Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

Abstract

Aim: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), 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. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining.

Results: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20+/-1.16 microg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28+/-3.11 microg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02+/-0.15 microg/mL, and to 3.63+/-0.30 microg/mL on d 28 (t = 11.748, P<0.01). Urea (4.72+/-1.03 micromol/L) was detected on d 20 (t = 4.272, P<0.01), and continued to increase to 10.28+/-1.06 micromol/L on d 28 (t = 9.276, P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24.

Conclusion: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCB-derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

Figures

Figure 1

Different morphology and differentiation of HUCB-derived MSCs. A: morphology of primary cultured HUCB-derived MSCs. MSCs are spindle-like and osteroclasts are big and round; B: morphology of sub-cultured HUCB-derived MSCs stained with HE. The fibroblast-like cells are growing as a whirlpool ( ×100); C: representative immuocytochemistry showing expression of CK-18 on HUCB-derived MSCs on d 28 (SABC×200); D: cells stained by PAS. Glycogen storage is seen as accumulated magenta staining, the round and epithelioid cells show magenta staining in the region (×400).