Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

Soria, Bernat; Roche, Enrique; Berná, Genoveva; León–Quinto, Trinidad; Reig, Juan A.; Martı́n, Franz

doi:10.2337/diabetes.49.2.157

Cited by 809 publications

(554 citation statements)

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“…Targeted intervention in apoptotic and/or oxidative stress and inflammatory signalling pathways could be one such approach. Such methodology will also be essential, in our opinion, for obtaining the large numbers of highly differentiated insulin-secreting cells envisaged for cell replacement therapy [44] regardless of whether they have been derived by conditional immortalisation of beta cells [4,5], from adult [6] or embryonal [7,8,9] stem cells or by any other means for creating surrogate beta cells.…”

Section: Discussionmentioning

confidence: 99%

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

et al. 2002

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Aims/hypothesis. Human islet cells survive poorly in culture and are overgrown by non-endocrine cells. The aims of this study were to sort human beta cells and to develop approaches for their improved survival in culture. Methods. Human islets were infected with recombinant adenovirus expressing green fluorescent protein (GFP) under the control of the rat insulin promoter such that only beta cells expressed GFP. GFP-positive beta cells were sorted by flow cytometry, and expression of select integrins evaluated by RT-PCR. Beta cells were cultured on different extracellular matrices for up to 15 days. Apoptosis was measured by annexin V binding and ELISA. Insulin secretion was measured by ELISA. Results. Sorted beta cells survived less well in culture than unsorted islet cells. This did not appear to be due to adenoviral infection and/or GFP expression. Purified beta cells expressed the integrins α3, α5, α6, αV, β1, but not β4. Of the various matrices tested, sorted beta cells attached and spread best on a lawn of lysed human bladder carcinoma cells (5637 cells). However, survival remained poor. Cell death was decreased but not prevented by continued presence of 10 mmol/l nicotinamide and apoptosis decreased by 24 h incubation with 20 µmol/l Z-VAD. Insulin secretion was maintained over 6 days following treatment with both agents. Conclusions/interpretation. Purification of human beta cells induces marked apoptosis limiting their function and survival in vitro. This was improved by matching the extracellular matrix to the specific expression of integrins and by addition of nicotinamide and Z-VAD. [Diabetologia (2002) 45:841-850]

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Section: Discussionmentioning

confidence: 99%

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

et al. 2002

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“…It has been shown that pancreatic cell types can be generated from mouse and non-human primate ESCs [10][11][12][13][14][15][16][17][18]. Based on these findings, recent studies have also suggested the potential of hESCs to differentiate into insulin-producing cells through spontaneous in vitro differentiation [5] or the use of a multi-stage protocol [6].…”

Section: Introductionmentioning

confidence: 99%

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

et al. 2007

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Aims/hypothesis The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation. Materials and methods The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation. Results Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice. Conclusions/interpretation Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.

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“…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.…”

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

2006

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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

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Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

Abstract: Embryonic stem (ES) cells display the ability to differentiate in vitro into

Cited by 809 publications

References 14 publications

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

Impact of integrin-matrix matching and inhibition of apoptosis on the survival of purified human beta-cells in vitro

Directed differentiation of human embryonic stem cells towards a pancreatic cell fate

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

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