Construction Of Tissue-engineered Limbal Graft With Acellular Limbal Matrix And Limbal Stem Cell-like Cells Derived From Embryonic Stem Cells

Limbus, the place harbouring limbal stem cells (LSCs) and the barrier structure between cornea and sclera, plays an important role in maintaining corneal transparency and normal physiological function in vivo. Severe ocular surface disorders could damage limbus, lead to LSCs deficiency and barrier structure destruction. Thus far, LSCs transplantation is the major therapeutic option, yet the extreme shortage of donor LSCs blocks its application. Tissue engineering might provide a new solution to this problem. To create a graft substitute sharing similar structure and function with native limbus, tissue engineered limbal graft is fabricated by expanding ex vivo cultured seed cells on scaffold. Abundant seed cells and suitable scaffold are the two key elements to construct the graft.Corneal epithelium which exposes directly to the external environment is easy to be damaged, so suitable substitutive cells of LSCs should have well self-renewal and proliferative capacity. Embryonic stem cells (ESCs), which possess inexhaustible proliferative capacity and totipotent differentiation ability, may supply an infinite cell source for constructing tissue engineered limbal grafts. Recently several studies showed that ESCs could differentiate into corneal epithelial cells-like cells, yet the proliferative capacity of these cells was limited. Hence, the proposal that inducing ESCs toward LSCs-like cells was needed. The specialized histostructure and basement membrane components of limbus play an important role in the maintenance of LSCs stemness. Human limbal matrix is the ideal scaffold for bioengineered LSCs graft, yet the shortage of donor limits its application. Recently, for the similar structure and composition with native tissue, natural biological scaffold has become more attractive. Our previous study proved that0.5%Sodium Dodecyl Sulfate (SDS) could decellularize satisfactorily the cell components, well preserve the structure of collagen and basement membrane in porcine cornea and form acellular porcine corneal matrix (APCM). Acellular porcine limbal matrix (APLM) which maintained the specific structure of limbus could be used to repair the damaged limbal stroma. However, whether APLM is a suitable scaffold for LSCs ex vivo expansion and transplantation still need further study.Hence, this study firstly explored the possibility of differentiating human ESCs in to LSCs-like cells in vitro and optimized the proposal of differentiation-the induced cells shared similar morphology and phenotype with human LSCs, possessed active proliferative capacity, and could differentiate into corneal epithelial cells-like cells in subsequent culture. Secondly, through analyzeing the components of APLM basement membrane, this research proved that APLM could support adherence, growth, and stemness maintenance of human LSCs, indicating that APLM might be a suitable scaffold for LSCs ex vivo expansion and transplantation. At last, this study proceeded the preliminary animal experiment by transplanting tissue engineered LSCs graft constructed with human ESCs derived LSCs and APLM, the results showed that grafts might help to reconstruct limbus and regenerate damaged oucalr surface in vivo. PartⅠ Differentiation of Human Embryonic Stem Cells into Limbal Stem Cells in vitro[Purpose]The aims of this research were to explore the feasibility of inducing human ESCs (hESCs) into LSCs-like cells in vitro and to optimize the proposal of differentiation.[Methods]1.Culture and identification of hESCs:Mouse embryonic fibroblasts (MEFs) were mitotically inactivated by mitomycin C for2hours and plated at a density of5×104/cm2in35mm culture dishes. The hESCs were cultured on a MEFs feeder cell layer at a density of100colonies/dish and passaged mechanically every6days. The expression of hESCs markers, OCT-4and SSEA-3, was examined by immunofluorescent staining.2. Primary culture and identification of LSCs:3T3fibroblasts were mitotically inactivated by mitomycin C and plated at a density of2.4×104cm2in35mm culture dishes. The corneal endothelium of human limbal rims was mechanically removed from the limbal stroma. Then the remaining limbal tissue was immersed in2.4U/ml dispase II for1.5hours at37℃, followed by mechanical separation of the limbal epithelium from the underlying stroma. The epithelium was trypsinized by0.25%trypsin-0.02%EDTA for10minutes, resuspended with DMEM/F12-LSCs medium or MCDB151-LSCs medium, and seeded at a density of5×103cells/cm2on a3T3feeder cell layer. Cells were cultured at37℃under5%CO2and95%humidity and fed with either DMEM/F12-LSCs medium or MCDB151-LSCs medium every other day. The expression of LSCs markers(p63α and ABCG-2) and corneal epithelial cells marker (CK12) was anaylzed by immunofluorescent staining.3. Preparation of conditioned medium(CM):When adjacent colonies began to merge, LSCs were given200μl/cm2either fresh DMEM/F12-LSCs medium or fresh MCDB151-LSCs medium every24hours. The supernatant were collected for5days, filtered through a0.22μm filter and stored at-80℃. Before use, the harvested supernatant were mixed with fresh media of the same kind at different ratios of3:1,1:1and1:3to acquire75%,50%and25%DMEM/F12-LSCs CM and MCDB151-LSCs CM. The supernatant from3T3fibroblasts was used as control.4. hESCs induction and measurement of differentiation markers:To obtain embryoid bodies (EBs), hESCs colonies were mechanically dissociated into small pieces and transferred into EBs medium for5days. Then EBs was plated into35mm culture dishes coated with IV collagen and supplied with DMEM/F12-LSCs CM or MCDB151-LSCs CM in different concentration. Based on the kind and concentration of CM, EBs were respectively designated as group75%DMEM/F12-LSCs CM (75DF), group50%DMEM/F12-LSCs CM (50DF), group25%DMEM/F12-LSCs CM (25DF), group75%MCDB151-LSCs CM (75M), group50%MCDB151-LSCs CM (50M), group25%MCDB151-LSCs CM (25M), group DMEM/F12-LSCs CM-3T3(DF-3T3) and group MCDB151-LSCs CM-3T3(M-3T3). The expression of p63a, ABCG-2and CK12in induced cells was anaylzed by real time-polymerase chain reaction (RT-PCR) and immunofluorescent staining.5、Proliferation of induced cells in vitro:Differentiated cells in75DF were consecutively subcultured on3T3feeder layer until no clone was formed anymore. Giemsa staining was used to examine the colony forming efficiency in different passages. The expression of p63α, ABCG-2and CK12in different passages was anaylzed by immunofluorescent staining.[Results]Both hESCs and LSCs could form colonies on feeder layers, and expressed their own specific markers. After being transferred into CM, EBs adhered to culture dishes within24hours. Most induced cells in75DF changed into cuboid cells and formed regularly arranged, cobble stone-like cell sheets;9days later stratified cell structures were seen in75DF. In MCDB151-LSCs CM groups, some spindle shaped cells were found in the periphery of the colonies after9days. The lower the CM concentration, the severer the morphological diversity was. The expression of p63α and ABCG-2peaked at the9th day, and their levels in75DF were obviously higher than those in other groups. CK12expression maintained at a low level during the first9days, and began to upregulate at day12. The result showed that more than80%induced cells in75DF were ABCG-2+cells and40%were p63α+. Proliferative examination revealed that induced cells could consecutively subcultured at least4passages in vitro, and possessed the potential to further differentiate into the corneal epithelial cells.[Conclusion]hESCs could differentiate into functional LSCs-like cells with conditioned medium. PartⅡPreparation and biological characteristics detection of acellular porcine limbal matrix[Purpose]The aim of this study was to explore the suitability of using acellular porcine limbal matrix (APLM) for LSCs expansion in vitro.[Methods] 1. Preparation of APLM and APCM:Sterile fresh porcine limbus (containing2mm peripheral cornea and1mm sclera) and central cornea (diameter7.5mm) were decellularized with a0.5%SDS solution for24hours in4℃and then washed8times with PBS for16hours to remove SDS. APLM and APCM were the anterior lamella of these decellularized tissues (0.5mm thick, including basement membrane).2. Preparation of denuded amniotic membrane (DAM):Sterile fresh amniotic membrane was trimmed into small pieces (5cm×5cm) and stored in1:1glycerol/DMEM solution at-144℃. Before use, cryopreserved amniotic membrane was thawed in room temperature for30minutes, and trypsinized in0.25%trypsin-0.02%EDTA at37℃for1hour. Finally the epithelial cells were carefully scraped to obtain DAM.3. Histological and immunological detection of scaffold materials: HE staining and DAPI staining were used to evaluate the effect of decellularization. The expression of collagen IV a2, laminin β1, perlecan, agrin, laminin a2, laminin γ3, tenascin-C in basement membrane of APLM, APCM, DAM and human limbus were detected by immunohistofluorescence.4. Fabrication and characteristics of LSCs tissue-engineered grafts: APLM, APCM and DAM were soaked in DMEM/F12-LSCs medium at37℃for24hours before cell seeding. LSCs were reseeded on the basement membrane of these scaffold materials at a density of1.5×103cell/mm2and cultured for21days. The histological structure of the constructed grafts was observed by HE staining. The expression of ABCG-2, p63a and CK12in seeded cells were analyzed by immunofluorescence and RT-PCR.[Results]HE and DAPI stainings showed that no visible cells or cell nuclear materials were found in these scaffold sections, and structure of collagen and basement membrane were well preserved. Collagen IV a2, laminin β1, perlecan and agrin expressed highly in the basement membrane of APLM, DAM and human limbus; however in APCM only perlecan expressed highly, the level of collagen IV a2and laminin was low, and no agrin expression was observed. Limbal-specific basement membrane components-laminin a2, laminin γ3and tenascin-C, were all detectable in APLM; yet none of them were found in APCM and DAM. LSCs could fabricate closely arranged and stratified epitheloid cell sheets on APLM, APCM and DAM. LSCs seeded on APLM showed4to5layers of cell-the basal layer was cuboid-shaped cells and the elongated cells located in suprabasal layers. Only2to3layers of cell were observed on APCM. LSCs expanded on DAM formed5to6layers of cell but without the cuboid-shaped cells at the bottom. Immunof luorescence staining showed that most cells on APCM and DAM expressed highly CK12, yet the expression of p63α and ABCG-2were low. On the other hand, CK12expression mainly focused on superficial cell layers on APLM and p63α and ABCG-2were only observed in basal cells. RT-PCR results revealed that the level of p63a and ABCG-2on APLM was almost2times higher than those on APCM or DAM; yet CK12expression in APLM was only40%of that in APCM or DAM.[Conclusion]The composition of APLM basement membrane was similar with that of native limbal basement membrane and might supply a suitable microenvironment for the expansion and preservation of LSCs population in vitro. PartⅢ Fabrication of tissue-engineered limbal grafts and animal transplantation[Purpose]To investigate the feasibility of constructing tissue-engineered LSCs grafts with hESCs derived LSCs and APLM and to evaluate the repair performance of the graft in vivo.[Methods]1.Fabrication of tissue-engineered LSCs grafts:hESCs were cultured in75%DMEM/F12-LSCs CM for9days, then induced cells were reseeded on APLM and DAM at a density of1.5×103cell/mm2. After21days culture, the histological structure of grafts was observed by HE staining.2.Transplantation of tissue-engineered LSCs grafts:To create LSCs deficiency model, the limbal tissue of rabbits was excised by limbal lamellar keratectomy and central corneal epithelium was removed by n-heptanal. Fifteen rabbits of LSCs deficiency were randomly divided into3groups,5in each group. Group A was given transplantation of tissue-engineered grafts constructed with hESC derived LSCs and APLM, group B was given transplantation of tissue-engineered grafts constructed with hESC derived LSCs and DAM, and group C was only given APLM transplantation. After operation, dexamethasone-tobramycin drops were used3times daily and ointment once at night. Animals were followed up with slit lamp microscope weekly and HE staining was proceeded1month after surgery.[Results]Induced cells formed4to5layers of cell on APLM and DAM. On APLM, the basal layer was cuboid-shaped cells, while the suprabasal layers were elongated cells. One week after transplantation, conjunctival congestion and corneal edema were obvious in all groups. Two weeks later, inflammatory reaction and corneal edema became slight in group A and B, while corneal opacity was still severe in group C. One month after surgery, in group A and group B the corneal transparency restored, fluorescein staining was negative, and only few neovascularization was observed; while neovascularization was apparent in group B. HE staining revealed that an intact stratified epitheloid structure was found in group A; the epithelial structure in group B was abnormal; no intact epithelial structure existed in group C.[Conclusion]The tissue-engineered LSCs grafts constructed with hESCs derived LSCs and APLM possessed the potential to repair damaged ocular surface in vivo.