OBJECTIVETo explore whether rat mesenchymal stem cells (MSCs) can be induced to develop into hepatocytes and the role of the microenvironment of hepatocytes growth in inducing MSCs differentiating into hepatocytes in vitro.

METHODSMesenchymal stem cells were collected from the aspirates from femurs of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase (ALP) staining. Rat hepatocytes were isolated by the modified two-step method described by Seglen. Two 6-well culture plates were piled up after the chambers' bottoms of the upper plate was removed. Then the upper and lower chambers were separated by a semi-permeable membrane. MSCs and hepatocytes of rats were plated separately in the upper and lower chambers of the two 6-well culture plates for co-culturing. MSCs cultured alone without co-culturing with hepatocytes served as controls. On days 1, 3, 7, 14, 21 and 28, mRNA of cytokeratin 18 (CK-18), alpha-fetoprotein (AFP) and albumin were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and immunocytochemistry staining of CK-18 AFP and albumin were also examined.

RESULTSThe shapes of MSCs co-cultured with hepatocytes changed and their sizes and numbers increased in the course of the culturing. When MSCs were co-cultured with hepatocytes for 2 weeks, colonies composed of polygonal cells resembling mature hepatocytes were found. In the controls, shapes of cells also changed and their sizes and numbers increased, but colonies composed of polygonal cells resembling mature hepatocytes were not found. Of the MSCs co-cultured with hepatocytes, on day 7, the mRNA of AFP was detected by RT-PCR, and it increased on day 14, and then decreased on day 21. mRNA of albumin and CK-18 were detected by RT-PCR from day 14 to day 28 in the co-cultured cells, but mRNA of AFP and CK-18 and albumin were not detected in the controls in the course of the culturing. Immunocytochemical analysis for CK-18, albumin, and AFP, showed positive staining reaction for AFP on day 7, for CK-18 and AFP on day 14 in the co-cultured cells but not in the controls.

CONCLUSIONSRat MSCs co-cultured with hepatocytes can differentiate into hepatocytes.