1. Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells.
- Author
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Tomasello L, Musso R, Cillino G, Pitrone M, Pizzolanti G, Coppola A, Arancio W, Di Cara G, Pucci-Minafra I, Cillino S, and Giordano C
- Subjects
- ATP Binding Cassette Transporter, Subfamily G, Member 2 genetics, ATP Binding Cassette Transporter, Subfamily G, Member 2 metabolism, Adult, Age Factors, Aged, Biomarkers metabolism, Cell Differentiation, Cell Movement, Cell Proliferation, Epithelial Cells metabolism, Epithelium, Corneal metabolism, Female, Fibroblasts metabolism, Gene Expression, HLA-DR Antigens genetics, HLA-DR Antigens metabolism, Humans, Leukocyte Common Antigens genetics, Leukocyte Common Antigens metabolism, Limbus Corneae metabolism, Male, Middle Aged, Nanog Homeobox Protein genetics, Nanog Homeobox Protein metabolism, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Octamer Transcription Factor-3 genetics, Octamer Transcription Factor-3 metabolism, Primary Cell Culture, Spheroids, Cellular cytology, Spheroids, Cellular metabolism, Stage-Specific Embryonic Antigens genetics, Stage-Specific Embryonic Antigens metabolism, Stem Cells metabolism, Epithelial Cells cytology, Epithelium, Corneal cytology, Fibroblasts cytology, Limbus Corneae cytology, Stem Cells cytology
- Abstract
Background: In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population., Methods: Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student's unpaired t test and considering p < 0.05 as statistically significant., Results: The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of "the stem gene profile"; 84 were differentially expressed and were involved in structural activity., Conclusions: The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.
- Published
- 2016
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