Hepatogenisis Of Adipose-derived Adult Stem Cells On Poly-lactide-co-glycolide Scaffolds (PLGA)

End stage liver disease accounts for many deaths each year in our country. Currently, the only solution is orthotopic liver transplantation (OLT). However, this therapy is limited by increasing shortage of donor organs, immunologic rejection and the side effect caused by years of immunosuppression. These limitations increased the requirement of new therapies for end stage liver diseases. Hepatic tissue engineering is focused on creating a whole, implantable, and functional liver through the way of tissue engineering to compensate or replace the function of disorder hepatic tissues. So, the study and application of hepatic tissue engineering brings new hopes to patients with end stage liver diseases.The study of hepatic tissue engineering is currently in it's primary stage. Before clinical application, many challenges must be overcome, such as the selection of cell sources, scaffold materials and neovascularization within tissue-engineered constrcut. First of all are the problems of cell sources and scaffolds.How to get large number of hepatocyte-like cells with function is one of the restrictive factors for the development of hepatic tissue engineering. Cell sources for hepatic tissue engineering include mature hepatocytes and stem cells (ES cellsand adult stem cells). Mature hepatocytes cultured in vitro are easy to lose activity and function and their in vitro expansions are limited. ES cells have limitations of difficult isolation, ethical issues and possibility of teratoma formation. So, adult stem cells, which can be easily isolated and expanded largely in vitro, attract attentions of many scholars. Bone marrow-derived mesenchymal stem cells (MSCs) have been considered as the most typical adult stem cells. Adipose tissue-derived MSCs (i.e. adipose-derived stem cells, ADSCs) have recently been shown to be very similar to bone marrow-derived MSCs in morphology and phenotype, and also have the capacity to differentiate into several types of cells including hepatocyte-like cells. Furthermore, compared to marrow-derived MSCs, ADSCs can be isolated more easily, largely, and via more minimally invasive procedures. So, ADSCs have the potential to become another promising cell source for hepatic tissue engineering.The study and application of appropriate scaffold materials is related to the success of tissue engineering. So, selection of scaffold materials is very important. Recently, studies of scaffold materials have also made great progress recently. Frequently used biomaterials for hepatic tissue engineering are poly-varepsilon-caprolactone (PCL), poly-lactide-co-glycolide (PLGA), collagen, laminin and fibronectin. PLGA, which has been approved by the United States Food and Drug Administration, is the most important and widely used synthetic polymer in tissue engineering.Until now, hepatic differentiation of ADSCs on any three-dimensional setting has not been reported. So, in this study, we selected human ADSCs (hADSCs) as cell sources and porous PLGA as scaffolds to investigate the effect of PLGA on hepatic differentiation of hADSCs in vitro and in vivo, with intention to select appropriate cell sources and scaffold materials for hepatic tissue engineering. hADSCs were firstly cultured with improved hepatic media in monolayer culture system. Then, the hepatic differentiation of hADSCs on porous poly-lactide-co-glycolide (PLGA) scaffolds in the same hepatic media was investigated. On base of this, we investigated the effect of the physical property of scaffolds and fibroblast coculture on hepatic differentiation of hADSCs. Furthermore, we investigated the in vivo influence of PLGA scaffolds on hepatic transdifferentiated hADSCs.Contents of this study were mainly as follows:Hepatic differentiation of hADSCs in two-dimensional (2D) monolayer culture systemhADSCs were isolated from human adipose tissue, and identified by morphology, surface markers and multiple differentiation potential. On base of this, hADSCs were seeded in monolayer culture system and treated with improved hepatic media containing hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) and FGF4, OSM (oncostatin M) was added to the hepatic media after 7 days of culture. After 14 days of induction, hADSCs differentiated into cells with several characteristics of hepatocytes, including cell morphology, genes and protein expression, and functionality.The growths of stem cells with and without differentiating are two different processes and most studies are focused on the latter. Moreover, studies have reported that HGF and FGF promote cell proliferation and differentiation as well. So, we examined hADSCs growth during hepatic differentiation process. Results of significant proliferation of hADSCs suggest we can expand these cells at the same time of hepatic differentiation. Findings above can contribute to the hepatic differentiation of hADSCs on porous PLGA scaffolds.Hepatic differentiation of hADSCs on three-dimensional (3D) porous PLGA scaffolds in vitroCurrently, studies of hepatic differentiation of ADSCs are limited to 2D culture system. And, differentiation of ADSCs into a liver cell lineage in any 3D setting has not been reported. So, after confirming the hepatic differentiation of ADSCs in conventional monolayer culture system with improved hepatic media, we investigate the hepatic differentiation of hADSCs with same hepatic media on porous PLGA scaffolds, with the goal to explore appropriate 3D scaffold for hepatic differentiation of hADSCs.Cytocompatibility of biomaterials with stem cells is the foundation of stem cell differentiation on scaffolds. Our studies by using DAPI staining, CCK8 assay and SEM thoroughly demonstrated that porous PLGA scaffolds were cytocompatible with hADSCs. Cell growth is a necessary and critical part of tissue regeneration. Results of CCK8 assay show hADSCs on 3D PLGA scaffold in the presence of former hepatic media also proliferated significantly and the proliferation is faster than in monolayer culture system. This not only confirmed the role of hepatic media containing HGF, bFGF and FGF4 in promoting cell proliferation, but also suggested 3D environment is more inducible for proliferation than 2D environment. The type of scaffolds affect cells'morphology on scaffolds, present data from SEM analysis represented an original morphological investigation of continuously differentiating hADSCs on PLGA scaffolds. hADSCs spread along the surface of the scaffold and gradually formed a confluent layer on top of the PLGA scaffolds. Morphology of cells become more multi-angular with time of culture. Numerous cytoplasmic extensions linked the cells on top of the cell sheet, and cells proliferated and differentiated into 3D tissue-like structures at 14 days after induction. Based on the studies above, we mainly investigate the ability of PLGA scaffolds to support the hepatic differentiation of hADSCs. Results show hADSCs can differentiate into cells with phenotypic characteristics of hepatocyte-like cells and these cells also acquired functional properties of glycogen deposit and Alb secretion.Effect of fibroblast coculture and physical properties of PLGA scaffolds on hepatic differentiation of hADSCs Many factors affect the hepatic differentiation of hADSCs on porous PLGA scaffold. Fibroblasts have the ability to secret many kinds of extracellular matrix and cytokines relevant to hepatogenic differentiation. So, we investigate whether coculture of hADSCs with fibroblasts without cell-cell contact play a role in hepatic differentiation of hADSCs by RT-PCR analysis of hepatic specific gene expression and real-time PCR analysis of Alb expression. Results show hADSCs cocultured with human dermal fibroblasts cells without hepatic media can even differentiate into cells with hepatic specific gene expression. In addition, coculture of hADSCs with human dermal fibroblasts in hepatic media can increase the hepatic differentiation of hADSCs without coculture. These results suggest that direct cell–cell contact may not be a necessary condition for the hepatogenisis of hADSCs induced by human dermal fibroblasts cells. Soluble factors such as HGF and FGF2 secreted by these cells might provide sufficient cues for hepatic differentiation by paracrine manner and may have an additive effect with hepatic media.Porosity and pore size are importantly physical properties of porous PLGA scaffolds, which mainly dependent on weight fraction and size of the porogens of NaCl particles respectively. The PLGA scaffolds used in former study were just prepared with 30~50μm size and 75% weight fraction of NaCl particles. For better design of the PLGA scaffolds in future, we varied the size and weight fraction of NaCl particles and explored their effect on hepatic differentiation of hADSCs by SEM examination of cell morphology and real-time PCR analysis of Alb expression. Compared to the groups of <50μm, 50~120μm, or >200μm size, scaffolds prepared with 120~200μm size of NaCl particles (75% weight fraction) were most beneficial for hepatic differentiation of hADSCs, suggesting smaller and larger pore size are both unfavorable to hepatic differentiation. Compared to the groups of 75% and 90% weight fraction, scaffolds prepared with 50% weight fraction of NaCl particles (50~120μm size) were most beneficial for hepatic differentiation of hADSCs, suggesting higher porosity impaired the hepatic differentiation. . In vivo infuence of PLGA scaffolds on hepatic transdifferentiated hADSCsIn vitro and in vivo properties of scaffolds varied with the microenvironment. So, after confirming the hepatic differentiation of hADSCs on PLGA scaffolds in vitro, it is necessary to investigate the in vivo influence of PLGA scaffolds on hepatic transdifferentiated hADSCs. hADSCs combined with PLGA scaffolds were cultured with hepatic media for 14 days in vitro and were implanted into 70% hepatectomized rats for 14 days. The partial hepatectomized microenvironment has been demonstrated to be beneficial for hepatic differentiation of stem cells by many studies. Results of the survival and hepatic phenotype maintenance of these cells suggest that PLGA scaffolds do not exert undesirable side effects on implanted cells in vivo. However, in this study, human Alb was not detectable in rat serum 14 days after implantation. This could be a result of specific implantation sites. It is also possible that the number of transplanted cells was not adequate.In conclusion, PLGA scaffolds supported the hepatic differentiation of hADSCs in vitro and in vivo, and hADSCs combined with PLGA scaffolds seems to be promising for hepatic tissue engineering. Results of this study should help in developing stem cell-based hepatic tissue constructs for clinical application.