Isolation And In Vitro Hepatogenic Differentiation Of Adult Liver-derived Mesenchymal-like Stem Cells

Background and Objection:The foundation of drug testing, hepatocyte transplantation and bioartificial liver application all lies in the source of ideal liver cells. Freshly isolated hepatocytes are highly variable in their quality are difficult to source, do not proliferate in vitro and usually lose their hepatic-specific function rapidly in in vitro cultures. Hence there is a need to identify alternative sources of hepatocytes for various application mentioned above.Several sources have been explored as alternative cells sources for primary hepatocytes such as tumor-derived cell lines, genetically modified immortalized cell lines, embryonic stem cells (ESCs) or pluripotent stem cells (iPSCs) derived hepatocytes. Tumor-derived or genetically modified immortalized cell lines have the capability of rapid and infinite proliferation. However, the tumorigenic potential limits their use for different applications. Embryonic stem cells (ESCs) are of interest to researchers for their pluripotency and their self-renewal ability. However, their high instability, high cost of culture and related ethical concerns also lead to their restricted application. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell, which can be generated directly from adult cells using techniques such genetic modification popularly described by Yamanaka. The major concern with the potential clinical application of iPSCs is their propensity to form tumors [1]. Like ESCs, teratomas have been shown to form when iPSCs are injected into animals. Teratoma formation is considered a major obstacle to stem cell-based regenerative medicine by the FDA (ref).Recently studies using adult stem cells have been gaining prominence. Adult stem cells (ASCs), found in the body are undifferentiated cells that multiply by cell division to replenish dying cells and regenerate damaged tissues. ASCs usually stay static and get activated upon tissue damage. They are of multi-differentiative potential, which is the ability to give rise to several distinct cell types. ASCs can be generated without genetic engineering, thus, are much safer to apply to clinical applications compared to ESCs and iPSCs. Bone marrow stem cells, epidermal stem cells and adipose stem cells have been wildly studied. It is reported that bone marrow stem cells could be differentiated into functional hepatocyte-like cells; however, the derived cells remained fetal[3-5].Cell sources mentioned above still cannot be able to provide an ideal alternative of liver cells. In recent years, researchers started to look for the presence of adult liver-derived stem cells. A study showed that they isolated stem/progenitor cell successfully from damaged rat livers (such as liver fibrosis or chronic liver failure) and another study demonstrated the generation of liver progenitor cells from healthy mature rat liver cells (Chen,2012#8).In this study, we aimed to find a safer liver cell source with better hepatic-specific function. Adult liver mesenchymal-like stem cells (rALMSC) were isolated from mature and healthy rat liver and differentiated in vitro into hepatocyte-like cells (rALMSC-H). Further studies were carried out to evaluate the biological characteristics and metabolic functions of rALMSC-H. rALMSC-Hs were also used for drug testing of paradigm hepatotoxicants to assess their utility for drug testing applications.Methods and materials1. Isolation and culture of rALMSCs. Primary hepatocytes were cultures in Dulbecco’s modified Eagle’s medium DMEM supplemented10%FBS until emergence of rALMSCs. Cells were trypsinized with0.25%trypsin-EDTA when they reached90%confluence, and replated2on collagen-coated flasks.2. Characterization of rALMSCs. Immunofluorescence and flow cytometry were used to characterise rALMSCs.3. Directed Differentiation of rALMSCs in vitro. Undifferentiated rALMSCs were incubated with basal differentiation media supplemented with EGF and FGF for pre-differentiation for2days. Thereafter, cells were subjected to differentiation induction for7days with basal differentiation media containing HGF and FGF-4. For subsequent maturation step, cells were treated with basal differentiation media supplemented with HGF, OSM for7days.4. Quantitative reverse-transcription polymerase chain reaction. Gene (ALB, HNF-4a, CYP1A2, CYP2B2, CYP3A2) expression was performed using rat specific primers and normalized to GAPDH. Data was plotted as a Log2transcript over GAPDH.5. Hepatocyte functional assays. The urea production was measured using the Urea Nitrogen Kit. CYP1A2activity was measured by liquid chromatography-mass spectrometry (LC/MS).6.Statistical Analysis. Differences between two groups were analyzed using the Student’s t test; the level of significance was defined as p<0.05. All statistical tests were performed using SPSS software version13.0Results:1. Isolation and characterization of rALMSC. rALMSCs began to appear and proliferate after5days of incubation in DMEM media. Mesenchymal-like stem cells with typical spindle shape were isolated and proliferated in vitro.2. Characterization of rALMSCs. Immunofluorescence stainings were performed for a-SMA and VIM protein. The positive expression for both proteins suggested the mesenchymal phenotype of rALMSC. rALMSC also expressed the mesenchymal markers, CD29, CD44and CD90(Thy-1) examined by flow cytometry. Additionally, the hepatocyte specific marker, ASGPR and hematopoietic marker, CD45were undetectable.3.Morphological changes upon differentiation rALMSC underwent distinct morphology change during the differentiation process. rALMSC started to lose their spindle shape after being exposed to differentiation media, and gradually adopted a polygonal or cuboidal morphology.4. Liver-specific gene expression. We compared the expression of ALB, HNF-4a, CYP1A2, CYP2B2and CYP3A2between undifferentiated, differentiated rALMSC and primary hepatocytes.The expression of ALB, CYP1A2, CYP3A2were significantly upregulated after differentiation and maturation. The expression of HNF-4a and CYP2B2were higher in rALMSC-H compared to rALMSC, but there were no significant differences of that between undifferentiated and differentiated rALMSC and primary hepatocytes.5. Synthetic and metabolic function of differentiated rALMSCs. rALMSC-H acquired the ability to synthesise albumin and produce urea. The level of urea production was significantly increased after in vitro differentiation. Activity of drug-metabolizing enzyme CYP1A2, one of the most important P450cytochromes in rat, was also significantly upregulated after hepatogenic differentiation.6. Hepatotoxicity testing. Differentiated rALMSC-H exhibited similar hepatotoxicity responses to chlorpromazine, ketoconazole and flutamide with hepatocytes, whereas hepatotoxicity responses to diclofenac and quinidine were not as sensitive as primary liver cells.ConclusionWe successfully isolated adult liver stem cells (rALMSC) from healthy rat liver without any genetic manipulation. rALMSC would be benefical for clinical applications for their higher safety compared to ESCs and iPSCs. In vitro differentiation studies demonstrated that rALMSC are able to differentiate into hepatocyte-like cells (rALMSC-H), which expressed ALB, HNF-4a, CYP1A2, CYP2B2and CYP3A2and acquired the ability to synthesize albumin and urea. Furthermore, rALMSC-H exhibited similar hepatotoxicity responses to hepatotoxic drug with primary liver cells. Hence, rALMSC can be considered as a potential new hepatocyte source for drug testing, cell therapy applications.