Differentiation of mouse embryonic stem cells to insulin-producing cells

Abstract

Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d. Differentiation induction by growth and extracellular-matrix factors, including laminin, nicotinamide and insulin, leads to the formation of ES-derived progeny that resembles cells committed to the pancreatic lineage. During differentiation, transcript levels of genes expressed in early pancreatic cells are upregulated. Continued differentiation results in the development of C-peptide/insulin-positive islet-like clusters that release insulin upon glucose stimulation. Differentiated ES cells that overexpress the pancreatic developmental control gene Pax4 develop insulin-secretory granules and reveal functional properties with respect to the pancreas-specific ATP-modulated K+ channel and the normalization of glycemia of streptozotocin-treated diabetic mice.

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

“…For immunolabelling see [40] and [8]. Bar=50 μm (a), 20 μm (b-g) produced during neuronal [18] and pancreatic [1,[7][8][9]19] differentiation of ES cells, respectively (see [19][20][21][22]). …”

“…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][3][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].…”

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

“…For RNA isolation, yeast cells were suspended in lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sodium N -lauroylsarcosinate, 1% mercaptoethanol). The isolation of total RNA was carried out according to Schroeder et al (2006). cDNA was synthesized following the recommendations provided in the instruction manual First Strand cDNA Synthesis (Fermentas, Germany).…”

Cellular Therapies for Type 1 Diabetes

“…Although the amount of insulin produced by these clusters was not sufficient to correct hyperglycemia, the implanted cells did impart a survival and health benefit to its recipient. Schroeder et al [99] generated C-peptide/ insulin-positive islet-like clusters that release insulin upon glucose stimulation through a three step culture process; these clusters were also able to induce the normalization of glycemia in STZ-diabetic mice.…”