| Embryonic stem (ES) cells are pluripotent cells derived from the inner cell masses (ICMs) of preimplantation embryos or primordial germcell (PGCs), capable of self-renewing, proliferating indefinitely and differentiating into a wide variety of cell types both in vitro and in vivo. This line of cells possess features of both embryo cells, with normal diploid karyotype and developmental toti-or pluripotency, and that of normal cultured cells, proliferating indefinitely, tolerant of cryopreservation and genetic operations. Cattle as one of the most important domestic animal, its ES cells have a broad application prospect. It has turned to be an urgent problem to resolve the difficulties confronted in the establishment of ES cell line and to define the suitable condition for the growth of ES cells. In this research, different types and densities of feeder cells, three kinds of treatment of embryos and multiple classes of culture media have been explored to investigate the effects on culture of bovine ES cells. The expression of differential expressed mRNA and protein in different morphology and passages of bovine ES cell were tested in the method of DDRT-PCR and Western blotting, so that to provide the experimental basis for establishing bovine ES cells culture system.Effects of different feeder layer cells on bESC-like cell growth1. Effects of murine embryonic fibroblasts (MEF) or bovine embryonic fibroblasts (BEF) as feeder layer cells on bESC-like cell growthBEF and MEF were used as feeder layers respectively for bESC-like cells culture. Formation of stem cells clones with morphological characteristics could be observed on both kinds of feeder cells. However, bESC-like cells on BEF layer could not grow as fast as that on MEF layer, and the colonies could not attach tightly. Disaggregation occurred till the fifth passage and all the clones disappeared. Whereas, ES cell colonies on MEFs could maintain undifferentiated state untill the10th passage.2. Effects of feeder layer density on embryo attachment rates and ES cell colony forming ratesThree densities of MEF feeder cells were used to compare the effects on embryo attachment and ES cell colony formation. The results showed that the best derivation and maintenance of bESC-like cells could be acquired at a feeder cell density of1.25×105cells/ml, with efficient attachment and the highest primary colony forming rate. Effects of embryo treatment and inoculation density on bESC-like cell isolation and culture1. Effects of embryo treatment on bESC-like cell isolation and cultureThe ICM used for bESC-like cells isolation were acquired from zona pellucida free blastocysts treated by pronase, naturally hatched blastocysts and mechanical isolated ICM. The result showed that the most efficient adherence was obtained in naturally hatched blastocysts, indicating this method to be beneficial for isolation and culture of bESC-like cells.2. Effect of inoculation density on bESC-like cell isolation and cultureNaturally hatched blastocysts were inoculated at a density of1-4embryos/well in24-well plates, the highest primary colony forming rates (76%) were acquired at the density of2embryos/well.Effects of culture medium formula on bESC-like cells cultureTo confirm the effects of serum and IGF1on bESC-like cells culture, three different kinds of culture media were investigated, with a MEF feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. The results showed that there was no significant difference on bESC-like cells culture between normal serum and stem cell-specific serum supplemented in the culture media. The bESC-like cells cultured in media with10ng/ml IGF1possessed the most vigorous clone colony growth and the largest formed clone. However, the cells showed a distinct differentiation at the fifth passage, suggesting that IGF1facilitated bESC-like cells growth, but was not in favor of maintenance of stem cells pluripotency.Verification of bESC-like cellImmunofluorescence assay of OCT-4, SSEA-1, SSEA-4and TRA-1-61and RT-PCT assay of Nanog, Oct-4and Sox-2has been taken in bESC-like cells cultured in DMEM/F12media with15%FBS,0.1mM β-mercaptoethanol,0.1mM non-essential amino acids, penicillin/streptomycin,10ng/ml LIF and10ng/ml bFGF, with a feeder cells density of1.25×105/ml and inoculation density of2embryos/well in24-well plate. These analysis all showed positive results. The alkaline phosphatase assay of bESC-like cells in different shapes and passages also showed positive results. These results confirmed that the cultured and collected bESC-like cells shared the features of stem cells.mRNA DDR-PCR assay and identification of bESC-like cells of different shapes1. mRNA expression detection of differential expression genesIn this research, differential expression genes of bESC-like cells in sheet, vesicular and lump shapes have been screened by DDRT-PCR assay, expression quantity was analyzed by Q-PCR. Six DNA fragment has been acquired and confirmed to be homologous to RPL9, LOC100850994,AMP,RPL31,Erbb2ip and CLIP1, respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis of the six differential genes showed that RPL9expression quantity in lump and vesicular shaped bESC-like cells were0.67(P<0.01) and1.46(P<0.01) times as that in sheet shaped cells, LOC100850994expression quantity in lump and vesicular shaped bESC-like cells were0.33(P<0.01) and2.73(P<0.01) times as that in sheet shaped cells, RPL31expression quantity in lump and vesicular shaped bESC-like cells were0.71(P<0.05) and1.58(P<0.01) times as that in sheet shaped cells,, respectively. There were no significant difference for AMP, Erbb2IP and CLIP1expression quantity between sheet shaped cells and lump shaped cells, whereas their expression quantity in vesicular cells were2.11(P<0.05),1.43(P<0.05) and1.61(P<0.05) times as that in sheet shaped cells respectively.2. Protein expression detection of differential expression genesProtein expression of differential expression genes were detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results showed that sheet shaped cells possessed the highest protein expression level of RPL9, lump shaped cells possessing the lowest level (P＜0.05). RPL31expression was almost same (P>0.05) between sheet and vesicular shaped cells but descended significantly in lump shaped cells (P＜0.05). There was no significant difference for AMP and CLIP1protein expression in the three shaped cells(P>0.05).mRNA DDR-PCR assay and identification of bESC-like cells of different passages1. Detection of differential genes expressionDifferential expression genes in bESC-like cells of the first, fifth and tenth passage were screened by DDRT-PCR assay, and the expression quantity was analyzed by Q-PCR. Seven DNA fragments had been acquired and confirmed to be homologous to IK,TKDP1,BZW, RPL31,PRL9,RP42and IGBP1respectively after sequencing and comparative analysis with the GenBank database. The results of real time PCR detection and SPSS statistical analysis showed that the expression level of IK, TKDP1, BZW, PRL9, RP42and IGBP1in the cells of the tenth passage descend significantly (P<0.05) compared with that of the first passage, and there was no significant difference for RPL31expression among them (P>0.05).2. Protein expression detection of differential expression genesProtein expression of differential genes was detected by Western blotting, with sheet shaped bESC-like cells as calibration and a-tubulin as internal reference. The results suggested that there was a consistently significant higher expression level of all the detected genes in the cells of the first passage than that of the tenth (P＜0.05).
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