| Detecting an ideal kind of seed cell instead of limbal stem cell(LSCs)to reconstruct enginerring corneal epithelium, which is crucial to reconstruct the ocular surface of total limbal stem cell deficiency (LSCD). It was widely believed that both epidermis and corneal epithelium were derived from ectoderm during embryogenesis. Epidermal stem cells (ESCS) and LSCS share several important attributes, although these two kind of stem cells differentiated in different direction during embryogenesis, the direction of both ESCS and LSCS differentiation is not irrevertible. The present study propose that ESCS is a suitable seed cell instead of LSCS to reconstruct the ocular surface of total LSCD patient,whose both eye was involved. To address this issue, isolation and identification of pure ESCS were done , it's pluripotency also tested in vitro .human amniotic membrane(AM) was seleted as a scalfford to carry ESCS for reconstructing enginerring corneal epithelium in vitro. Then treated the damaged corneas of total LSCD goat model by both allogeneic and autologous transplantation of cultivated ESCs AM sheets.1．Isolation and plurification of ESCS from Goat ear skin.ESCS was isolated by explant culture,and purified it through selecting holoclone of ESCS. The colongenic cell was observed post culture 7～12d, which could form colone in diamater approximately 50～100μm. Selected those colones under dissecting microscope and expanded it in gelatin-coated dish with serum-free medium . A single ESCS can form a holoclone (0.8～1.5mm2) containing cells from 1500 to 2000, the diameter of single ESCS is about 8～11μm, the time of cell cycle approximately 25～30h. The CEF of ESCs at 0 passage is 58.9%.2．Identification of ESCsThe expression attributes of ESCs(0～9 passages) was tested by immuno staining. ESCS at 0 pasages highly express Oct-4 (+++),middlely express CD71,PCNA,K19 (++),not express P63, and integrin-β1. The Oct-4 expression of ESCS decreased during subcultures in vitro ,and coculture with feeder cells is need to maintain the expression of Oct-4. ESCS were express K19,P63,PCNA,CD71,β1-integrin,α6-integrin. when cultured on gelatin substrate.The Oct-4 expression is asymmetric during the process of ESCS mitosis.Single cell could form artificial epidermis after long-term culture on gelatin substrate ,hair follicle was also observed. When ESCS coculture with artificial dermis containing collagen type. I. ,fibroblast and mesenchymal stem cells, recontructed skin share similar structure with normal skin,which could regenerate the acute full-thick skin wound functionly.Transmission electron microscope (TEM) show desmosome and hemidesmosome formed in artificial skin.3．Pluripotency of ESCS in Vitro ESCS treated with 2mmolβ-Me for 16 houre during pre-inducing. Then cultured for 48h in formal induction 53.1 percent of ESCS was induced into neuron ,which express NSE, NF-200.ESCS treated with 3μmol 5-Aza for 20h then cultured in medium containing cardicomyocyte condition medium for 54d, heart cell-like clusters was abserved, which express α-actin, and myoglobin. Induced heart muscle share similar structure with normal heart muscle tested by TEM and histological analysis.Differentiation of ESCs toward the osteoblast lineage can be induced by supplementing serum-containing media with 50μg/mL ascorbic acid 50mmol/L β-glycerophosphate ,and 1 umol/L dexamethasone. The induced obsteoblasts was characterized by the formation of discrete mineralized nodules that were stained with 1%. Alizarin Red S'. After treatment 20-24 hours single ESCs differentiated into polygonal cell( ALP positive ),and at the 21th day the outgrowth oppeared the positive cells stained with Alizarin Red S',mineral secretion of differentiated cells was observed by scan.electron microscope(SEM).4．ESCS differentiated well on human amniotic membrane ESCS (1×105,10×107) plated on denuded human amniotic membrane ,the culture was submerged in serum-free medium for a week, then lift for air-liquid surface cultured for two weeks. Th... |
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