Generation of functional hepatocyte-like cells from human bone marrow mesenchymal stem cells by overexpression of transcription factor HNF4α and FOXA2

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(hi Hep 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 hi Hep cells. Results: hi Hep 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. hi Hep 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 hi Hep cells were significantly higher than those in MSCs-Vector cells. The hi Hep 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 hi Hep cells from MSCs.     

Bone-marrow mesenchymal stem cells reduce rat intestinal ischemia-reperfusion injury,ZO-1 downregulation and tight junction disruption via a TNF-α-regulated mechanism

AIM: To investigate the effect of bone-marrow mesenchymal stem cells (BM MSCs) on the intestinal mucosa barrier in ischemia/reperfusion (I/R) injury. METHODS: BM MSCs were isolated from male Sprague-Dawley rats by density gradient centrifugation, cultured, and analyzed by flow cytometry. I/R injury was induced by occlusion of the superior mesenteric artery for 30 min. Rats were treated with saline, BM MSCs (via intramucosal injection) or tumor necrosis factor (TNF)-α blocking antibodies (via the tail vein). I/R injury was assessed using transmission electron microscopy, hematoxylin and eosin (HE) staining, immunohistochemistry, western blotting and enzyme linked immunosorbent assay.RESULTS: Intestinal permeability increased, tight junctions (TJs) were disrupted, and zona occludens 1 (ZO-1) was downregulated after I/R injury. BM MSCs reduced intestinal mucosal barrier destruction, ZO-1 downregulation, and TJ disruption. The morphological abnormalities after intestinal I/R injury positively correlated with serum TNF-α levels. Administration of anti-TNF-α IgG or anti-TNF-α receptor 1 antibodies attenuated the intestinal ultrastructural changes, ZO-1 downregulation, and TJ disruption. CONCLUSION: Altered serum TNF-α levels play an important role in the ability of BM MSCs to protect against intestinal I/R injury.