| Embryonic stem cells (ESCs) are totipotent cells derived from the inner cell mass (ICM) of pre-implantation embryos or primordial germ cells (PGC) of embryos. Theoretically undifferentiated ESCs could be passaged in vitro infinitely with normal karyotypes. They are totipotent cells and have capability of differentiating into three blastodermic layers cells. ES cells could differentiate into any types of body cells in vitro, and run cells or organ transplantation with it. Using ES cells could make chimera animals and modify genes, etc. It is becoming a useful tool for studying cell differentiation, the mechanism of development, gene function, drug-selection and so on. Now experts in many territories around the world pay attention to the progression about differentiations of human embryonic stem cells and the progressions in tissue engineering. But embryonic stem cells are critical for the culture conditions. They are easy to differentiate and difficult to keep normal karyotypes. All of these hold back the developed step of human embryonic stem cells study and using. The critical factors of establish human embryonic stem cells are:①safe and good feeder cells;②high-quailty blastocyst;③safe and stabilize culture method;④effective method for isolation of inner cell mass,⑤effective and safe method of passage. There are two kinds culture system due to source of inhibit factors: feeder system and feeder-free system. Feeder cell is necessary for establishing human stem cells, which had be proved. It had be reported that establishing human stem cell successfully with two different culture methods: intact embryo culture method and immunosurgery method, But it could not sure which is better. In immunosurgery method, effevtive serum concentration is very important for isolating inner cell mass well. After primary colon implanted, correct digest time can maintain ES indifferent condition. To find correct time, we detected the expression of alkaline phosphates (AKP), stage-specific embryonic antigen-4(SSEA-4) of inner cell mass in different culture days. Based on establishment mouse embryonic stem cells successfully, research on blastocyst qulailty, different culture method, effective isolation method and correct digest time, which related to factors affecting the establishment of human embryonic stem cell. To find safe,effective method and establishing human embryonic stem cell lines successfully in future.Methods1,Mouse embryonic fibroblast (MEF) feeder layer come from the 12.5-14.5 pregnancy mouse.2,All the embryos were recruited from Reproductive Medical center in the first Affiliated Hospital of Zhengzhou University from May 2005 to February 2007. Then cultured and graded it. All the patient were written consent to experiment on their discard embryos.3,All the blastocysts were randomly divided into 2 groups, one group was cultured on the feeder cell just after removing zona pellucida; the other group was isolated inner cell mass by immunosurgery, then ICM implanted on the feeder layer. Record the rate of adherence,primary clone,passage.4,Putting the blastocysts into 4 different antibody and complement serum concentration when isolating ICM by immunosurgery, Record trophectoderm and ICM condition.5,Cultured ICM in ES medium, changed the medium everyday. Detected AKP staining and the expression of stage specific embryonic antigen 4(SSEA-4) of ICM on D5,D6,D7,D8,D9,D10,D11,D12.Result1,The result of embryos collected and culturedExperiment totally collected 257 discard embryos on D3, then got 102 blastocysts after culture. The rate of blastocyst was 39.7%, 13 3-4AA/3-4BA grade blastocysts,23 3-4AB/3-4BB grade blastocysts and 15 3CC grade blastocysts were used in establishment ES lines experiment. The other 52 blastocysts were detected the expression of ES markers.2,Efficiency of result by the blastocysts quality3-4AA/3-4BA grade blastocyst: The number totally were 13, 12 implanted, and 11 adherenced, the rate of adherence was 91.7%, the number of primary clone were 4, the rate of primary clone was 33.3%, only 2 passaged, the rate of passage was 16.7%.3-4AB/3-4BB grade blastocyst: The number totally were 23, 20 implanted, and 14 adherenced, the rate of adherence was 70%, the number of primary clone were 5, the rate of primary clone was 25%, onlyl passaged, the rate of passage was 5%.3CC grade blastocyst: The number totally were 15, 5 implanted, and 0 adherenced, the number of primary clone were 0, none passaged.The data of 4CC grade was lower than the others, P<0.01, The passage rate of 3-4AA/3-4BA grade was higher than 3-4AB/3-4BB grade blastocyst, P<0.05, and the other datas were no different.3,Effects of different culture methods on primary cloneIntact embryo culture method: The number of blastocysts were 19, 10 adherenced, the rate of adherence was 52%, the number of primary clone were 3, the rate of primary clone was 15.8%, none passaged.Immunosurgery method: The number of blastocysts was 32, isolated ICM was 18, 15 adherenced , the rate of adherence was 83.3%, the number of primary clone were 6, the rate of primary clone was 333.%, 3 passaged.Campareing two methods, the datas of immunosurgery method were higher than Intact embryo culture method, P<0.05.4,Comparison for different serum concentrations isolating ICM by immunosurgery1:8 rabbit anti human serum with 1:8 guinea-pig serum: trophectoderm cells wereno change.1:4 rabbit anti human serum with 1:4 guinea-pig serum: few trophectoderm cells were engorged. 1:2 rabbit anti human serum with 1:2 guinea-pig serum: trophectoderm cells and ICM were both destroyed.1:2 rabbit anti human serum with 1:4 guinea-pig serum: trophectoderm cells were removed clear, and ICM was intacted.5,The expression of alkaline phosphates (AKP), stage-specific embryonic antigen-4 (SSEA-4) of inner cell mass in different culture daysAKP: ICM on D5-7 was stained, D8-10 color was week, while D11 and D12 ,ICM could not be stain.SSEA-4: ICM on D5-8 had green fluorescent light, D9-10 light was week, while D11 and D12 , only we could see ICM cell and no loght.Conclusion1,3-4AA/3-4BA grade and 3-4AB/3-4BB grade blastocysts had effect on establishment of human embryonic stem cell lines, while lower grade blastocyst was ineffective for its' few cell and the poor capability of multiplication .2,Immunosurgery method was a better culture method, it choosed the good blastocysts when ICM was isolated, and raised the rate of adherenced , the number of primary colon because of removing trophectoderm cells which could leads to differentiation when coculture.3,1:2 rabbit anti human serum with 1:4 guinea-pig serum was best immunosurgery serum concentration. It can remove trophectoderm cells effectively.4,The best digest time was D7-D10 of culture day, the colon can multiplicate well while avoiding differentiation. |
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