Research On The Construction Of Bio-engineered Human Salivary Gland Organoids

BackgroundsThe salivary gland diseases such as Xerostomia seriously affect the health and life quality.At present,there is no effective treatment to improve symptoms mainly by conservative treatment.The construction of stem cell therapy or bio-engineered salivary gland organ provides new methods and ideas for its treatment.In recent years,stem cell-related research of salivary gland origin has become a new hotspot,focusing on the large salivary gland.In contrast,based on advantages of high quantity,widely distribution,abundant sources,shallow anatomical sites,easy access,donor site concealment,and so on,our research team pioneered stem cell research derived from human minor salivary glands,isolated and identified the stem/progenitor cells and mesenchymal stem cells,two different types of adult stem cells.In this experiment,we applied the human minor salivary gland epithelial progenitor cells and mesenchymal stem cells to carry on the bio-engineered human salivary organoids ex and in vivo.Preliminary analysis of reconstructed the human salivary organoids' morphological structure and function were carried on in the histology?cytology and molecular biology.The establishment of ex and in vivo models of human salivary gland organs will have important theoretical significance and transforming value in the study of salivary gland development,physiological function,salivary ingredient analysis and pathogenesis of salivary gland diseases.Objectives1.The epithelial stem/progenitor cells and mesenchymal stem cells isolated and identified from the human minor salivary gland,were cultured in vitro three-dimensional system,to explore the feasibility of constructing functional bio-engineered human salivary gland organoids.2.The epithelial stem/progenitor cells and mesenchymal stem cells were transplanted into mice by isolation and identification from the human minor salivary gland,to reconstruct in vivo model of human salivary gland organoids.Contents1.Reconstruction of bio-engineeered human salivary gland organoids in vitroMethods The respective and mixed three-dimensional culture of human minor salivar y gland stem/progenitor cells and mesenchymal stem cells were performed in the ma trix Matrigel.Compared with their plate culture,the morphological changes were obs erved,gene expression changes of AMY1?AQP5?MUC5B?LZM?CK14?CKI5?C K19 and Ki67 were detected with RT-PCR method.ELISA kits were used to test th e AMY1 and LZM level of supernatants of plate and three-dimensional culture colle cted on the 6th?12th?18th?24th and 30th day.Samples collected were examinated under HE staining and immunofluorescence assay for the expression of target protein s such as AQP5,CK14,CK15,CK19,CD44,CD166,Calponin,Claudinl,NKCC1,E-cadherin,ZO-1,?-SMA and NF-H.Results Formation of duct and acinar-like structure can be observed in organoids wh en 36-48hours after mixture of hMSGSC and hMSGMSC were seeded on Matrigel,However,the branching morphology can not be seen when either type of adult stem cell independently cultured in Matrigel.Through label with different fluorescent pro teins and observation in the confocal microscope,duct and acinar-like structure is m ainly composed of hMSGSCs.The expressions of AMY1,AQP5,LZM and MUC5B in HMSGSC group were hi gher than HMSGMSC and HMSGSC+hMSGMSC Group.AMY1 level of HMSGSC+hMSGMSC group was higher compared to HMSGMSC group.The expressions of C K14,CK15 and CK19 in HMSGSC and HMSGSC+hMSGMSC groups were high,th eir expression in HMSGMSC group were extremely low.The expression of Ki67 in all groups decreased when cultured in Matrigel,compared to plate culture.HE staining of hMSGSC+hMSGMSC group in Matrigel showed inflated globula r clusters and the interconnected branching-like structure.HMSGSC group in plate c ulture and three-dimensional culture,and hMSGSC+hMSGMSC in three-dimensional culture showed expression of CK15?CK19?CK14 and CD44.Three-dimensional cul ture of hMSGSC and hMSGSC+hMSGMSC also showed high expression of AQP5.CK15 and CK19 are duct-specific markers,CD44 and AQP5 are acinar-specific mark ers in human minor salivary gland.CK14 is located in both acinar and duct of min or salivary gland.Plate and three-dimensional culture of hMSGMSC did not basicall y express target proteins above.The concentration of LZM in supernatant of HMSGSC +hMSGMSC Group was significantly higher than that of HMSGSC group.The LZM concentration of supern atant in hMSGMSC group was too low to be detected.The concentrations of AMY 1 in the supernatant of the HMSGMSC and HMSGSC +hMSGMSC group were hig her than that of the HMSGSC group,there was no significant difference in the secr etion concentration between the former groups in most phases.Conclusions The salivary gland organoids characteristic with acinar and duct-like bra nching morphology?related immunophenotype and certain secretory function can be observed when hMSGSC and hMSGMSC were cultured in vitro three-dimensional sy stem.2.Research on the construction of bio-engineered human salivary gland organoids in vivoMethods The single and mixed implantation of human minor salivary gland stem/pro genitor cells and mesenchymal stem cells were resuspended in SD rat tail collagen.After cultured in vitro for 3-5 days,they were transplanted into Subcutaneous tissue layer of the nude mice,accompanied by the blank rat tail collagen and fresh human minor salivary gland tissues as control groups.Samples were taken in 2 weeks and 4 weeks after in vivo transplantation,HE?PAS staining and immunofluorescence a ssay were performedResults Labeled minor salivary gland stem cells were found in the samples.There was a small amount of cells infiltration in the blank rat tail collagen group.HE and PAS staining showed that only the hMSGSC+hMSGMSC group had better vasculari zation in vivo,and showed the formation of more typical ductal structure,accompan ied by adjacent vacuolus acinar-like structure.The expression of AQP5 and CD 166(4 weeks)and Calponin in the grid like structure were observed by immunofluoresce nee staining.Duct structure showed expression of CK19.After the transplantation of human small salivary gland into nude mice,the atrophy of original acinar and duct s were significant,but its adjacent ductal structure was similar to that in hMSGSC+hMSGMSC group.Conclusions Mixture of hMSGSC and HMSGMSC can survive in vivo,and generate salivary gland organoids with typical ductal structure and vacuolus acinar-like struct ure.