A study of rat mesenchymal stem cells (MSCs) differentiating into liver cells when co-cultured with rat hepatocytes / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Mesenchymal 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>Rat MSCs co-cultured with hepatocytes can differentiate into hepatocytes.</p>

Hepatocyte growth factor induces differentiation of adult rat mesenchymal stem cells into a hepatocyte lineage in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To explore whether the mesenchymal stem cells (MSCs) of rats can be induced into hepatocytes and the condition of differentiation in vitro.</p><p><b>METHODS</b>Mesenchymal stem cells were collected from the femora of SD rats by density gradient centrifugation and identified by flow cytometric analysis and alkaline phosphatase (AKP) staining. MSCs were divided into 4 groups to induce differentiation with the different concentration of hepatocyte growth factor (HGF) in culture medium. The concentration of each group was group A 0 ng/ml, group B 10 ng/ml, group C 20 ng/ml and group D 40 ng/ml, respectively. The morphological changes of MSCs were observed by phase-contrast microscope. On day 1, 3, 7, 14, 21 and 28, mRNA of albumin, AFP and CK18 of MSCs of each group were examined by reverse transcription polymerase chain reaction (RT-PCR), and the expressions of them were also detected with immunohistochemistry technique.</p><p><b>RESULTS</b>Mesenchymal stem cells collected from the femora of SD rats expressed antigens of CD29, CD44 and CD90, but not CD34 and CD45. AKP staining was negative for all of MSCs. On day 7, AFP mRNA of MSCs in group C and D could be detected by RT-PCR, and increased on day 14, and then directed on day 21. Albumin and CK18 mRNA of MSCs in group C and D could also be detected from day 14 to day 28 by RT-PCR. On the contrary, mRNA of AFP, CK18 and albumin was not detected in group A and B of culture. Immunocytochemical analysis for CK18, albumin and AFP showed positive staining reaction for AFP on day 7, for CK18 and albumin on day 14 in group C and D, and negative staining reaction both in group A and B of culture.</p><p><b>CONCLUSION</b>MSCs of adult rats cultured in high concentration of HGF can differentiate into hepatocytes.</p>