Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures
| Autor: | Fernando Henrique Lojudice, Elizabeth Maria Costa de Oliveira, Tércio Genzini, Silvya Stuchi Maria-Engler, Leticia Labriola, Freddy Goldberg Eliaschewitz, Mari Cleide Sogayar, Cassio Negro Coimbra, Renato A. Mortara, Marcos Luiz Dos Mares Guia, Irene L. Noronha, Maria Lúcia Corrêa-Giannella, Karin Krogh, Irenice Cairo da Silva, Tatiana Caroline Silveira Corrêa, Christian Colin, Carlos Alberto Mayora Aita, Luciano Vilela, Marcelo Perosa de Miranda |
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| Rok vydání: | 2004 |
| Předmět: |
Time Factors
Endocrinology Diabetes and Metabolism Cellular differentiation Nerve Tissue Proteins Biology Stem cell marker Nestin Islets of Langerhans Endocrinology Intermediate Filament Proteins Humans Insulin RNA Messenger Progenitor cell Cells Cultured Homeodomain Proteins geography Microscopy Confocal geography.geographical_feature_category Reverse Transcriptase Polymerase Chain Reaction Stem Cells Cell Differentiation Islet Immunohistochemistry Molecular biology Culture Media Cell biology Drug Combinations Cell culture Trans-Activators Proteoglycans Collagen Laminin Beta cell Stem cell Biomarkers |
| Zdroj: | Journal of Endocrinology. 183:455-467 |
| ISSN: | 1479-6805 0022-0795 |
| DOI: | 10.1677/joe.1.05703 |
| Popis: | Strategies to differentiate progenitor cells into β cells in vitro have been considered as an alternative to increase β cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell’s fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional β cells. |
| Databáze: | OpenAIRE |
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