Culture, characterization and differentiation of cells from buffalo (Bubalus bubalis) amnion

Abstract

Stem cells present an important tool in livestock assisted reproduction and veterinary therapeutic field such as tissue engineering. We report for the first time isolation of pluripotent stem cell-like cells expressing pluripotency markers (alkaline phospahatase, OCT-4, NANOG and SOX-2) from the amnion of water buffalo (Bubalus bubalis). The cells showed no apparent abnormalities in their chromosomal profiles before and after cryopreservation. The cytochemical staining revealed that pluripotent cells were capable of undergoing directed differentiation in vitro into osteocytes. It could be inferred that amnion-derived pluripotent stem cell-like cells can be isolated, cultured for many passages and differentiated into mesoderm lineage, and may be an alternative source to mesenchymal stem cells. These cells can have applications in assisted reproduction, developmental biological and regenerative medicine.

Fig. 1

Isolation and culture of amnion cells from amniotic membrane. a A thin-walled amniotic membrane that surrounds the fetus, b Amnion cells resemble to fibroblasts like morphology (×100), c confluent culture (70–80 %) of amnion cells in primary culture (×100)

Fig. 2

Characterization of buffalo amnion cells at different passages of culture. a Expression of AP in primary tissue (×400), b at passage 5 exhibition of strong positive staining (red) in buffalo amnion cells (×100), b RT-PCR analysis of gene expression in amnion cells, where Lane1 100 bp ladder 2 OCT-4 3 NANOG 4 SOX-2 5 β-actin 6 Negative control 7 100 bp ladder

Fig. 3

In vitro induced differentiation of amniotic membrane derived amnion cells. a osteocytes (×100), b osteogenic differentiated cells depicting Alizarin red (×200)