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The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells.

ABSTRACT: While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting the RLMSC. The effects of RLMSC were examined when applied alone (n = 3) and in conjunction with a stratified culture of human limbal epithelial cells (HLE) grown on the opposing surface of the HAM (n = 3). Outcomes were monitored over 3 months in comparison with animals receiving no treatment (n = 3) or treatment with HLE alone on HAM (n = 3). Animals treated with RLMSC (n = 6) displayed faster re-epithelialization (∼90% versus 70% healing after 12 weeks), with best results being observed when RLMSC were pre-cultivated and implanted in the presence of HLE (p < 0.01; 90% healing by 7 weeks). While all animals displayed conjunctival cells on the corneal surface (by presence of goblet cells and/or keratin 13 expression) and corneal neovascularization, evidence of corneal epithelial regeneration was observed in animals that received RLMSC in the presence of HLE (by staining for keratin 3 and the absence of goblet cells). Conversely, corneal neovascularization was significantly greater when RLMSC were applied in the absence of HLE (<0.05; 90% of cornea compared with 20-30% in other cohorts). Nevertheless, neither human nuclear antigen nor rabbit Y chromosome were detected within the regenerated epithelium. Our results demonstrate that while cultured LMSC encourage corneal re-epithelialization, healing is improved by the pre-cultivation and implantation of these mesenchymal cells in the presence of limbal epithelial cells.

SUBMITTER: Nili E

PROVIDER: S-EPMC6767890 | biostudies-other | 2019 Sep-Oct

REPOSITORIES: biostudies-other

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The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells.

Nili Elham E Li Fiona J FJ Dawson Rebecca A RA Lau Cora C McEwan Blair B Barnett Nigel L NL Weier Steven S Walshe Jennifer J Richardson Neil A NA Harkin Damien G DG

Cell transplantation 20190617 9-10

While limbal epithelial cells are used for treating ocular surface wounds, the therapeutic potential of mesenchymal cells cultivated from the limbal stroma (LMSC) is less clear. We have therefore examined the effects of LMSC when applied to acute ocular surface wounds. LMSC derived from male rabbits (RLMSC) were applied to the ocular surface of female rabbits immediately following removal of the corneal and limbal epithelium. Human amniotic membrane (HAM) was used as the vehicle for implanting t ...[more]

PMID: 31208228

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Project description:PurposeTo propose an improved stem cell-based strategy for limbal stem cell deficiency (LSCD) treatment.DesignExperimental randomized or parallel-group animal study.SubjectsFifty adult male New Zealand white rabbits.MethodsHuman limbal stem/progenitor cells (LSCs) and limbal stromal stem/progenitor cells (LSSCs) were cultured in serum-free conditions and further differentiated into corneal epithelial cells and keratocytes, respectively. All cell types were characterized with lineage-specific markers. Gene expression analysis was performed to identify the potential function of LSSCs in corneal regeneration. Two LSCD models of rabbits for transplantations were used: transplantation performed at the time of limbal and corneal epithelial excision (LSCD model) and transplantation performed after clinical signs were induced in an LSCD model (pLSCD model). The pLSCD model better mimics the pathologic changes and symptoms of human LSCD. Rabbit models received LSC or LSC plus LSSC treatment. Corneal epithelial defects, neovascularization, and opacity were assessed every 3 weeks for 24 weeks. ZsGreen-labeled LSSCs were used for short-term tracking in vivo.Main outcome measuresRates of corneal epithelial defect area, corneal neovascularization and opacity scores, graft survival rate, and immunofluorescence staining of specific markers.ResultsBoth LSC transplantation and LSC plus LSSC cotransplantation effectively repaired the corneal surface in the LSCD model. These 2 strategies showed no significant differences in terms of graft survival rate or epithelial repair. However, corneal opacity was observed in the LSC group (in 3 of 8 rabbits), but not in the LSC plus LSSC group. Notably, when treating LSCD rabbits with distinguishable stromal opacification and neovascularization, cotransplantation of LSCs and LSSCs exhibited significantly better therapeutic effects than transplantation of LSCs alone, with graft survival rates of 87.5% and 37.5%, respectively. The implanted LSSCs could differentiate into keratocytes during the wound-healing process. RNA sequencing analysis showed that the stromal cells produced not only a collagen-rich extracellular matrix to facilitate reconstruction of the lamellar structure, but also niche factors that accelerated epithelial cell growth and inhibited angiogenesis and inflammation.ConclusionsThese findings highlight the support of stromal cells in niche homeostasis and tissue regeneration, providing LSC plus LSSC cotransplantation as a new treatment strategy for corneal blindness.

Project description:PurposeTo investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions.MethodsHuman LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) "3D limbal niche-mimicking" CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM).ResultsHuman LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-β4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM.ConclusionRHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.

Dataset Information

The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells.

Publications

The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells.

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