Isolation And Characterization Of Mouse Embryonic Stem Cells Expressing Green Fluorescence Protein

Objective: The target of this study is to investigate GFP-expressing cell rate in organ tissues derived from EGFP mouse, to observe the effects of the homemade mytomycin on growth of the MEFs in vitro, to isolate pluripotent mouse embryonic stem cells (ES cells) from blastocysts of EGFP mouse and to analysis some biological characteristics of ES cells. GFP-expressing ES cells have great advantage in researching cell differentiation.Methods : The cell suspensions derived from organ tissues of EGFP mouse using 0.05%Trypsin -0.02%EDTA solution was used to analysis GFP-expressing cells with a flow cytometry. In our experiment, the MTT methods was adopted. The mouse embryonic fibroblasts (MEF) were treated by different concentrations of the homemade mytomycin for different duration to observe the effects of division and vitality in vitro. The GFP-expressing embryonic stem cells were isolated from inner cell mass (ICM) which were separated from EGFP-transgenic mouse blastocysts of 4 dpc. The blastocysts from EGFP mouse were collected and cultured on mouse embryonic fibroblast (MEF) feeder. The culture medium is DMEM with 4500mg/1L glucose , supplemented with 10% fetal bovine serum (FBS), 2mmol/1 L-glutamine, 0.1mmol/1L B-mercaptoethanol, 1000IU/ml mouse leukemia inhibitory factor (mLIF) and penicillin and streptomycin. Inner cell mass (ICM) was transferred into liquid drops of 0.05%Trypsin-0.02%EDTA. After incubation for 3～5minutes, the disaggregated clumps of ICM were implanted into newly-prepared feeder layer and were cultured continuously. After 4-6 days, ES cells-like colonies were continually disaggregated and were cultured ES cells were freezed in passage 5. To testify those cell colonies, AKP staining and immunocytochemistry of Oct-4,SSEA-1 was carried out. These ES cells in passage 10 was also to analysis the GFP-expressing cell rate with a flow cytometry.Resu Its : The rate of GFP-expressing cells in organ tissues of EGFP mouse is different. The rate of GFP-expressing cells in the spleen ,brain, heart, thymus and marrow is 99.75%, 91.76%, 87.95%, 83.32%, 83.16%, respectively. The rate GFP-expressing cells in the intestinal epithelium, epidermis, liver and muscle is 26.67%, 36.21%, 46.51%, 52.88%, respectively. The results show that the mytomycin produced by Zhejiang Hisun Pharmaceutical Co.,Ltd (20μg/mL for 4h, 30μg/mL for 2 or 4 h) can inhibit effectively the division of the MEFs, but not affect vitality of the MEFs and that resultant ES cells express high levels of green fluorescent protein(GFP) with a flow cytometry analysis and maintain the developmental potential to form teratoma with all three embryonic germ layers. AKP staining and immunocytochemistry of Oct-4,SSEA-1 is positive.Conclusions:(1) The rate of GFP-expressing cells in organ of EGFP mouse is different.(2)The ES cell derived from EGFP mouse can be used to study differentiatation from ES cells to the spleen, brain, heart, thymus. (3) The homemade mytomycin produced by Zhejiang Hisun Pharmaceutical Co.,Ltd (30μg/mL for 2) can inhibit effectively the division of the MEFs, but not affect vitality of the MEFs.(4) ES cells colonies which express AKP, SSEA-land Oct-4 positive can be isolated from ICM of blastocyst of EGFP mouse. Differentiation experiment showed that ES cells have ability to form EBs and teratomas with three embryonic germ layers which express strong green fluorescent protein.