Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro

Abstract

Aim: To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (MSCs) into hepatocytes and to find a new source of cell types for therapies of hepatic diseases.

Methods: MSCs were isolated by combining gradient density centrifugation with plastic adherence. The cells were cultured in osteogenic or adipogenic differentiation medium and determined by histochemical staining. MSCs were plated in plastic culture flasks that were not coated with components of extracellular matrix (ECM). When MSCs reached 70% confluence, they were cultured in low glucose Dulbecco's modified Eagle's medium supplemented with 10 mL/L fetal bovine serum, 20 ng/mL hepatocyte growth factor (HGF) and 10 ng/mL fibroblast growth factor-4 (FGF-4). The medium was changed every 3 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Glycogen store of hepatocytes was determined by periodic acid-Schiff staining.

Results: By combining gradient density centrifugation with plastic adherence, we isolated a homogeneous population of cells from rat bone marrow and differentiated them into osteocytes and adipocytes. When MSCs were cultured with FGF-4 and HGF, approximately 56.6% of cells became small round and epithelioid on d 24 by morphology. Compared with the control, levels of AFP increased significantly from d 12 to 15.5+/-1.4 microg/L (t = 2.31, P<0.05) in MSCs cultured with FGF-4 and HGF, and were higher (46.2+/-1.5 microg/L) on d 21 (t = 41.926, P<0.01), then decreased to 24.8+/-2.2 microg/L on d 24 (t = 10.345, P<0.01). Albumin increased significantly on d 21 (t = 3.325, P<0.01) to 1.4+/-0.2 microg/mL, and to 2.1+/-0.7 microg/mL on d 24 (t = 3.646, P<0.01). Urea (2.3+/-0.4 mmol/L) was first detected on d 21 (t = 6.739, P<0.01), and continued to increase to 2.6+/-0.9 mmol/L on d 24 (t = 4.753, P<0.01). Glycogen storage was first seen on d 21.

Conclusion: The method combining gradient density centrifugation with plastic adherence can isolate MSCs. Rat MSCs may be differentiated into hepatocytes by FGF-4 and HGF. Cytokines may play a more important role in differentiation from rat MSCs into hepatocytes.

Figure 1

Different morphology and differentiation of rat MSCs. A: Morphology of rat MSCs stained with HE. The cells were fibroblast-like and grew as a whirlpool (×100); B: Appearance of osteocytes 21 d after induction. The positive cells were observed by stain for alkaline phosphatase. The gray parts were cytoplasm stained for alkaline phosphatase. The cells differentiated MSCs into osteocytes. The blue ones were nuclei stained with hematoxylin (×400); C: Oil red-O positive cells 6 d after adipogenic differentiation of rat MSCs. The red parts were lipid-rich vacuoles, the blue ones were nuclei stained with hematoxylin (×100); D: Cells stained by PAS. Glycogen storage was seen as accumulated magenta staining. The small round cells and epithelioid cells had magenta staining in the region (×200).