Hydroxyl Fasudil Induce Human Adipose Derived Stem Cells Into Epidermic Cells In Vitro

PartⅠ: The Separation, Evaluation and Culture of Adipose derived Stem Cells (ADSCs) and the Differentiation of the ADSCsObjective: To observe the possibility of the induction of ADSCs into epidermic cells,skeletogenous cells and adipose cells.Method: Human ventral adipose tissues were obtained by electric vacuum aspiration technique from a thirty-year old woman who underwent suction lipectomy sugery. The ADSCs were obtained by using enzyme digestion and then the cells were cultured in vitro. After generating, amplifying and purifying, the 3rd-5th generation of ADSCs were obtained. The growth character of ADSCs was observed and the growth curve was recorded accordingly. Then the express of surface antigen was detected by flow cytometry and immunohistochemistry assay. The 3rd generation of ADSCs was selected for different induction as follows: Culture solution B:[70% culture solution A(90% L-DMEM,10%FBS,enicillin100 U/ml, phytomycin100μg/ml,2mmol/L glutamine, pH=7.2) +30% supernatant fluid from fibroblast medium+10 ng/LEGF] to induce human ADSCs differentiated into epidermic cells; Culture solution C: ( DMEM/10%FBS, 0.1μmol/L desacort, 50μmol/L vitamin C, 10mmol/L sodiumβ-glycerophosphate, 100U/ml penicilin, 100U/ml phytomycin ) to induce human ADSCs differentiated into skeletogenous cells; Culture solution D:(DMEM +10%FBS + 500μmol/L IBMX +1μmol/L antinfan) to induce human ADSCs differentiated into adipose cells. After 20d, the changes of cell morphology after induction were observed through inverted phase contrast microscope, the express of CK19 was detected by immunohistochemistry assay in group B, the ALP assay in group C and the Oil red O staining in group D.Result: Many lipid droplets, a few red blood cells and endothelial cells were observed when the cells are cultured. After 24h, we could see a few larger cells attached and most of the monolayer cells were large and applanate, few were slender as fibroblast through inverted phase contrast microscope. Most of them are attached and almost shuttle-shaped or needle tip-shaped after 48h. 5 to 7 days later, the cells were fused to monolayer gradually and distributed into clustering. The express of CD44 and CD49d were examined to identify human ADSCs by flow cytometry and immunohistochemistry assay, and the express of CD44 and CD49d were positive while the express of CD34 was negative. After 20d-induction, CK19 of the group which is induced into epidermic cells was detected to be positive by immunohistochemistry, ALP of the group which is induced into osteoblast cells is positive and the Oil red O staining of the group which is induced into adipose cells is positive.Conclusion:1. We obtained human ADSCs from human adipose tissue, amplified and cultivated them in vitro and then identified them by cell morphology, cytochemistry and flow cytometry assay. It confirmed that what we isolated were human adipose derived stem cells.2. The proliferative activity of ADSCs was not declined obviously within the 10 passages.3. ADSCs can be induced to differentiate into osteoblast cells and adipose cells with the osteoinductive culture and adipoinductive culture.4. ADSCs can be induced to differentiate into epidermis cells in medium with supernatant fluid from fibroblast and EGF in vitro.PartⅡ: The effect of Hydrochloric Fasudil (HA1077) on the differentiation of Epidermic Cells from hunam ADSCsObjective: To observe the effect of HA1077(a blocking agent against Rho molecular signal path)on the differentiation of ADSCs into epidermic cells. Method: According to the method above, we obtained and cultured human ADSCs in vitro. The 3rd generation of ADSCs was chosen from the cells which grew well with a 90% fusion rate, then divided into 4 groups. Group 1 was continuously cultured with culture solution 1:(culture solution A);In group 2,3,and 4, solution 1 were washed with sterile PBS and the cells were cultured with the different medium respectively as follows: Culture solution 2: (culture solution1+20μmol/L HA1077); Culture solution 3:( 70%culture solution 1 +30%clear supernatant liquid from fibroblast nutritive medium +10 ng/LEGF); Culture solution 4: (20μmol/L HA1077+ culture solution 3). The culture solution was changed every 2-3 days until the fusion rate rising up to 90%. We observed their morphology and growth condition regularly through inverted phase contrast microscope. After 20 day-induction, flow cytometry assay (CK19)and immunohistochemistry (CK10, CK19) were investigating the express of the related antibody in group 1~4.Result: The positive rate of CK19 with flow cytometry in group 1~4 were as follows:(0.23±0.010)%,(0.35±0.020)%,(9.73±0.800)% and(17.65±0.998)%(n=3). We observed the 2rd group which was induced respectively with solution 2 on 20d. The cells are still fusiform fibroblast and rank tightly, and have no difference with the blank control group. We then observed the 4th group which was induced respectively with solution 4. The cells which are fusiform fibroblas become short and coarse gradually, and rank tightly, the morphous changes of cells in this group are earlier than others, so this group has a promote effect on differentiation of epidermic cells from hunam ADSCs. We found a positive expression of CK19 and CK10 in group 3 and group 4, and a negative expression of CK19 and CK10 in group 1 and group 2.Conclusion:1. The application of certain concentration of HA1077 in the conditioned medium can not induce the differentiation of human ADSCs into epidermic cells. 2. The application of certain concentration of HA1077 in the induced medium can promote the differentiation of human ADSCs into epidermic cells obviously.