title: Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures.
abstract: Strategies to differentiate progenitor cells into beta cells in vitro have been considered as an alternative to increase beta 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 beta cells.
authors.affiliation: Instituto de Química, Universidade de São Paulo, Departamento de Bioquímica CP26077, São Paulo 05513-970 SP, Brazil.
authors.forename: Silvya Stuchi
authors.initials: SS
authors.lastname: Maria-Engler
authors.forename: Maria Lúcia C
authors.initials: ML
authors.lastname: Corrêa-Giannella
authors.forename: Letícia
authors.initials: L
authors.lastname: Labriola
authors.forename: Karin
authors.initials: K
authors.lastname: Krogh
authors.forename: Christian
authors.initials: C
authors.lastname: Colin
authors.forename: Fernando Henrique
authors.initials: FH
authors.lastname: Lojudice
authors.forename: Carlos Alberto Mayora
authors.initials: CA
authors.lastname: Aita
authors.forename: Elizabeth Maria Costa
authors.initials: EM
authors.lastname: de Oliveira
authors.forename: Tatiana C Silveira
authors.initials: TC
authors.lastname: Corrêa
authors.forename: Irenice Cairo
authors.initials: IC
authors.lastname: da Silva
authors.forename: Tercio
authors.initials: T
authors.lastname: Genzini
authors.forename: Marcelo Perosa
authors.initials: MP
authors.lastname: de Miranda
authors.forename: Irene Lourdes
authors.initials: IL
authors.lastname: Noronha
authors.forename: Luciano
authors.initials: L
authors.lastname: Vilela
authors.forename: Cassio Negro
authors.initials: CN
authors.lastname: Coimbra
authors.forename: Renato A
authors.initials: RA
authors.lastname: Mortara
authors.forename: Marcos Mares
authors.initials: MM
authors.lastname: Guia
authors.forename: Freddy Goldberg
authors.initials: FG
authors.lastname: Eliaschewitz
authors.forename: Mari Cleide
authors.initials: MC
authors.lastname: Sogayar
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D004338:Drug Combinations; D018398:Homeodomain Proteins / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007328:Insulin / Q000032:analysis* / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000032:analysis*; D007515:Islets of Langerhans / Q000737:chemistry*; D007797:Laminin; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000032:analysis*; D064231:Nestin; D011509:Proteoglycans; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000737:chemistry; D013997:Time Factors; D015534:Trans-Activators / Q000032:analysis
chemical_list: D015415:Biomarkers; D003470:Culture Media; D004338:Drug Combinations; D018398:Homeodomain Proteins; D007328:Insulin; D007381:Intermediate Filament Proteins; D007797:Laminin; C577484:NES protein, human; D009419:Nerve Tissue Proteins; D064231:Nestin; D011509:Proteoglycans; D012333:RNA, Messenger; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C059485:matrigel; D003094:Collagen