Inducing Human Embryonic Stem Cells Into Fibroblasts For The Construction Of Tissue-engineered Skin

The skin plays an important role in maintaining stable internal environment and protecting the body from the external environment disturbance. However, the skin defects caused by trauma and ulcer make patients suffering. In clinic, autologous skin graft transplantation is applied to treat the skin defects. However, this method needs another donor site and surgical harvesting which may bring extra suffering to patients. Further more, it's limited for the treatment of large area of defects. The development of tissue-engineered skin (TES) is an optimised alternative to the treatment of skin defects. For the development of TES, the obtain of seed cells seems to be one of the most important issue. In this experiment, we studied how to induce human embryonic stem cells (hESCs) into fibroblasts, and accessed the possibility to use the induced fibroblasts as seed cells to construct TES.In the current study, we tested the proliferation, pluripotency and the undifferentiated state of hESCs firstly. Then, we observed teratoma formation about 8 weeks later after injecting hESCs into nude mice. And muscle-like cells, epithelioid and fibrous cells could be observed after attachment culture of embryoid bodies (EBs) which was formed in the suspension culture of hESCs. All of these results demonstrated the differentiation capability of hESCs in vivo and in vitro. Subsequently, we scraped fibrous cells above differentiated hESCs clones, and then obtained human embryonic stem cells derived mesenchymal stem cells (hE-MSCs) after continuous passages. The results of flow cytometry assay and three lineages of differentiation test indicated that hE-MSCs possesses the characteristics of mesenchymal stem cells. Then, we employed connective tissue growth factor CTGF to induce hE-MSCs into human embryonic stem cells derived fibroblasts (h-Fbs). The expression of relevant gene and protein in induced cells were evaluated by quantitative real-time PCR (qPCR) assay and ELISA assay respectively, and the result showed that h-Fbs exhibited the characteristics of fibroblasts. Finally, we constructed TES by seeding h-Fbs into rat tail collagen, and implanted the TES into skin defects on the back of nude mice in order to investigate the ability of defect repair. Gross inspection indicated that 20 d after implantation, the skin defect was completely repaired. Compared with the TES constructed with normal human skin fibroblasts (HSF), there was no difference for the effect of repaire.Conclusively, hESCs can differentiate into hE-MSCs by self-differentiation and could be induced into h-Fbs by CTGF. The TES from h-Fbs exhibited optimised results in skin defect repair.