| Skin is the body’s largest organ, is mainly responsible for the protection of the body, sweating, feeling hot and cold and pressure function. Skin covering the whole body, it makes the body of various tissues and organs from physical, mechanical, chemical and pathogenic microbial invasion. When the skin defect caused by external injury or disease, the harm can be fatal. Skin tissue repair and functional reconstruction are the core and focus of bio-medical research both at home and abroad. Amniotic fluid cells due to their special anatomical location, received the people’s attention for a long time. The study of amniotic fluid cells can be traced back to the early 20 th century. With the growing maturity of amniocentesis and cell culture conditions, the success rate for culture of amniotic fluid cells has also been improved, amniotic fluid cell research has also been a rapid development. Amniotic fluid cell plays an important role in chromosomal abnormalities, genetic disease and congenital metabolic disorders, and other diseases in the prenatal diagnosis. Recent studies have found that amniotic cells contain a variety of stem cell markers, h AFSCs can be induced to a variety of different types of cells by special induction micro-environment, and Amniotic fluid-derived stem cells become a new hot spot for stem cell research. So as a new source of stem cells, h AFSCs will open a new field of stem cell study and provide a new source of seed cells for tissue engineering.Our study is aim to establish a method to isolate and culture human amniotic fluid stem in vitro and to analyze its biological characteristics. According to the high proliferation and differentiation potential of amniotic fluid stem cells, a study on amniotic fluid stem cell differentiation was carried out. We will verify the h AFSCs derived kerationcytes in m RNA, protein and ultra-structural level. Based on above results, combined with the low immunogenicity of h AFSCs, we will study on the repair of skin damage by amniotic fluid stem cells. We will also use a mouse excisional wound model to observe the survival, migration and differentiation of the implanted cells and to explore the key molecular mechanism of wound healing in mice. The research will provide ideal seed cells for skin tissue engineering, and will put forward new protocol for the clinical treatment of large area burn patients, which will have innovative and potential application value. Part I The cultivation, identification and differentiation of human amniotic fluid stem cells into keratinocytes in vitroObjective: To establish a method to isolate, extract and purify of human amniotic fluid stem cells and to identify its biological characteristics. On the basis of this, to differentiate of amniotic fluid stem cells into keratinocytes, and to identify its biological characteristics.Methods: Samples of amniotic fluid were obtained following routine amniocentesis carried out on pregnant women after 19-22 weeks of gestation. After amniocentesis, immunoselection with an antibody specific for human c-Kit(CD117) was used to isolate h AFSCs. Cultures of h AFSCs were maintained in a humidified incubator under 5% CO2 at 37°C. RT-PCR detected the expression of embryonic stem cells and adult stem cell marker of different passages of amniotic fluid stem cells. Third-passage h AFSCs were seeded in 6-well plates at a density of 2 ×104 cells per well. In the experimental group, inducing medium keratinocyte basal medium-2 supplemented with factors and keratinocyte-conditioned medium at a ratio of 1:1 was used. BMP4 and vitamin C were added additionally when the epithelial clone formed. RT-PCR and immunofluorescence staining were used to detect the gene and protein level of AFS-K respectively. Flow cytometry were to detect the differentiation rate of h AFSCs. The transmission electron microscope was used to observe the ultrastructure of cells. H&E staining analyze the ability to form a completely pluristratified epithelium in 3D air-liquid tissue cultures. Immunohistochemical analysis the markers of different layers.Results:(1) h AFSCs express K19 and β1-integrin(markers of epithelial stem cells) as well as K8 and K18(markers of epithelium). In addition, h AFSCs expressed marker genes for embryonic stem cells, including Oct4, Sox2, C-Myc, Klf4, Rex-1 and Nanog. After 10 passages, subcultures of h AFSCs maintained a gene expression pattern similar to that of early-passage cells. h AFSCs were negative for the positive co-stimulatory molecules CD40, CD80 and CD86 but showed strong expression of the negative co-stimulatory molecules B7H1, B7H2, B7H3, B7H4 and BTLA. h AFSCs can inhibit the activity of human and mouse T lymphocytes. The h AFSCs B7H4-blocking antibody can significantly inhibit the suppression of T lymphocytes by h AFSCs.(2) Keratinocytes derived from h AFSCs(AFS-K) formed spontaneously with typical pavementous epithelial morphology. RT-PCR results identified h AFS-K as K5 and K14 positive. Flow cytometry analysis of K5- and K14-positive h AFS-K cells showed the differentiation rate to 35%, and immunostaining confirmed that the cells express the K5 and K14 proteins.(3) h AFS-K cells display features of keratinocyte precursor cells, with tonofibrils distributed in the cytoplasm, but these cells also have a prominent nucleolus and a high nucleus-to-cytoplasm ratio.(4) H&E staining of organotypic cultures of h AFS-K showed a pluristratified epithelium. Immunohistochemistry showed that K14 and K5 were present in the basal compartment. A reciprocal pattern was found for K10, for involucrin, and for markers of keratinocytes in suprabasal layers, which was present only in layers overlying the basal layer.Conclusions: h AFSCs have the potential to differentiate into epithelium, showed strong expression of the negative co-stimulatory molecules suggests that they may be involved in inhibiting lymphocyte activation and suppressing inflammatory responses. h AFS-K cells express the K5 and K14 proteins and have similar biological characteristics to keratinocytes. h AFS-K cells can form a completely pluristratified epithelium in 3D air-liquid tissue cultures. Taken together, the present study identifies h AFSCs may be a novel source for seed cells for epidermal regeneration. Part II The effects of h AFSCs transplantation in reparation of skin damage and its preliminary mechanismObjective: An injury model was generated and the h AFSCs were transplanted to the edge of the wound area. We aim to observe the recovery of the mice and the proliferation, differentiation of the h AFSCs and analyze the possible mechanism of the reparation of wound.Methods: A lentivirus containing the GFP gene was obtained at a titre of 6.5 ×106 IU/ml. An injury model was generated in BALB/c male mice. A full-thickness skin wound(1 cm diameter) was made on the back of the mouse. The wound received a total of 5×106 GFP-positive h AFSCs or fibroblasts, and then housed the mice individually. The wounds were measured using the UTHSCSA Image Tool. 4 days of granulation tissue and 14 days of repair tissue were harvested and then stained with H&E. We observed the inflammatory cells and neonatal vascular. Flow cytometry and immunofluorescence staining were to detect the survival and differentiation of the cells. Real-Time PCR analyzed the expression of repair-related and inflammatory factors in different days. On such a basis, the interfering RNA for B7H4 down-regulation was constructed. The same injury model was generated in BALB/c male mice. The wound received a total of 5×106GFP-positive B7H4 down-regulated h AFSCs, normal h AFSCs or fibroblasts. Immunohistochemistry and flow cytometry were to detect the amounts of infiltration of T lymphocytes. Real-Time PCR analyzed the expression of repair-related and inflammatory factors in different days. The data, which are presented as the mean± SD, were analyzed using the t-test and considered statistically significant at P < 0.05.Results:(1) Wounds treated with h AFSCs exhibited accelerated wound closure when compared to fibroblast-treated wounds or sham groups at day 7. At day 21, the wounds in h AFSCs-treated mice achieved almost complete wound closure, whereas no completely closed wounds were observed in the fibroblast-treated or sham group.(2) Histological analysis of wounds revealed enhanced re-epithelialization in h AFSCs-treated wounds when compared with fibroblast-treated wounds or the sham group. Analysis of wounds at day 4 indicated that granulation tissue in h AFSCs-treated wounds contained more fibroblasts and capillary-like structures but fewer infiltrated inflammatory cells. According to the analysis of wounds at day 14, h AFSCs-treated wounds appeared to enhance the formation of skin appendage-like structures.(3) A large number of GFP-positive cells were present in h AFSCs-treated wounds at day 7, with this proportion declining at day 14 and day 21. Immunofluorescence analysis revealed that the number of cells(including h AFSCs and fibroblasts) in the wound declined over time.(4) The m RNA levels of repair-related factors were highest in the h AFSCs group at day 7 and much higher than in the fibroblast and sham group but then reduced gradually through days 14 and 21. In contrast, the expression of factors in the fibroblast and sham groups reached a maximum at day 21 before the repair was complete, while the expression of inflammatory cytokines in the fibroblast and sham groups was much higher than in the h AFSCs-treated group on the first day. By the end of wound healing, the expression of factors in h AFSCs groups was the same as its expression in normal mouse skin.(5) B7H4 expression was downregulated in h AFSCs by using a lentiviral vector that carries an interfering RNA for B7H4. B7H4 downregulation in h AFSCs relieved the suppression of T-cell activation. The h AFSCs group repaired the skin damage faster than the B7H4-downregulated and fibroblast groups.(6) The fibroblast and B7H4-downregulated groups had more visible infiltrated CD3+ T cells than the h AFSCs group.(7) The expression of repair-related factors in h AFSCs was higher than in the B7H4-downregulated h AFSCs and fibroblasts by day 7, whereas the B7H4-downregulated h AFSCs and fibroblast groups reached the highest expression of these factors at days 14 or 21. However, the expression of inflammatory factors in h AFSCs was slightly higher than in B7H4-downregulated h AFSCs at day 7. In later stages, the expression of inflammatory factors in the h AFSCs group was more moderate than in the other two groups.Conclusion: h AFSCs exhibited accelerated wound closure, provide a moderate inflammation reaction micro-environment and enhance the formation of skin appendage-like structures. B7H4-downregulated h AFSCs relief of accelerated repair and had more visible infiltrated T cells. B7H4 can regulate low immunogenicity and lead h AFSCs to create a mild repair micro-environment to effectively promote wound repair.Part III The differentiation of human amniotic fluid stem cells into sweat gland cellsObjective: To differentiate of amniotic fluid stem cells into sweat gland cells, and to identify its biological characteristics. On the basis of it, we further explored the sweat gland injury repairation in vivo.Methods: The amniotic fluid stem cells were cultured as previously described. Real-time PCR detected several different lines of amniotic fluid stem cells in sweat gland related genes, and analyze its differentiative potential into sweat gland cells. Third-passage h AFSCs were seeded in 6-well plates at a density of 2 ×104 cells per well. When the sweat gland cells reached confluence, the conditioned medium(CM) was collected every day. The collected CM was filtered through a 0.22μm filter before use. The sweat gland cells differentiation medium consists of a 1:1 ratio of SGM and CM supplemented with 50 ng/ml of EGF and 20ng/ml of Shh. The morphologic changes of differentiated cells were observed under inverted microscope. The differentiated amniotic fluid stem cells were analyzed at day7, 14, 21 and 28 respectively. Real-time PCR and immunofluorescence staining were used to detect the gene and protein level of AFS-SG respectively. Flow cytometry were to detect the differentiation rate of h AFSCs. The transmission electron microscope was used to observe the ultrastructure of cells. H&E staining analyze the ability to form a sweat gland-like structure in 3D tissue cultures. Immunofluorescence staining analysis sweat gland related markers. Sweat gland damage model is constructed on the shoulder part of the nude mice. 2X105 AFS-SG cells were injected. PBS injection group and consubstantial to side without any treatment as blank control groupResults:(1) h AFSCs express K18, CD24 and K19 highly and express EDA, EDAR and K8 weakly, while negative for CEA.(2) During the differentiation process, the h AFSCs exhibited changes in their morphology from mesenchymal like cells to epithelioid cells. The observed h AFS-SG cells were larger in size than the cells before culture in this medium, and observation shows obvious cell-cell contacts, similar to SG cells. The expression of the EDA, EDAR, K8 and CEA m RNAs was significantly increased. Flow cytometry was used to quantify the differentiation efficiency of the h AFS-SG cells which to be 30%.(3)Transmission electron microscopy confirmed that microvilli could be observed on the membrane of the h AFS-SG cells. These results suggest that the AFS-SG cells have the secretory function.(4) h AFS-SG cells formed sweat gland-like structures which expressed the sweat gland secretory markers EDA, EDAR, CEA, K8, K18, Na-K-ATPase and SMA.(5) The injury area can form the sweat gland like structure, expression of K14, K8, and CEA.Conclusions: h AFSCs have the potential to differentiate into sweat gland cells. AFS-SG cells express the EDA, EDAR, K8 and CEA proteins and have similar biological characteristics to sweat gland cells. AFS-SG cells had a typical cellular structure for sweat gland cells: microvilli. The AFS-SG cells have the ability to form the entire tubular structure of a sweat gland in vitro and in vivo. |
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