Generation of pancreatic insulin-producing cells from embryonic stem cells — ‘Proof of principle’, but questions still unanswered

Abstract

Abbreviations E embryonal day EB embryoid body EMT epithelial-mesenchymal transition ES embryonic stemThe generation of insulin-producing cells from differentiating mouse embryonic stem (ES) cells was described some years ago [1], but subsequent studies could not replicate the results. Instead, using the same differentiation protocol, it was found that insulin immunoreactivity occurred as a consequence of insulin uptake from the medium [2], neuronal cells were formed [2-4], or insulin was released as an artefact from differentiated ES cells [2,3]. Functional pancreatic cells, however, were successfully generated using lineage selection strategies based on pancreas-specific promoters [5,6], by modified protocols in combination with transgene expression [7][8][9], or by addition of a phosphoinositol-3 kinase inhibitor [10]. The differentiated cells showed properties of (neonatal) beta cells, such as insulin transcripts and C-peptide/insulin co-expression, insulin-secretory granules, ion channel activity of embryonal beta cells, and normalisation of blood glucose level after transplantation into diabetic mice [5][6][7][8][9][10]. Most of the differentiation protocols required a long cultivation period, including 4-5 days of embryoid body (EB) formation, followed by 3-4 weeks of differentiation, and some protocols required genetic manipulation. Recently, a relatively short procedure of pancreatic differentiation by a three-step experimental approach was published [11]. The strategy is based on the combined treatment by activin A, all-trans-retinoic acid, and other factors such as basic fibroblast growth factor (bFGF), which induced murine ES cells to differentiate into insulin-producing cells within 2 weeks. The authors presented results on insulin transcripts, C-peptide/insulin co-expression, glucose-induced insulin release, and the normalisation of glycaemia following transplantation into diabetic mice. Neuronal vs pancreatic differentiation in vivo and of ES cells in vitroOn looking more closely at the C-peptide-positive cells differentiated according to the protocol of Shi et al. [11], it became obvious that both pancreatic and neuronal differentiation had been induced. In part, the clusters showed the typical morphology of grape-like structures, but also neuronal cell types (Fig. 1a). Immunohistochemical stainings revealed varying levels of co-expression of C-peptide, a byproduct of pro-insulin synthesis (used as a marker for insulin-producing cells), and the neuron-specific beta-III tubulin (Fig. 1b-g). Such ectoderm-derived insulin-producing (C-peptide-positive) cells have been generated from ES cells via EB formation, as well as from monolayer cultures

Embryonic stem cells to beta-cells by understanding pancreas development

“…Nestin, however, is broadly expressed and commonly interpreted as a marker of neuroprogenitors or neural stem cells (Cattaneo and McKay 1990;Lendahl et al 1990). Based on the recognition that many transcription factors regulating beta-cell development and insulin gene expression are also required for neuronal differentiation (Rolletschek et al 2006), Nestin was considered as a potential marker of pancreatic progenitor cells (Cattaneo and McKay 1990). …”

Bone marrow-derived cells and the vasculature in diabetes: from biomarker to treatment?

“…Clinicians may feel some cynicism at the thought of transplanting beta cells produced ex vivo from ESCs to achieve injection-free, safe, curative therapy, but the concept of regenerative medicinegrowing back missing cells, organs and tissues by reawakening differentiation from endogenous precursors-has been taken up with renewed optimism [2,3]. Whatever one's opinion, the research and the debate have proved exciting, and a significant proportion has been played out in Diabetologia [4][5][6][7][8][9][10][11].…”

Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry

“…Previous studies (Boyd et al, 2008;D'Amour et al, 2006;Hori et al, 2002;Kroon et al, 2008;Leon-Quinto et al, 2004;Mfopou et al, 2005;Miralles et al, 2006;Soria et al, 2000;Vaca et al, 2006) and our own data (Blyszczuk et al, 2003;Schroeder et al, 2006) have successfully shown the differentiation of ES cells into pancreatic cells with functional properties, thus demonstrating the proof-of-principle for the derivation of insulin-producing cells. However, strategies to select for nestin-positive cells (Lumelsky et al, 2001) showed a lower and insufficient functional level of insulin-secretion (Hansson et al, 2004;Rajagopal et al, 2003;Sipione et al, 2004) [for discussion, see (Boyd et al, 2008;Kania et al, 2004;Rolletschek et al, 2006)]. …”