Construction of tissue engineered skin with human amniotic mesenchymal stem cells and human amniotic epithelial cells

摘要

OBJECTIVE: To establish a new model for construction of tissue engineered skin with human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial cells (hAECs). MATERIALS AND METHODS: hAMSCs and hAECs were isolated from amniotic membrane. The morphology and phenotype of hAMSCs and hAECs were confirmed by microscope and flow cytometry, respectively. Then, we performed RT-PCR and immunofluorescence staining to assess the expression of stem cells and keratinocyte markers. Moreover, cell co-culture was performed to observe the growth and phenotype characteristics of hAMSCs and hAECs in vitro. In addition, tissue engineered skin with hAMSCs and hAECs was constructed and assessed with histological methods. RESULTS: hAMSCs and hAECs were successfully isolated, exhibiting fibroblast-like morphous and cobblestone-shape epithelial morphous, respectively. The surface biomarker analysis showed that hAMSCs and hAECs were both positive for CD73, CD90 and CD105, and negative for CD34 and HLA-DR. The RT-PCR showed that hAMSCs expressed stem cell marker Nanog and c-MYC, and keratinocyte marker K19, β1 integrin and K8, whereas hAECs expressed stem cell marker KLF4 and c-MYC, and keratinocyte marker K19, β1 integrin, K5 and K8. The expression of keratinocyte proliferation antigen K14 was also found on hAECs. Furthermore, we found co-culture has no impact on the phenotype of hAMSCs and hAECs, but increased the proliferation activity of hAECs and decreased the proliferation activity of hAMSCs. Finally, the histological analysis showed that the tissue engineered skin exhibited similar structure as normal skin. CONCLUSIONS: Tissue engineered skin with hAMSCs and hAECs was successfully constructed and shown a similar feature as a skin equivalent. The tissue engineered skin might have good application prospects in regenerative medicine.