| Stem cells (SC) of the corneal epithelium have been found in the limbal palisades of Vogt, between the conjunctiva and limbus. Complete loss of the corneal limbal epithelium leads to re-epithelialization by bulbar conjunctival cells, which is followed by neovascularisation, chronic inflammation, causing a pronounced decrease in visual acuity and severe discomfort. Transplantation of of limbal stem cells (LSCs) is necessary to restore vision and the corneal surface in these diseases.The isolation, in vitro cell culture, and transplantation of LSCs for clinical applications has been well documented, although there are still many unsolved problems. All cornea transplantations reported to date used freshly cultured cells, which were usually cultured on NIH 3T3 feeder cells, applied directly to cornea-damaged patients or experimental animals. A disadvantage of this approach is that it limits the clinical practice. To resolve this, we report the use of cryopreserved LSCs cultured in a feeder-free system to generate the applicable artificial corneal epithelium, and the allograft transplantation of these corneal membranes in LSCD models. We used a series of methods to determine the outcome of the ex vivo-expanded stem cell allograft for LSCD, to provide reference experiences and data for clinical application. Many reports of human adult stem cell plasticity have focused on hematopoietic, mesenchymal, skeletal muscle, and neural stem cells. In contrast, little is known about the plasticity of LSCs. In the present study, we investigated the plasticity of LSCs in vitro and in vivo to further demonstrate the potential therapeutic benefits of these cells. The details of the research include the following three points:1. The isolation and identification of goat LSCs(1) Comparing the methods of digestion and explant, we find the digestion was much better than explant in terms of ration of stem cell, efficiency, proliferation and purification.(2) We selected the optimal the concentration of collagen IV and time of adherence, the results showed the optimal concentration is 20μg/mL, and the optimal adherence time is 20min.(3)We selected the optimal concentration of EGF and Insulin on LSCs were 20ng/mL and 10μg/mL respectively. We also optimized the culture system including serum, and established the serum free and feeder free culture system of LSCs, which was applied for a patent and the applied number was 200710018160.9.(4) The LSCs were subcultured 28 times in serum free system, and 6×107 LSCs were cryopreserved in liquid nitrogen. The studied the relative characteristics of thawed LSCs, the results showed that the stem cell characteristics were well maintained. The cryopreserved system set up in this research could satisfied with the needs of LSCs transportation, preservation and proliferation.2. Differentiation properties of LSCs in vitro(1)The thawed LSCs can be induced into neurons by theβ-mercaptoethanol(β-Me). Being treated with medium supplemented with 1 m mol/L pre-induced for 24h and then with 5 m mol/Lβ-Me formal induced for 18h, serum free medium cultured for 7d,LSCs differentiated towards neural cells which expressed the NSE marker of neural cells.(2)The thawed LSCs would differentiated towards myocardium when exposed to 10μmol/L 5-Azacitidine for 24h,then cultured in myocardium conditional medium for 25d, the cells congregated together and became longer in shape which expressedα-actin myocardial protein.(3)Differentiation of thawed LSCs towards the osteoblast lineage can be induced by supplementing with 0.1μmol/L dexamethasone,10m mol/Lβ-glycerophosphate and 50μg/mL ascorbic acid, the induced cells formed nodules after cultured 21 days, and the nodules were staining positive with Alizarin Red.(4)The thawed LSCs were induced by 2m mol/L glutamine, 0.01% soybean trypsin inhibitor, 10m mol nicotin and 5ng/ml hepatocyte growth factor for 21d ,the cell were induced to pancreatic cells which staining positive with insulin antibody.3. Construction, transplantation and assay of tissue engineered corneal epithelium(1)Seed the thawed LSCs on denuded amniotic membrane, the cell membrane was cultured in serum free medium for 12-14 days, the constructed artificial epithelium with multiple layers of cells which had the similar structure as normal corneal epithelium.(2)The artificial corneal epitheliums were transplanted into the limbal stem cells deficiency (LSCD) eyes. We surveiled the clinical outcomes, after 3 months, 100% (15/15) LSCD model transplanted with artificial epitheliums goat corneal type epithelium; after 6 months, 20% (3/15) LSCD models corneal epitheliums get transparency basically, and 80% (12/15) started to get transparency; We observed 6 transplanted goats for 12 months, 33.3%(2/6)reconstructed corneal epitheliums successfully which had transparent epitheliums and immune type as normal control, 66.7%(4/6)reconstructed partially. The negative control without transplantation corneal surface was entirely covered with conjunctiva and blood vessels, and the cornea lost vision completely.(3)We supposed the mechanism of transplanted LSCs reconstructed the corneal epithelium as the following: the therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of thawed LSCs, the allograft plays a key role to corporate with autologous progenitor cells to repair the damaged goat cornea.(4)The effects of reconstruction were evaluated by a series methods such as clinical observation, histological and immunofluorescence examination, detected transparency of corneal epitheliums and the SRY gene of transplanted seed cells. We first used the Bio-Tek Instrument to evaluate the transparency of corneas, this is a feasible and objective method to evaluate the effect of reconstruction used for animal experiments.
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