OBJECTIVETo optimize the protocols for isolation, in vitro culture, identification and induction of hepatic differentiation of rat bone marrow mesenchymal stem cells (BMSCs).

METHODSRat BMSCs were separated and purified by differential adherent culture for 1.5 h with the first medium change at 12 h. The surface markers of BMSCs were detected by flow cytometry. The cells were induced to differentiate into adipogenic, osteogenic, and chondrogenesis lineages. A 3-step protocol including sequential addition of growth factors, cytokines and hormones was used to induce the BMSCs to differentiate into hepatocyte-like cells.

RESULTSThe cells isolated using this protocol were positive for CD29, CD44, and CD90 and negative for CD29 and CD45. The adipogenic, osteogenic, and chondrogenic differentiation of the BMSCs were verified by Oil red, Alizarin red, and toluidine blue staining. The BMSCs induced with the 3-step protocol differentiated into hepatic-like cells that expressed hepatocyte-specific proteins (ALB and AFP) and genes.

CONCLUSIONThe optimized protocol allows simple and efficient isolation of highly purified populations of BMSCs, which can be induced into hepatic lineages in specific microenvironment.