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 cells in vitro have been considered as an alternative to increase 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 longterm islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semiquantitative 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 nestinpositive 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 shortterm 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 cells.

Generation and expansion of multipotent mesenchymal progenitor cells from cultured human pancreatic islets

“…30 To characterize the phenotype of proliferating-islet-derived cells in the initial days of culture, we analyzed the expression of nestin, an intermediate filament present in several undifferentiated precursor cells that has been found to be expressed in cultured islet cells under different conditions. [10][11][12] Nestin expression has been reported to precede the appearance of b-cells during embryogenesis 13 and to represent a possible marker of adult b-cell precursors. 11,36 In our study, we observed that hPIDM cells appear to emerge from a mixed cell population.…”

“…6 Additional studies suggested that progenitor cells may reside within the pancreatic ductal epithelium 2-7 or in the acinar tissue. 8,9 Nevertheless, the exact nature and localization of adult pancreatic stem/ progenitor cells remains controversial [9][10][11][12][13][14][15][16] and their existence in vivo, at least in mice, has recently been questioned. 6 Recent evidence has shown that human embryonic stem cells are able to differentiate into insulin-producing cells in vitro, thus potentially leading to an unlimited source of cells for transplantation.…”

“…17,18 Some of these studies have noted that cultured islet cells may express nestin, an intermediate filament expressed in several precursor/stem cells. [10][11][12] The association of nestin-positive cells with human pancreatic islets is of interest in light of evidence that nestin expression precedes the appearance of b-cells during embryogenesis. 13 In addition, although no direct origin of b-cells from nestinpositive cells has been demonstrated in postnatal life, 14 recent reports have shown that nestin-positive progenitor cells isolated from human fetal pancreas may have phenotypic markers identical to mesenchymal stem cells and are able to proliferate and differentiate into insulin-producing cells in vitro.…”

Intermediate filament protein nestin is expressed in developing meninges

“…The change mechanism of the intracellular organisations is still not fully understood in various situations such as mitosis, differentiation and different pathological conditions of dynamic structure of intermediate fi lament proteins (26,27). The word nestin is an acronym of neuroepithelial stem cell protein (28) and it is a type VI intermediate fi lament protein forming bundles in neuroepithelial stem/progenitor cells in the growing central nervous system of rat embryos and in humans (20,30). Nestin was expressed mostly in the progenitor cells of developing and innovating tissues in early periods of neural development and in CNS stem cells.…”

“…Nestin was fi rst identifi ed in mitotically active central and peripheral nervous system stem/progenitor cells that give rise to both neurons and glia during early neurogenesis (13,14). It is also detected in some adult stem/progenitor cells; such as newborn vascular endothelial cells (15)(16)(17), striated muscle precursor cell (18), hair follicle precursor cell (19), islet precursor cell (20) and liver oval cell (21). Nestin may represent the proliferation, migration and multi-differentiated characteristics of multi-lineage progenitor cells (11).…”