Teresa Nieto-Miguel

In Vitro Simulation of Corneal Epithelium Microenvironment Induces a Corneal Epithelial-like Cell Phenotype from Human Adipose Tissue Mesenchymal Stem Cells

Abstract Purpose: Transplantation of autologous corneal stem cells in not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore the ocular surface in these patients, an autologous extraocular source of stem cells is desirable to avoid dependence on deceased donor tissue and host immunosuppression of allogenic transplants. While bone marrow-derived mesenchymal stem cells (MSCs) can acquire certain characteristics of corneal epithelial cells, subcutaneous adipose tissue (AT) is more readily available and accessible. The aim of this study was to determine if extraocular human AT-derived MSCs (hAT-MSCs) can acquire in vitro some features of corneal epithelial-like cells. Methods: hAT-MSCs were isolated from human lipoaspirates and expanded up to 3–4 passages. We studied the immunophenotype of MSCs and demonstrated its multipotent capacity to differentiate toward osteoblasts, adipocytes and chondrocytes. To test the capacity of differentiation of hAT-MSCs toward corneal epithelial-like cells, hAT-MSCs were cultured on substrata of plastic or collagen IV. We used basal culture medium (BM), BM conditioned with human corneal epithelial cells (HCEcBM) and BM conditioned with limbal fibroblasts (LFcBM). Results: The hAT-MSCs incubated for 15 days with HCEcBM acquired more polygonal and complex morphology as evaluated by phase-contrast microscopy and flow cytometry. Additionally, the expression of transforming growth factor-β receptor CD105 and corneal epithelial marker CK12 got increased as evaluated by flow cytometry, real-time reverse-transcription polymerase chain reaction, western blot and immunostaining. These changes were absent in hAT-MSCs incubated with unconditioned BM or with LFcBM. Conclusions: Corneal epithelial-like cells can be induced from extraocular hAT-MSCs by subjecting them to an in vitro microenvironment containing conditioning signals derived from differentiated human corneal epithelial cells. Our results suggest that hAT-MSCs could provide a novel source of stem cells that hold the potential to restore sight lost in patients suffering from bilateral ocular surface failure due to LSCD.

Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH–G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH–G 20:80 membranes than other proportions. Consequently, CH–G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH–G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH–G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

Consecutive expansion of limbal epithelial stem cells from a single limbal biopsy.

Abstract Purpose: Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm2 limbal explant (LE). Methods: LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy. Results: LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased. Conclusion: One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.

Stem Cell Therapy for Corneal Epithelium Regeneration following Good Manufacturing and Clinical Procedures

Objective. To evaluate outcomes of cultivated limbal epithelial transplantation (CLET) for management of ocular surface failure due to limbal stem cell deficiency (LSCD). Design. Prospective, noncomparative, interventional case series and extensive comparison with recent similar studies. Participants. Twenty eyes with LSCD underwent CLET (11 autologous; 9 allogeneic) and were followed up for 3 years. Etiologies were divided into 3 prognostic categories: Group 1, chemical injuries (7 eyes); Group 2, immune-based inflammation (4 eyes); and Group 3, noninflammatory diseases (9 eyes). Intervention. Autologous and allogeneic limbal epithelial cells were cultivated on amniotic membranes and transplanted. Evaluations were based on clinical parameters, survival analysis, and in vivo confocal microscopy (IVCM). European Union Tissues/Cells Directive and good manufacturing procedures were followed. Main Outcome Measures. Improved clinical parameters, absence of epithelial defects, and improved central corneal epithelial phenotype. Results. Success rate was 80% at 1-2 years and 75% at 3 years. Autografts and allografts had similar survival. Success rate was significantly lower in prognostic Group 1 (42.9%) than in Groups 2-3 (100% each). All clinical parameters improved substantially. By IVCM, 80% of cases improved in epithelial status. Conclusions. CLET improved corneal epithelium quality, with subsequent improvement in symptoms, quality of life, and vision. These results confirm that CLET is a valid therapy for ocular surface failure.

Therapeutic Effect of Human Adipose Tissue‐Derived Mesenchymal Stem Cells in Experimental Corneal Failure Due to Limbal Stem Cell Niche Damage

Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue‐derived mesenchymal stem cells (hAT‐MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT‐MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E‐cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT‐MSC transplantation. For the first time, we demonstrated that hAT‐MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT‐MSCs as a novel treatment of ocular surface failure due to LSCD. hAT‐MSCs represent an available, non‐immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160–2174

Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions

ABSTRACT Purpose: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.