Abstract: Objective To optimize the protocols for isolation, in vitro culture, identification and induction of hepatic
differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Methods Rat 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. Results The 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. Conclusion The optimized protocol allows simple
and efficient isolation of highly purified populations of BMSCs, which can be induced into hepatic lineages in specific
microenvironment.