| In this study, to explore the effects and mechanism of Wnt/β-catenin signaling pathway on the maintenance of pluripotency of Buffalo Embryonic Stem Cell-Like Cells (Buffalo ES cell-like cells), the GSK3inhibitors BIO and CHIR99021were used to culture for the buffalo ES cell-like cells. These efforts will provide a chemical screening platform to optimize the culture condition for establishing buffalo ES cells lines.Four experiments were included in this study.The experiment1, effect of GSK3inhibitor on primary colony formation of buffalo ESC-like Cells was investigated. The percentage of Inner cell mass (ICMs) attachment and primary colony formation were observed and found that there was no significant difference in the ICMs attachment rate among of the0.5μg/mL BIO,5mmol/L CHIR99021and the control groups (91.18%and92.98%vs94.59%, P>0.05), moreover, treating with CHIR99021could obtained more primary colony formation rate comparing with the control group (77.71%vs55.41%, P<0.05),but the group treated with BIO was not significantly higher than that of control group (72.06%vs55.41%, P>0.05).The experiment2, effect of GSK3inhibitor on the proliferation of buffalo ESC-like Cells primary colonies was studied. Firstly, the mitosis index of buffalo ES cell-like Cells primary colonies was detected by Brdu. The results indicated that the proliferation rate of primary colonies in the group treated with CHIR99021was significantly higher than that of the control group on day1, day3, day4and day5(P<0.05), but the group treated with BIO was evidently higher than that of control group only on day1(P<0.05), and there was no significant difference in other days (P>0.05). Secondly, the average diameter of Buffalo ES cell-like primary colonies was measured and found that the average diameter of primary colonies in the group treated with CHIR99021was remarkably bigger than that in control group (203.41μm vs128.01μm, P<0.05), but it was no significant difference between the group treated with BIO and control group (166.64μm vs128.01μm, P>0.05). Furthermore, based on qRT-PCR analysis, the expression of proliferation marker PCNA was significantly up-regulated in the both of CHIR99021and BIO groups (P<0.05). Additionally, the passaging times of buffalo ES cell-like cells derived from the control, BIO and CHIR99021groups were observed, and found that both of the control and BIO groups are currently maintained for10passages, and the group of CHIR99021is presently maintained for11passages.The experiment3, effect of GSK3inhibitor on maintaining pluripotency of buffalo ESC-like Cells was detected. The mRNA expression level of Oct4、 Sox2、Nanog were analysed by qRT-PCR. The results showed that treating buffalo ESC-like Cells with CHIR99021significantly up-regulated the expression of Oct4、Sox2(P<0.05),but had no effect on Nanog expression (P>0.05).However, in the treatment group of BIO, the expression of Oct4was obviously increased (P<0.05),and the expression of Sox2and Nanog were significantly decreased (P<0.05).The experiment4,effect of GSK3inhibitor on Wnt/β-catenin signaling pathway in buffalo ESC-like Cells was studied. The relative protein level of β-catenin (the downstream effector of Wnt/β-catenin signaling pathway) was detected by immunofluorescence staining, and found that treating buffalo ESC-like Cells with CHIR99021and BIO significantly increased the relative protein level of β-catenin in buffalo ESC-like Cells (P<0.05). The mRNA expression level of c-Myc (the downstream target gene of Wnt/β-catenin signaling pathway) was analysed by qRT-PCR, and showed that the expression of c-Myc was obviously increased when the buffalo ESC-like Cells treated with CHIRR99021and BIO(P<0.05).In conclusions, these results suggest that treating buffalo ES cell-like cells with GSK3inhibitors BIO and CHIR99021can promote proliferation of buffalo ES cell-like cells and maintain their undifferentiated state, and up-regulate the expression levels of β-catenin and c-Myc in buffalo ES cell-like cells, moreover, CHIR99021is better than BIO. These indicate that Wnt/β-catenin signaling pathway plays an important role in regulation of self renewal of buffalo ES cell-like cells. |
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