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World J Gastroenterol. Jun 14, 2005; 11(22): 3479-3484
Published online Jun 14, 2005. doi: 10.3748/wjg.v11.i22.3479
Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro
Xin-Qin Kang, Wei-Jin Zang, Tu-Sheng Song, Xiao-Li Xu, Xiao-Jiang Yu, Dong-Ling Li, Ke-Wei Meng, Sheng-Li Wu, Zhi-Ying Zhao
Xin-Qin Kang, Wei-Jin Zang, Xiao-Li Xu, Xiao-Jiang Yu, Dong-Ling Li, Division of Cardiovascular Physiology and Pharmacology, Medical School of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Wei-Jin Zang, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Xin-Qin Kang, Tu-Sheng Song, Department of Biology, Medical School of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Ke-Wei Meng, Sheng-Li Wu, Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Zhi-Ying Zhao, Department of Anatomy, Medical School of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Wei-Jin Zang, Box 77, Division of Cardiovascular Physiology and Pharmacology, Medical School of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China. zwj@mail.xjtu.edu.cn
Telephone: +86-29-82655003 Fax: +86-29-82655190
Received: June 29, 2004
Revised: June 30, 2004
Accepted: August 5, 2004
Published online: June 14, 2005
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 µg/L (t = 2.31, P<0.05) in MSCs cultured with FGF-4 and HGF, and were higher (46.2±1.5 µg/L) on d 21 (t = 41.926, P<0.01), then decreased to 24.8±2.2 µg/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 µg/mL, and to 2.1±0.7 µg/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.

Keywords: Mesenchymal stem cell; Differentiation; Hepatocyte