Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System

Abstract

Objective: 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.

Materials and methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Buffalo; Embryonic Stem Cells; FGF-2; LIF; Y-27632.

Fig.1

The growth rate of buffalo ES cell colonies in the presence of LIF and FGF-2 either alone or in the presence of Y-27632 (Y=Y- 27632). ES; Embryonic stem, LIF; Leukemia inhibitory factor and FGF-2; Fibroblast growth factor.

Fig.2

Alkaline phosphatase and immunofluorescence staining for characterization of buffalo ES cells at passage 20. AP; Alkaline phosphatase, ES; Embryonic stem, SSEA; Stage-specific embryonic antigen and TRA; Tumor rejection antigen.

Fig.3

A. Compact embryoid bodies, B, C. Cystic embryoid bodies produced from buffalo ES cells, D, E and F. Different cell types produced by spontaneous differentiation (lipid-like cells, epithelial- like cells and hepatocyte-like cells, respectively, scale bar=200 μm). ES; Embryonic stem.

Fig.4

The effects of fibroblast growth factor 2 (FGF-2, 5 ng/ml) and leukemia inhibitory factor (LIF, 1000 U) on buffalo ES cells cultured on gelatin coated dishes on, A. Mean area of buffalo ES colonies and B. Expression of pluripotency genes. C; Control, F; FGF-2, L; LIF, FL; FGF-2+LIF and ES; Embryonic stem.

Fig.5

The effects of Y-27632 (10 μM) on buffalo ES cells cultured on gelatin coated dishes on A. Mean area of buffalo ES cellcolonies and B. Expression of pluripotency genes. FGF-2; Fibroblast growth factor 2, LIF; Leukemia inhibitory factor, FL; FGF-2+LIF, FLY; FGF-2+LIF+Y-27632 and ES; Embryonic stem.

Fig.6

The effects of bFF feeder layer and gelatin coated dishes either alone or feeder-CM, on buffalo ES cell colonies. A. Mean area of buffalo ES cellcolonies, B. Expression of pluripotency genes for normal and high concentration of fibroblast cells (3x10⁴ and 5x10⁴ cells per cm², respectively) in feeder layer and C. Phase-contrast image after 24 hours and 6 days of culture, A. Gelatin coated dishes, B. Normal concentration of bFF cells in feeder layer, C. High concentration of bFF cells in feeder layer, D. 50% feeder-CM in gelatin coated dishes and E. 100% feeder-CM in gelatin coated dishes. bFF; Buffalo Fetal fibroblast, CM; Conditioned media and ES; Embryonic stem.