ABSTRACT
In order to retain an undifferentiated pluripotent state, embryonic stem [ES] cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase [AP] and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 [FGF-2], leukemia inhibitory factor [LIF] and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells
ABSTRACT
This research studies the effects of activation and inhibition of WntSA signaling pathway in buffalo [Bubalus bubalis] embryonic stem [ES] cell-like cells. To carry on this experimental study, the effects of activation and inhibition of WntSA signaling in buffalo ES cell-like cells were examined using Bio [0.5 mM] combined with WNT3A [200 ng/ml], as an activator, and Dickkopf-1 [Dkkl, 250 ng/ml], as an inhibitor, of the pathway. ES cells were cultured up to three weeks in ES cell medium without fibroblast growth factor-2 [FGF-2] and leukemia inhibitory factor [LIF], but in the presence of Bio, WNTSA, Bio+WNT3A and Dkkl. The effects of these supplements were measured on the mean area of ES cell colonies and on the expression levels of a number of important genes related to pluripotency [Oct4, Nanog, Sox2 and c-Myc] and the Wnt pathway [p-cateniri]. ES cell colonies cultured in ES cell medium that contained optimized quantities of LIF and FGF-2 were used as the control. Data were collected for week-1 and week-3 treated cultures. In addition, WNT3A-transfected ES cells were compared with the respective mock-transfected colonies, either alone or in combination with Dkkl for expression of fi-catenin and the pluripotency-related genes. Data were analyzed by ANOVA, and statistical significance was accepted at P=O.05. Among various examined concentrations of Bio [0.5-5 mM], the optimum effect was observed at the 0.5 mM dose as indicated by colony area and expressions of pluripotency-related genes at both weeks-1 and -3 culture periods. At this concentration,the expressions of Nanog, Oct3/4, Sox2, c-Myc and fl-catenin genes were nonsignificant-ly higher compared to the controls. Expressions of these genes were highest in the Bio+WNT3 A treated group, followed by the WNT3A and Bio-supplemented groups, and lowest in the Dkkl-treated group. The WNT-transfected colonies showed higher expressions compared to both mock and Dkkl-treated mock transfected colonies. WNT3A functions to maintain the pluripotency of ES cell-like cells both as an exogenous growth factor as well as an endogenously expressed gene. It complements the absence of FGF-2 and LIF, otherwise propounded essential for buffalo ES cell culture. WNT3A antagonizes the inhibitory effects of Dkkl and acts in combination with its activator, Bio, to activate the Wnt signaling pathway