[Human bone marrow multipotent adult progenitor cells differentiate into hepatocyte-like cells with hepatocyte growth factor plus fibroblast growth factor-4 in vitro].

OBJECTIVE

To investigate the possibility of the human bone marrow multipotent adult progenitor cells (hMAPCs) to differentiate into hepatocytes with hepatocyte growth factor (HGF)/ fibroblast growth factor-4 (FGF-4) in vitro.

METHODS

(1) Obtaining the hMAPCs. Bone marrow was obtained from volunteers and then centrifuged through density gradient centrifugation methods. The collected mononuclear cells were cultured through adheret culture to get mesenchymal stem cells (MSCs). The hMAPCs were obtained through collecting and isolating the MSCs by magnetic activated cell sorting (MACS) through depletion selection by use of CD45 and GlyA microbeads. (2) Differentiation of the hMAPCs with HGF+FGF-4. Group A: HGF (20 ng/ml) + FGF-4 (10 ng/ml) induced hMAPCs; group B (positive control group): L-02 human hepatocytes(cell lines); and group C (negative control group): the undifferentiated hMAPCs. (3) The expressions of albumin (Alb), alpha fetoprotein (AFP), cytokeratin-18 (CK-18), and cytokeratin-19 (CK-19) were detected with immunocytochemistry to identify the characteristics of the differentiated cells at different times and the ratio of the positive cells was determined. (4) ALB, AFP, CK-18, and CK-19 expressions of the differentiated cells were detected by RT-PCR assay to investigate the mRNA transcriptions of characteristic hepatic proteins. (5) Alb expressions of the differentiated cells at different times were detected by Western blot on the 21st and 35th days.

RESULTS

(1) The results of immunocytochemistry. The staining of Alb, CK18 were essentially positive in group A. As an early marker of immature hepatocytes, AFP staining was positive on the 7th day but negative in later differentiating periods in group A. (2) The results of RT-PCR. On the 7th day, the differentiated hMAPCs expressed AFP mRNA but were negative in later differentiating periods. On the contrary, the mRNA of Alb and CK-18 were positive at all times. (3) The results of Western blot assay. Alb protein was positive on the 21st day and 35th day.

CONCLUSIONS

Under some definite inducing conditions hMAPCs can differentiate into hepatocyte-like cells. They may serve as a potential cell source for liver engineering.