101 AMNIOTIC FLUID STEM SELLS IN BUFFALO (BUBALUS BUBALIS): IDENTIFICATION OF PLURIPOTENCY MARKERS, CHARACTERIZATION, AND GUIDED DIFFERENTIATION INTO NEUROGENIC, ADIPOGENIC, AND OSTEOGENIC LINEAGES

Abstract

Amniotic fluid (AF) holds great promise as a stem-cell source. We report expression of various pluripotency molecular and immunocytochemical markers and chemically induced differentiation of bubaline (Bubalus bubalis) AF cells into neurogenic, adipogenic, and osteogenic lineages. Expression of pluripotecy-related markers, namely Oct-4 (also known as POU5F1), Sox-2, nestin, fibroblast growth factor (FGF-5), Nanog, alkaline phosphatase (AP), 18S rRNA, stem cell factor (SCF), cyclin A, β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and GATA-4, was observed in the cells, with amplicons of 314, 277, 307, 210, 317, 180, 162, 216, 421, 178, 180, and 334 bp, respectively. A semiquantitative real-time PCR analysis revealed the overexpression of Oct-4, Nanog, Sox-2, SCF, nestin, and FGF-5. Furthermore, the immunocytochemical staining confirmed the expression of Oct-4, Nanog, Sox-2, SSEA-1, SSEA-4, tumor rejection antigens (TRA-1-60 and TRA-1-81), and AP in AF cells. The undifferentiated cells were found to have a normal karyotype before and after their cryopreservation. We conclude that AF cells may contribute toward establishing nonembryonic pluripotent mesenchymal stem cells for various therapeutic and reproductive biotechnological applications in this species, and possibly in other livestock species.