RESUMEN
PURPOSE: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. METHODS: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. RESULTS: The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. CONCLUSION: This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.
Asunto(s)
Lesiones de la Cornea , Células Madre Embrionarias/trasplante , Lesiones Oculares/terapia , Trasplante de Células Madre , Heridas no Penetrantes/terapia , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Lesiones Oculares/metabolismo , Proteínas del Ojo/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Queratina-15/metabolismo , Queratina-3/metabolismo , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Adhesión del Tejido , Fijación del Tejido , Heridas no Penetrantes/metabolismoRESUMEN
The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.