Hepatocyte growth factor and basic fibroblast growth factor induce differentiation of human bone marrow mesenchymal stem cells into hepatic lineage cells

Yi ZHANG; Yuanmei CHE; Yang WANG

ABSTRACT

BACKGROUND:

Within the bone marrow stroma there exists a subset of non-hematopoietic stem cells referred to as marrow stromal cells or mesenchymal stem cells. Mesenchymal stem cells (MSCs) are a group of cells with highly capability of self-renew and potential of multilineage differentiation, these properties make them present a promising prospect for clinical practice. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. However, the culture system has not been developed.

OBJECTIVE:

To explore whether human MSCs are able to differentiate into functional hepatocyte-like cells with hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) in vitro.

DESIGN:

Open study.

SETTING:

Department of Infectious Disease and Institute of Urology Surgery, First Affiliated Hospital, Nanchang University.MATERIALS The study was performed in the Institute of Urolgoy Surgery, the First Affiliated Hospital of Nanchang University from July 2004 to March 2005. Bone marrow was donated by healthy adult with informed consent. DMEM/F12 medium (Gibco); insulin, transferrin, human epidermal growth factor (EGF); human HGF; monoclonal antibodies against human AFP; FITC-conjugated rabbit anti-mouse IgG (Sigma); human bFGF (Invitrogen); monoclonal antibodies against human CK18 and CK19 (Chemicon); fetal bovine serum (Si jiqin, Hangzhou).

METHODS:

Bone marrow (10 mL) in this study was aspirated from the iliac crest of healthy donors. MSCs were isolated by density gradient centrifugation in combination with plastic adherence. For hepatic differentiation, the 4th- to 8th-passage human MSCs seeded on 24-well tissue culture plates coated with 0.1% gelatin, at 1×104 MSCs/mL, were serum deprived for 2 days, in DMEM/F12 supplemented with 10 μg/L EGF, 10 μg/L bFGF, 5 mg/L insulin and 5 mg/L transferrin. Differentiation was induced by treating MSCs with differentiation medium, consisting of DMEWF12 supplemented with 10 μg/L bFGF, 20 μg/L HGF, 5 mg/L insulin, 5 mg/L transferrin. Medium changes were performed every three days. MSCs without HGF and bFGF in medium served as the control. In the differentiating period, the concentration of AFP in the suernatant was determined dynamically by radioimmunoassay (RIA). The hepatic surface phenotype including AFP, CK18 and CK19 were identified by immunofluorescent staining at day 0, 7, 14, 21 and 28. Glycogen storage was detected by Periodic Acid-Schiff (PAS) staining.MAIN OUTCOME

MEASURES:

① the morphological changes of induced MSCs; ② the concentration of AFP in the supernatant; ③ the hepatic surface phenotype; ④ glycogen storage.

RESULTS:

① After 14 days ofinduction, the fibroblast-like morphology of human MSCs was lost and cells became broadened and fiattened. After prolonged culture, polygonal cells were seen and further matured hepatocyte-like colonies were seen by day 28. ② The concentration of AFP in the supernatant was first detected on day 14, at a concentration of 0.1 μg/mL, and increased to 0.4 μg/mL by day 17, then decreased to 0.3 μg/mL by day 21. ③ Immunofiuorescent staining showed the expression of AFP and CK18 until day 14. The expression of CK19 was detected by day 28. ④ Glycogen storage could be detected by day 21.

CONCLUSION:

Human bone marrow MSCs are able to differentiate into functional hepatocyte-like cells and may sere as a new source of cells for cell therapy of hepatic diseases.