Characterization of ? cells developed in vitro from rat embryonic pancreatic epithelium

Abstract

The present study evaluates the development and functional properties of β cells differentiated in vitro. The authors have previously demonstrated that when E12.5 rat pancreatic rudiments are cultured in vitro in the absence of mesenchyme, the majority of the epithelial cells differentiate into endocrine β cells. Thus, depletion of the mesenchyme provokes the expansion of endocrine tissue at the expense of exocrine tissue. The potential use of this procedure for the production of β cells led the authors to characterize the β cells differentiated in this model and to compare their properties with those of the endocrine cells of the embryonic and adult pancreas. This study shows that the β cells that differentiate in vitro in the absence of mesenchyme express the homeodomain protein Nkx6.1, a transcription factor that is characteristic of adult mature β cells. Further, electron microscopy analysis shows that these β cells are highly granulated, and the ultrastructural analysis of the granules shows that they are characteristic of mature β cells. The maturity of these granules was confirmed by a double‐immunofluorescence study that demonstrated that Rab3A and SNAP‐25, two proteins associated with the secretory pathway of insulin, are strongly expressed. Finally, the maturity of the differentiated β cells in this model was confirmed when the cells responded to stimulation with 16 mM glucose by a 5‐fold increase in insulin release. The authors conclude that the β cells differentiated in vitro from rat embryonic pancreatic rudiments devoid of mesenchyme are mature β cells. Dev Dyn 1999;214:116–126. © 1999 Wiley‐Liss, Inc.

Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

“…Note that the eGFP staining was found both in nuclear and cytoplasmic compartments, while insulin staining was restricted to the cytoplasmic compartment. Scale bar 50 μm simulate pancreatic differentiation [27,35]. To evaluate the transduction yield of both vectors, double immunofluorescence detection of insulin and eGFP was performed after 3 and 7 days of culture.…”

Epidermal Growth Factor Increases Undifferentiated Pancreatic Embryonic Cells In Vitro

“…These experiments indicated that the development of the endocrine tissue can occur in the absence of signals from the mesenchyme that surrounds the epithelium (13). It has also been shown in vitro that when e12Ϫe13 pancreatic epithelium is cultured in the absence of mesenchyme, pancreatic endocrine cells develop properly (6,7). In fact, in vitro, more insulin-expressing cells develop in the absence of mesenchyme, when epithelial cell proliferation is low, than in the presence of mesenchyme, when proliferation is high, suggesting that signals from the mesenchyme activate the proliferation of immature epithelial cells and repress their differentiation into endocrine tissue (6).…”

Control of β-Cell Differentiation by the Pancreatic Mesenchyme

“…This required an in vitro model permissive for ␤-cell development both with and without mesenchyme. Previously, we had shown that in collagen gel, ␤-cells developed properly from embryonic pancreatic epithelium when cultured without mesenchyme (20), whereas this culture condition was poorly permissive for ␤-cell development when the epithelium was cultured with mesenchyme (21). Here, we developed an in vitro model in which ␤-cells can develop both with and without mesenchyme.…”