| Objective:1.To isolate,cultivate and identify human adipose-derived stem cells(ADSCs) which from adipose tissue masses and processed lipoaspirate.To observe their biological characteristics and activities as stem cells.2.To explore the possibility and the best method of ADSCs transdifferentiated into corneal epithelial-like cells by single inducing and co-culture inducing in simulating the vegetating microenvironment of corneal epithelial cells.Methods:1.The subcutaneous tissue masses-derived and processed lipoaspirate-derived ADSCs were isolated and purifed with improved methods,and cultured with modified medium in vitro.2.The growth curves were drawn by MTT colorimetry.Phenotypes including CD29,CD34,CD49d,CD105,CD106 were identified by flow cytometry at passage 3,4 and 5.Adipogenesis and osteogenesis tests were put into practice,and identified by Oil Red O stain and alkaline phosphatase activity tests.Contrasted the differences of phenotypes,proliferation and differentiation abilities were compared between 2 sources of ADSCs.3.The human ADSCs were marked with CFDA SE.And rat corneal epithelial cells (CECs) were isolated by tissue culture process,and made the corneal stroma tissue masses.They would be used into co-cultured induction in the following steps.4.The human ADSCs and rat CECs were placed together in the same plate which grown on slips,and co-cultured 3 weeks,observed the migration ability of human ADSCs. 5.Single induced group of ADSCs were cultured with several kinds of mediums, including Keratinocyte medium(KM),Dulbecco's Modified Eagle Medium/F12(DMEM/F12) and KM+DMEM/F12(1:1),and stimulated with different concentrations of growth factors.Observed the morphological changes under phase contrast microscope and electron microscope.Immunohistochemical and immunofluorescent staining of AE5(CK3/CK12),the specific protein of cornea, were taken at the same time.Contrasted the transdifferentiated ability of ADSCs in different mediums and with different concentrations of growth factors.6.Placed rat CECs or corneal stroma tissue masses into the upper section of Transwell co-culture system,while human ADSCs into the under section,and co-culture with KM for 30 days,added 40ng/mL EGF in them or free.Observed the expressions of AE1(broad spectrum of CytoKeratins) and AE5 by immunofluorescent staining. Evaluated the expressing differences of AE1 and AE5 between the groups of EGF-added and free,single and co-cultured induction,and CECs co-culture induction and corneal stroma tissue masses co-culture induction.7 The 2 sources of single induced groups for 30 days,and contrasted their expressing differences of AE1 and AE5.Results:1.Large amounts of ADSCs were obtained from both 2 kinds of isolated processes, tissue masses and processed lipoaspirate,and with high purity.However,the operation of the latter process was more time- and strength-saving,while gained more cells.The phenotypes of ADSCs at passage 3～5 are CD34~-,CD106~-,CD29~+,CD49d~+ and CD105~+.CD29~+,CD49d~+ cells rates of subcutaneous tissue masses-derived ADSCs at passage 3 accounted for 82.5%and 8.0%,and gradually increased to 89.4%,70.1% at passage 5,while CD105~+ cells rates of each passage ADSCs were about 80%. Processed lipoaspirate-derived ADSCs at passage 3～5 expressed more stable phenotype,which spread approximately 99%,40%,80%of CD29~+,CD49d~+,CD105~+; And compared with the subcutaneous tissue-derived ADSCs,the positive rate of CD29 at passage 3～5 and CD49d at passage 5 had a significant difference(P＜0.01).MTT colorimetry showed,ADSCs proliferative activity improved gradually following the increased of passage.After two weeks' adipogenic/osteogenic induction in vitro,the oil red O staining and alkaline phosphatase activity test showed positive results. Positive rates of subcutaneous tissue-derived ADSCs were 81.6%,26.5%,while another derivative were 64.6%,29.3%.2.CFDA SE-labeled ADSCs migrated obviously,and gradually enveloped and replaced the ECEs with induced by poor nutrition of CECs.The AE5 expression rate of ADSCs cultured in KM system presented a dose-dependence of EGF;while bFGF inhibited the differentiation.ADSCs cultured in the other two systems were negative either with or without growth factor.3.Among the different induced groups that cultured with KM,AE5-positive rate of the corneal stroma Transwell co-culture induction group which added EGF reached top to 97.7%,and AE1-positive expression was 0%,which prompted to mature corneal epithelial-like cells.The single induction group was the second top,that AE5~+ cells occupied 75.4%(add EGF),46.0%(EGF free),AE1~+ cells accounted for 86.0%(add EGF),96.5%(EGF free),premature.ADSCs co-culture induced by the corneal stroma with EGF free showed the least positive rate of AE5(2.4%),AE1(51.2%).4.subcutaneous tissue-derived ADSCs were single induced with KM and added 40ng/mL EGF,which expression was 100%AE5~+ rate,and 0%AE1~-,on the other hand,EGF free group were 16.6%,97.7%.It suggested that the differentiation capacity of this source was stronger than liposuction group.Conclusion:1.We could isolate more quantity and purer ADSCs from subcutaneous tissue and Processed lipoaspirate adipose liquid with improved process methods.And it was evidenced with multi-lineage differentiation potential abilities and could be used in engineering researches,cell and gene therapies in the future.2.Following the results of our study,the KM contained 40ng/mL EGF co-culture corneal stroma co-induction in Transwell system could be the best way to make ADSCs have a transdifferention to corneal epithelial-like cells.In addition,the subcutaneous adipose tissue-derived ADSCs have the better differential ability that could be more suitable for future clinical treatment than processed lipoaspirate-derived ADSCs.Howere, processed lipoaspirate-derived ADSCs have less simple isolated process and are more adequate,so it may be more suitable for laboratory research.3.CFDA SE is a stable cell tracer,and can be used as the tracer of in vivo animal experiments in future.
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