Investigative ophthalmology & visual science · 2014
Generation of a biomimetic human artificial cornea model using Wharton's jelly mesenchymal stem cells.
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Garzón Ingrid, Martín-Piedra Miguel Angel, Alfonso-Rodríguez Camilo, González-Andrades Miguel, Carriel Victor, Martínez-Gómez Carlos, Campos Antonio, Alaminos Miguel
Аннотация
PURPOSE: Human Wharton's jelly stem cells (HWJSCs) are able to differentiate into skin and oral mucosa epithelial-like cells. In this work, we demonstrate for the first time the capability of HWJSCs to differentiate in vitro into cornea epithelial-like cells in a three-dimensional model.
METHODS: First, primary cell cultures of HWJSCs, corneal epithelial cells, and corneal keratocytes were cultured and three-dimensional orthotypic and heterotypic human cornea models were generated with fibrin-agarose scaffolds. Then, in vitro differentiation of HWJSCs and corneal epithelial cells was performed with keratinocytic inductive medium in a three-dimensional system that allowed interaction between stromal and epithelial compartments. Histological, histochemical, and immunofluorescence analyses were used to determine the differentiation status of each sample.
RESULTS: Our results demonstrated that HWJSCs were able to differentiate into corneal epithelial-like cells, with results similar to the native cornea. Heterotypical corneas generated with HWJSCs showed adequate differentiation of the epithelium and stroma, and were similar to orthotypic and native corneas in the expression of epithelial markers (CK3/12, PKG, ZO1, and CX43) and extracellular matrix components (proteoglycans, collagen, elastic and reticular fibers). Immunofluorescence analysis confirmed the presence of crystallins Cry-αA, Cry-αB, Cry-β, and Cry-ζ with moderate or weak expression of Cry-βγ3 and Cry-λ1 (key proteins involved in cornea transparency) in both models.
CONCLUSIONS: Our findings suggest that HWJSCs can be considered an alternative cell source for cornea regeneration and may offer a solution for patients with limbus stem cell deficiency.
METHODS: First, primary cell cultures of HWJSCs, corneal epithelial cells, and corneal keratocytes were cultured and three-dimensional orthotypic and heterotypic human cornea models were generated with fibrin-agarose scaffolds. Then, in vitro differentiation of HWJSCs and corneal epithelial cells was performed with keratinocytic inductive medium in a three-dimensional system that allowed interaction between stromal and epithelial compartments. Histological, histochemical, and immunofluorescence analyses were used to determine the differentiation status of each sample.
RESULTS: Our results demonstrated that HWJSCs were able to differentiate into corneal epithelial-like cells, with results similar to the native cornea. Heterotypical corneas generated with HWJSCs showed adequate differentiation of the epithelium and stroma, and were similar to orthotypic and native corneas in the expression of epithelial markers (CK3/12, PKG, ZO1, and CX43) and extracellular matrix components (proteoglycans, collagen, elastic and reticular fibers). Immunofluorescence analysis confirmed the presence of crystallins Cry-αA, Cry-αB, Cry-β, and Cry-ζ with moderate or weak expression of Cry-βγ3 and Cry-λ1 (key proteins involved in cornea transparency) in both models.
CONCLUSIONS: Our findings suggest that HWJSCs can be considered an alternative cell source for cornea regeneration and may offer a solution for patients with limbus stem cell deficiency.