Parthenogenetic Embryonic Stem Cells Derived Seed Cells For Tissue-engineered Skin

The skin is the largest organ in human beings. It plays a significant role in mediating temperature and prevention against the environment. However, the skin defects due to trauma, inflamation or tumor excision, diabetes and varicosity may affect both patients’ health condition and mood. Currently, the most commonly applied method for the treatment of skin defect is autologous graft transplantation. However, the source of autologous skin graft is limited and is incapable of covering large area of defects. Furthermore, autologous graft transplantation may cause donor site deformity and the patients will suffer more from the harvesting operation for autologous skin grafts.Tissue-engineered skin (TES) has emerged as an optimized alternative for the treatment of skin defects. Until now, kinds of TES product, including Transcyte, Apligraf and Dermagraft, have been authorized by FDA. TES is a combination of seed cells and matrix. Generally, seed cells are harvested from circumcised infant foreskin, which consists mainly of dermal fibroblasts. But the resource of circumcised infant foreskin specimens is limited and it takes a longer time to obtain enough cells due to the relatively low proliferaive capacity of cells. Morever, high price of currently applied TES products is a heavy burden to the patients and society.Embryonic stem cells (ESCs) derivatives are considered to be good candidate for the construction of TES grafts. However, harvesting of ESCs destroys viable embryos, which may lead to significant political and ethical concerns over their application. Parthenogenetic embryonic stem cells (pESCs) could be obtained from artificially activated parthenogenetic embryo without destroying viable placenta formation. pESCs have only one set of maternal genome. Therefore, pESCs may trigger less immune rejections than ESCs do. In the current study, we investigated the differentiation ability of pESCs into fibroblasts in vitro and their application for the construction of TES. Finally we evaluated the skin defects repair capacity of the constructed TES in in vivo model.ESCs and pESCs were expanded on fibroblastic feeder cells. After EBs formation and MSCs differentiation, the Parthenogenetic mesenchymal stem cells (pMSCs) and embryonic mesenchymal stem cells (eMSCs) were directed toward differentiation into fibroblasts. The molecular characteristics of cell differentiation were assessed by quantitative real-time Polymerase chain reaction (PCR), Enzyme-linked immuno sorbent assay (ELISA), immunofluorescent staining and flow cytometry. TES grafts were constructed and the functional skin defect repair ability were assessed by in vivo model.The results showed that we generated a population of cells that presented characteristics similar to those of fibroblasts. The growth factors gene expression levels in pESCs-derived fibroblasts were similar to those of mouse fibroblasts. Vimentin immunofluorescent staining was positive with the pESCs-derived and ESCs-derived fibroblasts, whereas the fibroblasts were negative for cytokeratin. After seeded into collagen hydrogels, the fibroblasts derived from pMSCs and eMSCs formed TES. Three weeks after grafting onto the mice skin defects, the TES grafts showed satisfying defect repair capacity. There was no significant difference between pESCs-derived fibroblasts (pESCs-d Fs) group and mouse fibroblasts group (P>0.05).(1) It had a high expression of sternness marker Nanog, Oct3/4and SSEA-1in cytoplasm in both pESCs and ESCs, saying that pESCs and ESCs remained in undifferentiated state. The teratoma formation results indicated that both pESCs and ESCs kept pluripotency into three germ layers. The quantitative real-time PCR results showed that the ectoderm, mesoderm and endoderm gene was expressed successively in EBs. We expanded the cells migrated from EBs to obtain mesenchymal stem cells (MSCs).(2) We characterized the cells by molecular method. Flow cytometry results indicated that the acquired pMSCs and eMSCs expressed the MSCs surface antigen. We induced the pMSCs and eMSCs into osteogenic, chondrogenic and adipogenic differentiation. Alizarin red and Von Kossa results demonstrated accumulation of mineralization. Similarly, Safranin O and Oil O staining confirmed the formation of cartilage matrix and lipid droplets individually. We added bFGF and BMP-4into medium for fibroblastic differentiation. We acquired the fibroblasts characterized by Growth Factors and positive Vimentin expression, negative Cytokeratin expression.(3) We constructed the TES with pESCs-d Fs seeded into collagen, ESCs-derived Fibroblasts (ESCs-d Fs) seeded into collagen. Compared with the TES of Fibroblasts and collagen, they have the similar effects. There was a1-2d advancement in skin healing compared with the TES without cells. There was no significant difference between pESCs-d Fs and Fibroblasts (.P>0.05). In total, pESCs can be induced to differentiate into fibroblasts, which can be used as seed cells. This technique could be enriched to provide TES seed cells.