Hepatic Differentiation of Bone Marrow Mesenchymal Stem Cells in Vitro

Objective:To explore the feasibility of differentiation of rat BM-MSCs into hepatocyte-like cells induced by hepatocyte growth factor(HGF) and granulocyte colony stimulating factor (G-CSF) in vitro. Methods: Rat BM-MSCs were collected and identified. And then BM-MSCs were divided into the following groups and induced into hepatocyte-like cells. Go served as a negative control, with no HGF or G-CSF. G1 was a positive control, with 20ng/ml HGF. G2 contained 20 ng/ml HGF and 5 ng/ml G-CSF. G3 was 20 ng/ml HGF with 10ng/mlG-CSF while G4 contained 20ng/ml HGF and 20ng/ml G-CSF. In order to identify hepatocyte-like cells, glycogen synthesis was detected by PAS staining and expression of AFP and ALB mRNA levels were investigated with reverse transcription polymerase chain reaction (RT-PCR). Results: Cells showed morphologic changes of hepatocyte, especially in G3 and G4. Shape of cells in G0 showed no obvious change. Positive staining was found G1, G2, G3 and G4 on PAS response on day 7 and 14, while negative staining was found in G0. At each time point, there was significant difference in the rate of positive dyeing between G1, G2, G3 and G4(P0.05). Multiple comparison showed no significant difference between G1 and G2, or G3 and G4(P0.05). Significant difference was found between G2 and G3 or G4(P0.05). The rate of positive dyeing in G3 or G4 was higher than that of G2. AFP and ALB mRNA expression levels were detected by RT-PCR. AFP mRNA positive expression emerged on day 7, decreased on day 14 and disappeared on day 21 in G1, G2, G3 and G4.ALB mRNA positive expression emerged on day 14 and arrived at peak on day 21.G0 showed no expression of AFP or ALB. There was significant difference in expression level of AFP or ALB in G1-4 at different time point(P0.05), showing an downtrend in AFP, and increasing tendency in ALB. Multiple comparison of AFP or ALB showed no significant difference between G2 and G1 at the same time(P0.05).Statistical result was the same with the comparison of target genes between G4 and G3. At each time point, there was significant difference in expression level of AFP and ALB between G2 and G3 or G4(P0.05). G3 and G4 expressed more AFP or ALB than G2.Conclusion: Under the induction by HGF and G-CSF in vitro, BM-MSCs could differentiate into hepatocyte-like cells. Under application of HGF and appropriate dose of G-CSF, BM-MSCs could differentiate into hepatocyte-like cells more effectively than using HGF alone.