Biological Characteristics Identification And In Vitro/in Vivo Hepatogenic Differentiation Of Human Minor Salivary Gland-Derived Mesenchymal Stem Cells And Epithelial Progenitor Cells

Objective1. To isolate and culture the mesenchymal stem cells and epithelial progenitor cells derived from human minor salivary glands. To further identify the relative stem cell markers of these two cell types. To assess the biology characters of these two cell types by cell cycle MTT detection, doubling time, cloning efficiency etc.. To investigate the differentiation potential toward hepatocytes of these two cell types through2D and3D inducing methods in vitro.2. To establish2/3partial hepatectomy models in mice for in vivo differentiation assay. To label the mesenchymal stem cells and epithelial progenitor cells with fluorescent dye CM-Dil in order to trace them in vivo. To examine the distribution and differentiation status of human minor salivary gland derived mesenchymal stem cells (MSG-MSCs) and epithelial progenitor cells (MSG-EPpiCs) transplanted in acute liver injury models, discus the hepatic differentiation potential of these two cell types in vivo.Methods1. Immunofluorescence (IF) of human minor salivary gland tissues collected from clinical surgeries was performed to assess whether there were hypothetical stem cells inside minor salivary glands. MSG-MSCs and MSG-EPpiCs were cultured by explant culture method with different culture medium. Cells were purified and amplified through passaging. Phenotypic identifications were performed on these cells by IF and flow cytometer (FCM). Analyze the biology characters of MSG-MSCs and MSG-EPpiCs through cell cycle MTT detection, doubling time, cloning efficiency, and osteogenic, chondrogenic, adipogenic differentiation assay were performed on MSG-MSCs to confirm its multiple differentiation potential. Compare MSG-MSCs, MSG-EPpiCs and BMSCs on sternness and hepatic-associated gene expressions by QPCR, in order to deep investigate the stem characters and hepatic differentiation potential of these two cell lines.2.2D and3D hepatic differentiation induction systems were performed on MSG-MSCs and MSG-EPpiCs in vitro. The changes of cell growth and morphology were observed during induction, and the hepatic differentiation results were assessed by IF, ICG uptake and QPCR.3. Using CM-Dil dye to label MSG-MSCs and MSG-EPpiCs; Establish2/3partial hepatectomy models in mice, and randomly divided into three groups:MSG-MSCs group(n=15,3of them were used for FCM), MSG-EPpiCs(n=12) and control group(n=3, normal mouse). CM-Dil-labeled MSG-MSCs and MSG-EPpiCs were transplanted by tail-intravenous injection immediately after partial hepatectomy.1week,2weeks,3weeks, and4weeks after surgery tissue specimens were collected separately. The distributions of transplanted MSG-MSCs cells2weeks after surgery were examined by fluorescence microscope and FCM, and the remaining liver cells obtained from FCM were cultured again to identify the vitality of transplanted cells. IF identification was performed on liver samples obtained from mice models to observe the expression of liver-associated markers.Results1. IF showed that human minor salivary glands express ALB, AFP, CK18, CK19, LGR5and LGR6. Both MSG-MSCs and MSG-EPpiCs grew well in each culture medium, and they were stably subcultured though at least25passages in vitro. IF and FCM showed that the MSG-MSCs’expression profiles were similar to the well-accepted MSCs markers; while MSG-EPpiCs express epithelial progenitor cell markers such as cytokeratins, ABCG2, PLET-1etc.; Both cells were positive to LGR5and LGR6in IF; FCM showed that some MSG-MSCs had specific expression on SSEA-1(12.95%±2.65%), while some MSG-EPpiCs had specific expression on SOX2(19.2%±8.94%). Doubling time of MSG-MSCs and MSG-EPpiCs at P5were19.77h and24.93h, and cloning efficiency were41%±5.4%and10.7%±3.0%, respectively. MSG-MSCs could be induced into adipogenic, osteogenic, and chondrogenic lineages. QPCR showed that in comparison with BMSCs, LGR5were high expressed in MSG-MSCs and LGR6were high expressed in MSG-EPpiCs.2. MSG-MSCs and MSG-EPpiCs could be induced into hepatic lineage in both2D and3D differentiation system. MSG-MSCs were better induced in2D condition, and MSG-EPpiCs preferred3D condition. ICG uptake assays were both positive. IF and QPCR results showed that both MSG-MSCs and MSG-EPpiCs expressed liver-associated markers.3.2weeks after MSG-MSCs transplanted into mice, CM-Dil signals still could be seen in liver, spleen, thymus, heart, lung, pancreas, kidney and skin. The positive signals were most abundant in liver tissue, followed by the lung and spleen. Liver tissue culture revealed that most adherent cells were CM-Dil labeled human cells, and they were still1GR5positive. Liver tissue slices IF showed that both MSG-MSCs and MSG-EPpiCs could express liver-associated markers in vivo, and some MSG-EPpiCs migrated into biliary ducts and expressed CK19.Conclusion1. Both MSG-MSCs and MSG-EPpiCs could be obtained from human minor salivary glands. They both showed high abilities of self-renewal, expressed stem cell markers, in which MSG-MSCs high expressed LGR5and MSG-EPpiCs high expressed LGR6, and this was for the first time that found stem cells obtained from human minor salivary glands involved in Wnt signal pathways.2. Both MSG-MSCs and MSG-EPpiCs could be induced into hepatic lineage in both2D and3D differentiation systems, and expressed liver-associated markers.3. In2/3partial hepatectomy mouse models, MSG-MSCs and MSG-EPpiCs could migrate toward and resident in the injured liver, expressed hepatocyte-associated markers and participated in the process of liver regeneration. MSG-EPpiCs could also involve in biliary ducts regeneration, express biliary epithelial marker CK19.