Low serum concentration facilitates the differentiation of hepatic progenitor cells

OBJECTIVE: To investigate the effect of serum culture conditions on the differentiation of hepatic progenitor cells (HPCs) in vitro.

METHODS: This study was conducted at the Stem Cell Biology and Therapy Laboratory, The Children's Hospital of Chongqing Medical University, Chongqing, China from August 2009 to June 2010. Differentiation of HPCs was induced with dexamethasone, hepatic growth factor, and fibroblast growth factor-4 in 10% fetal bovine serum (FBS), 2% FBS, 2% horse serum (HS) conditions. First, we investigated the indirect synthesis of albumin by the albumin promoter-driven Gaussia luciferase assay, and cell proliferation by cell counting. Then, we performed reverse transcriptase-polymerase chain reaction and immunofluorescence to detect hepatic related markers Delta-like protein (DLK), cytokeratin 18 (CK18), tyrosine aminotransferase (TAT), albumin (ALB), a-fetoprotein (AFP), and glucuronosyltransferase 1A (UGT1A). Lastly, we used indocyanine green (ICG) uptake assay and periodic acid-Schiff (PAS) staining to determine the function of induced hepatocytes.

RESULTS: The proliferation of HPCs was inhibited in 2% serum culture. Induced HPCs in 2% serum exhibited higher expression of ALB, CK18, UGT1A, and TAT, whereas lower expression of DLK and AFP, compared with 10% FBS. Interestingly, 2% serum culture improved ICG uptake function, but repressed PAS staining of induced HPCs. Generally, 2% HS was better at inducing mature hepatic markers and the function of HPCs than 2% FBS.

CONCLUSION: Two percent low serum concentration conditions, especially 2% HS, improve the hepatic differentiation of HPCs, and PAS staining may not be used in induction testing of low serum concentrations.