Hepatic Differentiation Of Mesenchymal Stem Cells Research Articles

Effect of valproic acid on the hepatic differentiation of mesenchymal stem cells in 2D and 3D microenvironments.

Mesenchymal stem cells (MSCs) have multi-lineage differentiation potential which make them an excellent source for cell-based therapies. Histone modification is one of the major epigenetic regulations that play central role in stem cell differentiation. Keeping in view their ability to maintain gene expression essential for successful differentiation, it was interesting to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, in the hepatic differentiation of MSCs within the 3D scaffold. MSCs were treated with the optimized concentration of VPA in the 3D collagen scaffold. Analyses of hepatic differentiation potential of treated MSCs were performed by qPCR, immunostaining and periodic acid Schiffassay. Our results demonstrate that MSCs differentiate into hepatic-like cells when treated with 5mM VPA for 24h. The VPA-treated MSCs have shown significant upregulation in the expression of hepatic genes, CK-18 (P < 0.05), TAT (P < 0.01), and AFP (P < 0.001), and hepatic proteins, AFP (P < 0.05) and ALB (P < 0.01). In addition, acetylation of histones (H3 and H4) was significantly increased (P < 0.001) in VPA-pretreated cells. Further analysis showed that VPA treatment significantly enhanced (P < 0.01) glycogen storage, an important functional aspect of hepatic cells. The present study revealed the effectiveness of VPA in hepatic differentiation within the 3D collagen scaffold. These hepatic-like cells may have an extended clinical applicability in future for successful liver regeneration.

The impact of oxidative DNA changes and ATM expression on morphological and functional activities on hepatocytes obtained from mesenchymal stem cells

Resistance to oxidative damages in undifferentiated mesenchymal stem cells (MSCs) in comparison with the undifferentiated progenitor cells may differ depending on several factors. This study was carried out to examine the impact of hepatogenic differentiation process of MSCs on oxidative DNA damage markers. Hepatic differentiation of MSCs was carried out using a two-step conventional protocol and the cells were processed for characterization using morphological and biochemical markers. During the course of differentiation cellular levels of reactive oxygen species (ROS), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and expression of ataxia-telangiectasia mutated (ATM) protein were estimated at time intervals (10, 20 and 30 days). The results showed a decrease in cellular ROS (13%, P < 0.05) at early stages of hepatogenic differentiation. Similarly, there was a small but significant decrease in 8-OH-dG level and ATM expression in differentiated hepatocytes. Despite the small changes in oxidative damage factors and ATM expression during the differentiation process, the hepatocytes obtained were morphologically and biologically intact.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P &lt; 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture

Extensive cell–cell or cell–matrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is significant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet configuration, with or without the presence of small intestinal submucosa (SIS). After 4 weeks of differentiation with growth factors bFGF, HGF, and OsM, we obtained hepatocyte-like cells that expressed a subset of hepatic genes, secreted albumin and urea, stored glycogen, and showed inducible CYP3A4 mRNA levels. When these cells were implanted into livers of hepatectomized rats, they secreted human albumin into the bloodstream. The hepatic differentiation of MSC was faster in cell pellets without SIS. The plausible explanations for this finding may be related to the mass transport issues of the two different pellets and the role of cell–cell contact over cell–matrix interactions. The findings of this study should help in the design of optimal culture configurations for efficient hepatic differentiation of adult stem cells.