The Experimental Study On Construction Of Tissue-engineered Corneal Epithelium By Culturing Human Amnion-derived Mesenchymal Stem Cells In Vitro

AIM:1. TO compare the effect of different protocols of dissociating mesenchymal stem cell from human amniotic membrane tissue.2. Explored the optimal conditions of the water-soluble quantum dots (QDs) labeled human amniotic mesenchymal stem cells (HAMSCs) in order to be better to mark the HAMSCs which is induced to differentiate in vivo.3. Investigated the possibility of construction of tissue-engineered corneal epithelium by cultureing HAMSCs in vitro.METHODS:1. Fresh human amniotic membrane organization is cut into four explants of 6cm×6cm,and treated with 4 tissue dispersion protocols:GroupⅠ:scrape technique+ collagenaseⅡ; GroupⅡ:scrape technique+collagenaseⅣ; Group III:Shred+collagenaseⅡ; GroupⅣ:Shred+collagenaseⅣ. Cell viability was measured by CCK-8, in combination with morphological observations and HE staining. Immunochemical and immunofluorescence staining, flow cytometry were used for analyzing HAMSCs markers expression. Osteogenic and adipogenic differentiation potential test were taken to analyze the HAMSCs'differentiation capability.2. HAMSCs of passage 3 at 80%-90% confluence in cell culture plate were respectively incubated with 10 nmol/L QDs in 2 ml cell culture medium for 20min,30min,40min,60min. QDs are endocytosed by HAMSCs and distributed in the cytosol, and the amount of QDs are observed by laser confocal scanning microscopy (LSM)in HAMSCs. Compared respectively the amount of QDs retained in HAMSCs after conventional culture 14d in vitro.3. HAMSCs of passage 3 at 80%-90% confluence were cultured on rabbit corneal stroma which removed corneal epithelial cells. Then moved the rabbit corneal stroma on which HAMSCs were co-cultured at an air-liquid interface up to 14 days and analyzed by histochemical and immunochemical staining of CK3+12 on days 14.RESULTS:1. GroupⅠand groupⅡ's number of HAMSCs survive are more than group III and groupⅣ(P<0.05). HAMSCs obtained by all four methods expressed the markers of stem cell, including CD29,CD44,CD73,CD9,CD 105 positive and CD31,CD34,CD45,HLA-DR negative expression in flow cytometry, vimentin,SSEA-3,SSEA-4,OCT-4,telomerase-positive in immunohistochemistry and immunofluorescence results. HAMSCs yield from two protocols with scrape technique were purer than those with shred technique. Adipogenic and osteogenic induced differentiation experiment was a success.2. The amount of QDs in HAMSCs which were tracked with 10nmol/L QDs after 30min and 40min was superior to all other mark time experimental groups.3. A stratified epidermal structure resembling native corneal epithelium was established on day 14 of culture HAMSCs on rabbit corneal stroma. Moreover, the immunostaining studies revealed that HAMSCs are positive expression in this experimental group.CONCLUSIONS:1. Scraped the human amniotic membrane tissue before putting into collagenase digestion medium improve the HAMSCs viability and purity of dissociated HAMSCs. Collagenase II and IV have little difference in the effect of HAMSCs dispersion.2. 10nmol/L QDs and 30min or 40min mark time is the optimal marker conditions which was used to tracked HAMSCs for live-cell imaging and tracing and dynamics studies in vivo.3. The HAMSCs may be an easily accessible alternative therapeutic source of allogeneic adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders.