Author/Authors :
Zandi، Mohammad نويسنده Department of Animal and Poultry Science and Fisheries, Agricultural Institute, Iranian Research Organisation for Science and Technology (IROST), Tehran, Iran , , Shah، Syed Mohamad نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India , , Muzaffar، Musharifa نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India , , Singh، Manoj Kumar 1972- نويسنده , , Palta، Prabhat نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India , , Singla، Suresh Kumar نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India , , Manik، Radhey Sham نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India , , Chauhan، Manmohan Singh نويسنده Embryo Biotechnology Laboratory, Animal Biotechnology Centre, National Dairy Research Institute (NDRI), Karnal, India ,
Abstract :
Background: This research studies the effects of activation and inhibition of Wnt3A
signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells.
Materials and Methods: To carry on this experimental study, the effects of activation
and inhibition of Wnt3A 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 (Dkk1,
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, WNT3A, Bio+WNT3A and Dkk1. 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 (B-catenin). 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 Dkk1 for expression of B-catenin and the pluripotency-related genes.
Data were analyzed by ANOVA, and statistical significance was accepted at P < 0.05.
Results: 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 B-catenin genes were nonsignificantly
higher compared to the controls. Expressions of these genes were highest in the
Bio+WNT3A treated group, followed by the WNT3A and Bio-supplemented groups, and
lowest in the Dkk1-treated group. The WNT-transfected colonies showed higher expressions
compared to both mock and Dkk1-treated mock transfected colonies.
Conclusion: 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 Dkk1 and acts in combination with its activator,
Bio, to activate the Wnt signaling pathway.
