Generation of induced pluripotent stem cells from mouse buccal mucosa fibroblasts

Abstract

Objective To generate induced pluripotent stem cells (iPSCs) from mouse buccal mucosa fibroblasts (BFs) by genetic reprogramming. Methods iPSCs were established from mouse BFs via retroviral gene transfer with three reprogramming factors (Oct4, Sox2, and Klf4) . The properties of iPSCs were characterized by alkaline phosphatase staining assay, karyotype analysis, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) , immunofluorescence and bisulfite genomic sequencing. In vitro and in vivo studies were also performed to test their differentiation and pluripotent capability. Results The resulting iPSCs were found to resemble mouse embryonic stem cells (ESCs) with similar clone growth and high positive stain of alkaline phosphatase. They had normal karyotype, and expressed high levels of ESCs-like genes (Dppa3, Nanog, Rex1, Sox2, Klf4, Oct4 and c-Myc) and protein markers (Oct4, Sox2, and Nanog) . Bisulfite sequencing demonstrated that the methylation of the endogenous Oct4 and Nanog promoters in iPSCs and the control ESCs clones was low or absent, while a high methylation level was found in mouse embryonic fibroblasts (MEFs) . By in vitro induction, iPSCs could produce mineralized nodules, form adipocyte-like cells with lipid droplets, and change to chondrocytes. Moreover, they can differentiate into teratomas composed of tissues of the three germ layers in vivo. Conclusions iPSCs were successfully established from the mouse BFs with ESCs-like properties and their pluripotency was verified. These iPSCs may serve as seed cells for tooth regeneration. Key words: Induced pluripotent stem cells; Fibroblasts; Cellular reprogramming; Regeneration