310 EFFECT OF TGF-β1 ON THE SELF-RENEWAL OF BUFFALO (BUBALUS BUBALIS) EMBRYONIC STEM CELLS

Abstract

Substantial species differences exist in the type of signalling pathways that govern self-renewal of embryonic stem (ES) cells in a pluripotent state. Members of TGF-βsuper-family, which includes more than 30 proteins in mammals, play an important role in self-renewal of these cells in several species. Production of ES cells from buffalo, a very important farm animal in many developing countries, could have a big influence on their genetic modification through transgenesis and cloning. The aim of this study was to examine the role of TGF-β1 in self-renewal of buffalo ES cells, because no information is available on any signalling pathway in ES cells of this species. Buffalo ES cells derived from in vitro produced embryos between passage 41 and 64 that had been characterised using transcription-based markers OCT-4, NANOG, SOX-2, FOXD-3, REX-1, STAT, and NUCLEOSTAMIN and surface markers SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81 were cultured on buffalo fetal fibroblast feeder layer in ES cell culture medium (Knockout-DMEM + 15% Knockout Serum Replacer + 5 ng mL–1 of fibroblast growth factor-2 + 1000 IU mL–1 of murine leukemia inhibitory factor) and TGF-β1 (0, 0.1, 1.0, or 10 ng mL–1) or its inhibitor SB431542 (0, 10, 25, or 50 μM). The medium containing TGF-β1 or SB431542 at respective concentrations was changed every 24 h. The percentage of colonies that appeared morphologically normal after 144 h of culture and the colony size at 24, 72, and 144 h of culture were recorded. The percentage of ES cell colonies found to be morphologically normal (rounded or dome shaped, compact, with defined edges, and with no apparent signs of cell death) was significantly higher (P ≤ 0.05) with 1.0 ng mL–1 of TGF-β1 than that with 0 (control), 0.1, or 10 ng mL–1 of TGF-β1. Although the percent increase in ES cell colony size was significantly higher (P ≤ 0.05) with 1.0 ng mL–1 of TGF-β1 than that of controls or with other concentrations of TGF-β1 at 72 h, the increase in colony size was not significantly different among the 4 groups at 144 h of culture. The percentage of ES cell colonies found to be morphologically normal was not significantly different for 0 (control), 10, or 25 μM of SB-431542 but decreased significantly (P ≤ 0.05) with 50 μM of SB-431542. During the 144-h period of culture, the percent increase in colony size was significantly lower (P ≤ 0.05) with 10 μM SB-431542 than that in the controls, whereas with 25 or 50 μM SB-431542, the colony size decreased (P ≤ 0.05) instead of increasing. Buffalo fetal fibroblast feeder layers expressed TGF-β1 strongly at passage-3, -5, and -7, as examined by RT-PCR. These results suggest that although TGF-β signalling appears to be active in buffalo ES cells, TGF-β1 supplementation of culture medium does not improve the self-renewal of buffalo ES cells, at least in the presence of fibroblast growth factor-2 and leukemia inhibitory factor. This work was supported by NAIP grant No. C4/C-2067 from ICAR, India.