Hypoxia-Induced miR-210 Overexpression Promotes the Differentiation of Human-Induced Pluripotent Stem Cells to Hepatocyte-Like Cells on Random Nanofiber Poly-L-Lactic Acid/Poly (ε-Caprolactone) Scaffolds

An alternative treatment to liver transplantation includes the use of differentiated stem cells. Hypoxia has been shown to endow human-induced pluripotent stem cells (hiPSCs) with enhanced hepatic differentiation. We have investigated a new strategy for hepatocyte differentiation from hiPSCs using a three-step differentiation protocol with lentiviral overexpression of hypoxia-microRNA-210 of cells grown on a hybrid scaffold. We analyzed the transduction of the miR-210 lentiviral and definitive endoderm and pluripotency gene markers, including SRY-box 17 (SOX17), forkhead box A2 (FOXA2), and octamer-binding transcription factor 4 (OCT-4) by Real-Time PCR and fluorescent microscope. The scanning electron microscopy (SEM) examined the 3D cell morphological changes. Immunocytochemistry staining was used together with assays for aspartate aminotransferase, alanine aminotransferase, and urea secretion to analyze hepatocyte biomarkers and functional markers consisting of α-fetoprotein (AFP), low-density lipoprotein (LDL) uptake, fat accumulation, and glycogen. The flow cytometry analyzed the generation of reactive oxygen species (ROS). Compared to cells transfected with the blank lentiviral vectors as a control, overexpressing miR-210 was at higher levels in hiPSCs. The expression of endodermal genes and glycogen synthesis significantly increased in the differentiated lentiviral miR-210 cells without any differences regarding lipid storage level. Additionally, cells containing miR-210 showed a greater expression of ALB, LDL, AST, ALT, urea, and insignificant lower AFP and ROS levels after 18 days. However, SEM showed no significant differences between cells under the differentiation process and controls. In conclusion, the differentiation of hiPSCs to hepatocyte-like cells under hypoxia miR-210 may be a suitable method for cell therapy and regenerative medicine.

Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells

Background Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. Methods We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. Results We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. Conclusion This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

Sodium Butyrate Pre-Treatment Enhance Differentiation of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) into Hepatocytes and Improve Liver Injury

Objective: The treatment of liver failure by stem cell transplantation has attracted growing interest. Herein, we aim to explore the role of sodium butyrate (NaB) in the hepatic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro and vivo. Materials & Methods: We isolated BM-MSCs from the mononuclear cell fraction of rabbit bone marrow samples, and identified the cells by Immunophenotypic analysis. We investigated the effects of different concentrations and induction conditions. The histone deacetylase inhibitor NaB induced hepatic differentiation of BM-MSCs under liver-specific factors induction in vitro. Morphological features, liver-specific gene and protein expression, and functional analyses in vitro and vivo were performed to evaluate the hepatic differentiation of BM-MSCs. Results: Our results showed that pre-treated NaB inhibited the expression of liver-specific protein in a dose-dependent manner. The induction efficiency of NaB with 24h pre-treatment was higher than that of NaB continuous intervention. 0.5 mM 24h NaB pre-treated cells can improve liver tissue damage in vivo. And the liver ALB, AAT and the serum TP were significantly increased, while the serum ALT was significantly reduced. Conclusion: Continuous NaB treatment can inhibit BM-MSCs proliferation in a dose-dependent manner at a certain concentration range. 0.5 mM 24h pre-treatment of NaB enhanced differentiation of BM-MSCs into hepatocytes and improves liver injury in vitro and vivo.

Regulation of Liver Regeneration by hepatocyte O-GlcNAcylation in mice

The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here we report that O-GlcNAcylation, an intracellular posttranslational modification (PTM) regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration (LR) following partial hepatectomy (PHX). We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-Seq studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and IHC analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes. These studies show that O-GlcNAcylation plays a critical role in the termination of LR via regulation of HNF4α in hepatocytes.

miR-27b antagonizes BMP signaling in early differentiation of human induced pluripotent stem cells

Human induced pluripotent stem (hiPS) cells are feasible materials for studying the biological mechanisms underlying human embryogenesis. In early embryogenesis, definitive endoderm and mesoderm are differentiated from their common precursor, mesendoderm. Bone morphogenetic protein (BMP) signaling is responsible for regulating mesendoderm and mesoderm formation. Micro RNAs (miRNAs), short non-coding RNAs, broadly regulate biological processes via post-transcriptional repression. The expression of miR-27b, which is enriched in somatic cells, has been reported to increase through definitive endoderm and hepatic differentiation, but little is known about how miR-27b acts during early differentiation. Here, we used miR-27b-inducible hiPS cells to investigate the roles of miR-27b in the undifferentiated and early-differentiated stages. In undifferentiated hiPS cells, miR-27b suppressed the expression of pluripotency markers [alkaline phosphatase (AP) and nanog homeobox (NANOG)] and cell proliferation. Once differentiation began, miR-27b expression repressed phosphorylated SMAD1/5, the mediators of the BMP signaling, throughout definitive endoderm differentiation. Consistent with the above findings, miR-27b overexpression downregulated BMP-induced mesendodermal marker genes [Brachyury, mix paired-like homeobox 1 (MIXL1) and eomesodermin (EOMES)], suggesting that miR-27b had an inhibitory effect on early differentiation. Collectively, our findings revealed a novel antagonistic role of miR-27b in the BMP signaling pathway in the early differentiation of hiPS cells.

Challenges for the Applications of Human Pluripotent Stem Cell-Derived Liver Organoids

The current organoid culture systems allow pluripotent and adult stem cells to self-organize to form three-dimensional (3D) structures that provide a faithful recapitulation of the architecture and function of in vivo organs. In particular, human pluripotent stem cell-derived liver organoids (PSC-LOs) can be used in regenerative medicine and preclinical applications, such as disease modeling and drug discovery. New bioengineering tools, such as microfluidics, biomaterial scaffolds, and 3D bioprinting, are combined with organoid technologies to increase the efficiency of hepatic differentiation and enhance the functional maturity of human PSC-LOs by precise control of cellular microenvironment. Long-term stabilization of hepatocellular functions of in vitro liver organoids requires the combination of hepatic endodermal, endothelial, and mesenchymal cells. To improve the biological function and scalability of human PSC-LOs, bioengineering methods have been used to identify diverse and zonal hepatocyte populations in liver organoids for capturing heterogeneous pathologies. Therefore, constructing engineered liver organoids generated from human PSCs will be an extremely versatile tool in in vitro disease models and regenerative medicine in future. In this review, we aim to discuss the recent advances in bioengineering technologies in liver organoid culture systems that provide a timely and necessary study to model disease pathology and support drug discovery in vitro and to generate cell therapy products for transplantation.