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Silvya Stuchi Maria-Engler¹, Maria Lúcia C Corrêa-Giannella², Letícia Labriola¹, Karin Krogh¹, Christian Colin¹, Fernando Henrique Lojudice¹, Carlos Alberto Mayora Aita¹, Elizabeth Maria Costa de Oliveira¹, Tatiana C Silveira Corrêa¹, Irenice Cairo da Silva¹, Tercio Genzini³, Marcelo Perosa de Miranda³, Irene Lourdes Noronha³, Luciano Vilela⁴, Cassio Negro Coimbra², Renato A Mortara⁵, Marcos Mares Guia^,4, Freddy Goldberg Eliaschewitz³

¹ Instituto de Química, Universidade de São Paulo, Departamento de Bioquímica CP26077, São Paulo 05513-970 SP, Brazil
² Laboratório de Nutrição Humana e Doenças Metabólicas LIM-25, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil
³ Albert Einstein Hospital, São Paulo, Brazil
⁴ Biomm S/A, Montes Claros, Minas Gerais, Brazil
⁵ Departamento de Micro, Imuno e Parasitologia, Escola Paulista de Medicina-UNIFESP, São Paulo, Brazil

(Requests for offprints should be addressed to M C Sogayar; Email: mcsoga{at}iq.usp.br)

(S S Maria Engler is now at Faculdade de Ciencias Farmacêuticas, Departamento de Análises Clínicas e Toxícológicas, Universidade de São Paulo, São Paulo, Brazil)

In memoriam

Strategies to differentiate progenitor cells into ß cells in vitro have been considered as an alternative to increase ß 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 ß cells.

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