Pei-Yi Xie1, Xiao-Jun Hu2, Ruo-Mi Guo3, Xiao-Chun Meng4, Peng-Fei Pang2, Zhi-Yang Zhou4, Dan Li2, Hong Shan5. 1. Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China; Department of Radiology, the Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China. 2. Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China; Interventional Medicine Center, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China; Interventional Radiology Institute, Sun Yat-sen University, Zhuhai 519000, China. 3. Department of Radiology, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China. 4. Department of Radiology, the Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, China. 5. Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China; Interventional Medicine Center, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China; Interventional Radiology Institute, Sun Yat-sen University, Zhuhai 519000, China. Electronic address: shanhong@mail.sysu.edu.cn.
Abstract
BACKGROUND: Our previous study showed that overexpression of hepatocyte nuclear factor 4α (HNF4α) could directly promote mesenchymal stem cells (MSCs) to differentiate into hepatocyte-like cells. However, the efficiency of hepatic differentiation remains low. The purpose of our study was to establish an MSC cell line that overexpressed HNF4α and FOXA2 genes to obtain an increased hepatic differentiation efficiency and hepatocyte-like cells with more mature hepatocyte functions. METHODS: Successful establishment of high-level HNF4α and FOXA2 co-overexpression in human induced hepatocyte-like cells (hiHep cells) was verified by flow cytometry, immunofluorescence and RT-PCR. Measurements of albumin (ALB), urea, glucose, indocyanine green (ICG) uptake and release, cytochrome P450 (CYP) activity and gene expression were used to analyze mature hepatic functions of hiHep cells. RESULTS: hiHep cells efficiently express HNF4α and FOXA2 genes and proteins, exhibit typical epithelial morphology and acquire mature hepatocyte-like cell functions, including ALB secretion, urea production, ICG uptake and release, and glycogen storage. hiHep cells can be activated by CYP inducers. The percentage of both ALB and α-1-antitrypsin (AAT)-positive cells was approximately 72.6%. The expression levels of hepatocyte-specific genes (ALB, AAT, and CYP1A1) and liver drug transport-related genes (ABCB1, ABCG2, and SLC22A18) in hiHep cells were significantly higher than those in MSCs-Vector cells. The hiHep cells did not form tumors after subcutaneous xenograft in BALB/c nude mice after 2 months. CONCLUSION: This study provides an accessible, feasible and efficient strategy to generate hiHep cells from MSCs.
BACKGROUND: Our previous study showed that overexpression of hepatocyte nuclear factor 4α (HNF4α) could directly promote mesenchymal stem cells (MSCs) to differentiate into hepatocyte-like cells. However, the efficiency of hepatic differentiation remains low. The purpose of our study was to establish an MSC cell line that overexpressed HNF4α and FOXA2 genes to obtain an increased hepatic differentiation efficiency and hepatocyte-like cells with more mature hepatocyte functions. METHODS: Successful establishment of high-level HNF4α and FOXA2 co-overexpression in human induced hepatocyte-like cells (hiHep cells) was verified by flow cytometry, immunofluorescence and RT-PCR. Measurements of albumin (ALB), urea, glucose, indocyanine green (ICG) uptake and release, cytochrome P450 (CYP) activity and gene expression were used to analyze mature hepatic functions of hiHep cells. RESULTS: hiHep cells efficiently express HNF4α and FOXA2 genes and proteins, exhibit typical epithelial morphology and acquire mature hepatocyte-like cell functions, including ALB secretion, urea production, ICG uptake and release, and glycogen storage. hiHep cells can be activated by CYP inducers. The percentage of both ALB and α-1-antitrypsin (AAT)-positive cells was approximately 72.6%. The expression levels of hepatocyte-specific genes (ALB, AAT, and CYP1A1) and liver drug transport-related genes (ABCB1, ABCG2, and SLC22A18) in hiHep cells were significantly higher than those in MSCs-Vector cells. The hiHep cells did not form tumors after subcutaneous xenograft in BALB/c nude mice after 2 months. CONCLUSION: This study provides an accessible, feasible and efficient strategy to generate hiHep cells from MSCs.