups (P>0.05). The results indicated that the MEF inactivated by 10μg/mL mitomycin C for 30 min were inhibited efficiently and maintained not to proliferate for 11 days at least.3. Isolation and culture of ES cells from mouse blastocytsWhen intact blastocyts from 3.5 dpc mouse cultured on the mitomycin C-inactivated MEF in Doulbecco's modified minimal essential medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 10ng/mL rmLIF,0.1mmol/L 2-mercaptoethanol, 80U/mL penicillin and 100U/mL streptomycin, the zona pellucida disengaged and removed from their blastocyts within 24h, but few blastocyts without zona pellucida attached to culture dish. The ratio of attached blastocyts was 39% and 74% at 48 h and 72 h of culture, respectively. The cloning ratio of ES cells was only 8 percent of growing ICM, and the ES clonies disappeared after going 5 passage at best.4. Proliferation of ES cell D3 lineES cell D3 line maintained the flourish and undifferentiated state with typical clonies at 48 h on the mitomycin C-inhibited MEF in DMEM supplemented with 15% Knockout Serum Replacement (KSR) or 15% fetal bovine serum (FBS). They propagated rapidly and needed to replace fresh medium every day and to subculture at 2-day intervals, whereas did not maintain the efficient proliferation and survive supplemented wth 15% newborn calf serum (NBS). The clonies of ES cells were flatter on the mitomycin C-inhibited STO fibroblasts, not easily differentiated from the feeder layer cells surround them, than that on the MEF in DMEM supplemented 15%FBS.5. Spontaneous differentiation of ES cell D3 line in vitroES cell D3 line via suspension culture can congregate small mass within 24 h, and large numbers of aggregates called embryoid bodies (EBs) gradually formed after 2 to 3 days of culture. During the 6th to the 8th days o...
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