Studies On The Isolation And Culture Of Naive/Primed Buffalo Embryonic Stem-Like Cells Derived From Blastocysts At Different Stages

The establishment of Embryonic Stem Cells (ESCs) line is effected by its source of cells and the environment of cultivation. The major factor influences the efficiency of blastocyst-derived ESCs which is the stage of blastocysts. Among the blastocysts form and implant to the uterine wall, pluripotent cells of blastocysts are in a transform process of self-renewal and differentiation, there are different stages of pluripotent cells, which is likely to eventually determine the state of pluripotent cells. Therefore, this study was to explore the expression pattern of pluripotent genes and the effect of isolating and culturing of buffalo ES-like cells in different stages of embryos. This study identified pluripotent state of buffalo ES-like cells in order to determine which stage of blastocysts is more suitable for isolating and culturing of buffalo ES-like cells.Firstly, the expression patterns of pluripotent genes in different stages of early embryos (Morula, Early blastocyst, Blastocyst, Expanded blastocyst, Hatched blastocyst) were analysised by qRT-PCR and immunofluorescence staining. Results showed that:pluripotent genes presented in the morula and early blastocyst mainly, the naive-related pluripotent genes KLF4, TBX3 and REX-1 high expressed in the two stages. The blastocyst was a relatively quiet period of gene expression and most of the expression of pluripotent genes were reduced. In expanded blastocyst, the expression of KLF4, TBX3 and REX-1 was low, but it highly expressed primed-related gene FGF5. Cultured the buffalo embryos with 2i (1 ?mol/mL PD0325901,3 ?mol/mL CHIR99021) in day 5 had no effect on the formation of buffalo blastocysts, the number of cells in blastocysts and the content of the ICM cells. The pluripotent genes expression pattern in 2i treatment group were similar to the control group, but the expression levels were significantly lower (P<0.05), the silent stage of blastocysts pluripotency was delay to expaned blastocyst and hatched blastocyst after 2i treatment.Secondly, the effect of different stages of blastocysts on isolating and culturing of buffalo ES-like cells were compared, and the biological characteristics of ES-like cells were analysed. Results showed that:the rate of adherent and original colony formation of the ICM which derived from expaned blastocyst and hatched blastocyst were significantly higher than early blastocyst and blastocyst, and the diameter of buffalo ES-like cells which derived from hatched blastocyst in 2i treatment group was significantly greater than the control group (P<0.05). The expression of core transcription factor in primary buffalo ES-like cells from different stages of blastocysts was analysed. Results found that the relative expression of NANOG and SOX2 in ES-like cells which derived from expanded blastocyst were significantly higher than hatched blastocyst. The expression of naive-related genes KLF4, TBX3 and REX-1 in 2i treatment groups were significantly higher than the control group (P<0.05). Furthermore, the expression of TBX3 and REX-1 in ES-like cells which derived from hatched blastocyst were significantly higher than expanded blastocyst. The results of analysing the biological characteristics in passaged buffalo ES-like cells showed that buffalo ES-like cells growed as colonies, expressed pluripotent genes OCT4, NANOQ SOX2, c-MYC, CDH1, KLF4 and TBX3. Immunofluorescence staining found that it expressed OCT4 and SOX2, weakly expressed SSEA1, SSEA4 and TRA-1-60. For experiment of differentiation in vitro, buffalo ES-like cells could form simple embryoid bodies which expressed three germ layer marker genes GATA4, TUBB3, ACTA2 and AFP. It could form fibroblast-like, epithelial-like and neuron-like cells after differentiation.Finally, in order to identify the pluripotent state of buffalo ES-like cells, the activity of X chromosome was detected, the expression of pluripotent-related genes were analysed, the reaction of different pluripotent signal to buffalo ES-like cells was analysed respectively. Results showed that the female buffalo ES-like cells inactivated one X chromosome randomly. It expressed naive-related gene KLF4, TBX3, primed-related gene FGF5 and ACTA2 but not naive-related gene REX-1. Both immunofluorescence staining and western blot analysis found that the buffalo ES-like cells expressed FGF5 but not REX-1. Buffalo ES-like cells could maintain its pluripotency in the signal of LIF and bFGF best, then in the primed-related signal bFGF+Noggin, it could not maintain its pluripotency in the naive-related signal LIF+BMP4. The gene expression patten and reaction to different plutipotent signal indicated buffalo ES-like cells were more similar to primed state stem cells.In conclusion:(1) the expression of pluripotent gene in buffalo early embryo presents in the morula and early blastocyst mainly, and the blastocyst is a relatively silent pluripotent stage.2i treatment can delay the pluripotent silent period to expanded blastocyst stage. (2) Expaned blastocyst and hatched blastocyst are suitable for the derivation of buffalo ES-like cells, moreover,2i treatment in buffalo embryos has positive effect on keeping the expression of naive-related genes in primary colony of buffalo ES-like cells. (3) The buffalo ES-like cells are similar to the primed state after passaged cultivation.