| In this study,the effects of the different fetal genders and seasons on the proliferation of buffalo fetal Bone marrow mesenchymal stem cells(BMSCs)were compared,and then the biological characteristics identification of the BMSCs was also detected.Meanwhile,the role of TGF-?1 in the maintenance of mesenchymal properties on buffalo fetal BMSCs was explored.Besides,the developmental potential of the reconstructed embryos from buffalo fetal BMSCs after nuclear transfer was investigated.These efforts would contribute to improve the culture system of buffalo fetal BMSCs,and provide a scientificfoundation for the selection of donor cells on buffalo nuclear transfer.At first,the effects of the different fetal genders and seasons on the growth characteristics of buffalo BMSCs were compared.The results showed that the primary buffalo fetal BMSCs which were isolated from different fetal genders and seasons all represented typical MSCs morphology.It was found that there was no significant difference in the proliferation of buffalo fetal BMSCs from different fetal genders(P>0.05).However,the proliferation of buffalo fetal BMSCs was signally affected by the culture seasons,and the proliferation ratio of BMSCs respectively isolated from the seasons of spring,summer and autumn was significantly faster than that of the winter(P<0.05).In cell colony experiment,the results found that the average number of cell colonies of buffalo fetal BMSCs among the spring,summer and autumn seasons showed no significant difference(71.33±1.25 vs 69.67±3.09 vs 73.33±1.24,P>0.05),but they were significantly higher than that of winter(71.33±1.25,69.67±3.09,73.33±1.24 vs 59.33 ± 2.62,P<0.05).The result of RT-PCR analysis was found that buffalo fetal BMSCs expressed the mRNA of CD73,CD105,Oct4.Cellular immunofluorescence staining result showed BMSCs also expressed Oct4,Sox2 and Nanog.Furthermore,the result of flow cytometry experiment confirmed that BMSCs expressed the surface antigen of CD44,CD29.And the third generation of BMSCs could be induced to differentiate into adipocytes,osteoblasts and chondrocytes through directional differentiation in vitro.Then,the effect of TGF-?1 in the maintenance of mesenchymal properties on buffalo fetal BMSCs was explored.It was found when buffalo fetal BMSCs were sub-cultured in vitro,the proliferation ability of BMSCs gradually became weaker with the increasing of passage,simultaneously,the cell morphology was irregular,and the mRNA expression of Oct4,Sox2 and Nanog was decreased,finally,the pluripotency of BMSCs was lost.The qPCR results showed that the expression of mesenchymal genes in BMSCs such as N-cadherin decreased sharply when cultured to the 10th generations(P<0.05).However,the proliferation speed of BMSCs was significantly accelerated after adding appropriate concentration of TGF-?1 to the cell culture medium.The qPCR results showed that the expression of N-cadherin and fn1 were significantly up-regulated,while the expression of epithelial marker gene such as E-cadherin was significantly down-regulated,moreover,the expression levels of EMT-related ' regulatory genes as Snail were enhanced,meanwhile the expression of pluripotency genes such as Sox2,Nanog was also significantly up-regulated(P<0.05).Cell adhesion experiments showed a decrease about cell adhesion in the TGF-?1 treated group,thus the BMSCs relatively had more mesenchymal cells characteristics.The result of karyotype analysis showed that the rate of normal karyotype in TGF-?1 treated group was significantly increased for the 10th generation of BMSCs compared with the control group(P<0.05).In addition,the developmental potential of the reconstructed embryos from both of buffalo fetal BMSCs and BFFs after nuclear transfer was compared.Theresults found that the blastocyst rate of BMSCs-NT was higher than that ofBFFs-NT,but it was not significant difference(P>0.05).Moreover,no significant difference was found in the mRNA expression of Oct4 of the blastocyst between two groups(P>0.05),while the mRNA expression of Nanog and Sox2 genes in the blastocyst of BMSCs-NT group were significantly higher than those in BFFs-NT group(P<0.05).The immunofluorescence staining result showed that Oct4 fluorescence degree of blastocyst in BMSCs-NT group was stronger than that in BFFs-NT group,the expression of Sox2 in two groups was weaker,and the expression of Nanog was not obvious in the blastocyst of two groups.These results demonstrated that:(1)The proliferate ability of buffalo fetal bone marrow mesenchymal stem cells is affected by different seasons,and the seasons of spring,summer and autumn are more suitable to isolate the buffalo fetal BMSCs.(2)Treating with appropriate concentration of TGF-?1 on buffalo fetal BMSCs is beneficial to maintain the characteristics of mesenchymal stem cells and karyotype stability.(3)The blastocysts rate of the reconstructed embryos from BMSCs after nuclear transfer is similar to that from BFFs,while the expression of pluripotency gene in BMSCs-NT blastocyst is higher than that in BFFs-NT. |
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