The Assessment of Apoptosis during Mouse Embryonic Stem Cells Differentiation towards the Primordial Germ Cells in Alginate Microspheres

Abstract

Introduction: Previous studies have shown the differentiation of Primordial germ cells (PGCs) from embryonic stem cells (ESCs). However, some PGCs die during the differentiation. Alginate biopolymers have shown to closely mimic an in vivo environment and they are suitable for stem cells viability. The aim of the present study was to asses ESCs apoptosis during differentiation into PGCs in alginate microspheres. Materials and Methods: Mouse ESCs were cultured in ESCs medium, alginate biopolymer and bone morphogenetic protein 4 (BMP4) were utilized as a differentiation factor. The expression of genes B-cell lymphoma 2 (Bcl2), BCL2 Associated X Protein (BAX), octamer-binding transcription factor 4 (Oct4), and VASA as a specific gene for PGCs, were analyzed in the time of differentiation. Results: Expression of apoptosis gene, BAX, in cells entrapped in alginate microspheres; in addition, they were significantly down-regulated and expression of anti-apoptosis gene, Bcl2, was not up-regulated in the entire groups. VASA gene expression was significantly up-regulated in alginate groups. The highest ratio of cells differentiated to putative PGCs belonged to the cells in alginate group without BMP4; based on the analysis of the flow cytometry. Immunocytochemistry staining as well demonstrated differentiation in these cells. Conclusion: It was magnificently attained that down-regulation of apoptosis gene and up-regulation of differentiation gene in alginate beads revealed the survival ability of the putative PGCs in alginate compare to ESCs medium. It was obtained that the improvement of alginate microspheres could assist ESCs differentiation towards the putative PGCs.