Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

Ghoubay-Benallaoua, Djida; de Sousa, Céline; Martos, Raphaël; Latour, Gaël; Schanne-Klein, Marie-Claire; Dupin, Elisabeth; Borderie, Vincent

Ghoubay-Benallaoua, Djida; de Sousa, Céline; Martos, Raphaël; Latour, Gaël; Schanne-Klein, Marie-Claire; Dupin, Elisabeth; Borderie, Vincent.

Afiliación

Ghoubay-Benallaoua D; Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
de Sousa C; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.
Martos R; Etablissement Français du Sang-Ile-de-France, Paris, France.
Latour G; Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.
Schanne-Klein MC; Laboratoire Imagerie et Modélisation en Neurobiologie et Cancérologie, Univ. Paris-Sud, CNRS, Université Paris-Saclay, Orsay, France.
Dupin E; Laboratoire d'Optique et Biosciences, Ecole polytechnique, CNRS, INSERM U1182, Université Paris-Saclay, Palaiseau, France.
Borderie V; Institut de la Vision, Sorbonne Universités, INSERM, CNRS UMR 7210, UPMC Univ Paris 06, Paris, France.

PLoS One ; 12(11): e0188398, 2017.

Article en En | MEDLINE | ID: mdl-29149196

RESUMEN

Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

Asunto(s)

Separación Celular/métodos; Sustancia Propia/citología; Células Epiteliales/citología; Epitelio Corneal/citología; Limbo de la Córnea/citología; Células Madre/citología; Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética; Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo; Adipocitos/citología; Adipocitos/efectos de los fármacos; Adipocitos/metabolismo; Biomarcadores/metabolismo; Diferenciación Celular; Proliferación Celular; Condrocitos/citología; Condrocitos/efectos de los fármacos; Condrocitos/metabolismo; Sustancia Propia/efectos de los fármacos; Sustancia Propia/metabolismo; Medios de Cultivo/química; Medios de Cultivo/farmacología; Células Epiteliales/efectos de los fármacos; Células Epiteliales/metabolismo; Epitelio Corneal/efectos de los fármacos; Epitelio Corneal/metabolismo; Fibroblastos/citología; Fibroblastos/efectos de los fármacos; Fibroblastos/metabolismo; Expresión Génica; Humanos; Queratinocitos/citología; Queratinocitos/efectos de los fármacos; Queratinocitos/metabolismo; Limbo de la Córnea/efectos de los fármacos; Limbo de la Córnea/metabolismo; Proteínas de Neoplasias/genética; Proteínas de Neoplasias/metabolismo; Nestina/genética; Nestina/metabolismo; Neuronas/citología; Neuronas/efectos de los fármacos; Neuronas/metabolismo; Factor de Transcripción PAX6/genética; Factor de Transcripción PAX6/metabolismo; Complejo Represivo Polycomb 1/genética; Complejo Represivo Polycomb 1/metabolismo; Cultivo Primario de Células; Factor de Transcripción SOX9/genética; Factor de Transcripción SOX9/metabolismo; Esferoides Celulares/citología; Esferoides Celulares/efectos de los fármacos; Esferoides Celulares/metabolismo; Células Madre/efectos de los fármacos; Células Madre/metabolismo

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Células Madre / Separación Celular / Limbo de la Córnea / Epitelio Corneal / Sustancia Propia / Células Epiteliales Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2017 Tipo del documento: Article País de afiliación: Francia Pais de publicación: Estados Unidos

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