Detecting de novo insulin synthesis in embryonic stem cell-derived populations

Kiernan, Eadaoin Mc; Barron, Niall; O’Sullivan, Finbarr; Barham, Paul; Clynes, Martin; O’Driscoll, Lorraine

doi:10.1016/j.yexcr.2006.12.013

Cited by 5 publications

(4 citation statements)

References 24 publications

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“…Several investigators had argued that the presence of insulin in such cells does not indicate intrinsic insulin production. They suggest that insulin from the utilized culture media can be absorbed by and sequestrated in these cells (15, 26, 32). Immunofluorescence staining was positive for insulin, c-peptide, and glucagon with coexpression of insulin and c-peptide by the same cells confirming that proinsulin synthesis was occurring in the cells and not being derived from any insulin in the culture media.…”

Section: Discussionmentioning

confidence: 99%

Insulin-Producing Cells from Adult Human Bone Marrow Mesenchymal Stem Cells Control Streptozotocin-Induced Diabetes in Nude Mice

et al. 2013

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Harvesting, expansion and directed differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) could provide an autologous source of surrogate β-cells that would alleviate the limitations of availability and/or allogenic rejection following pancreatic or islet transplantation. Bone marrow cells were obtained from three adult type 2 diabetic volunteers and 3 non-diabetic donors. After 3 days in culture, adherent MSCs were expanded for 2 passages. At passage 3, differentiation was carried out in a 3-staged procedure. Cells were cultured in a glucose-rich medium containing several activation and growth factors. Cells were evaluated in-vitro by flow cytometry, immunolabelling, Rt-PCR and human insulin and c-peptide release in responses to increasing glucose concentrations. One thousand cell-clusters were inserted under the renal capsule of diabetic nude mice followed by monitoring of their diabetic status. At the end of differentiation, ~5–10% of cells were immunofluorescent for insulin, c-peptide or glucagon; insulin and c-peptide were co-expressed. Nanogold immunolabelling for electron microscopy demonstrated the presence of c-peptide in the rough endoplasmic reticulum. Insulin-producing cells (IPCs) expressed transcription factors and genes of pancreatic hormones similar to those expressed by pancreatic islets. There was a stepwise increase in human insulin and c-peptide release by IPCs in response to increasing glucose concentrations. Transplantation of IPCs into nude diabetic mice resulted in control of their diabetic status for 3 months. The sera of IPC-transplanted mice contained human insulin and c-peptide but negligible levels of mouse insulin. When the IPCs-bearing kidneys were removed, rapid return of diabetic state was noted. BM-MSCs from diabetic and non-diabetic human subjects could be differentiated without genetic manipulation to form IPCs which, when transplanted, could maintain euglycaemia in diabetic mice for 3 months. Optimization of the culture conditions are required to improve the yield of IPCs and their functional performance.

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Section: Discussionmentioning

confidence: 99%

Insulin-Producing Cells from Adult Human Bone Marrow Mesenchymal Stem Cells Control Streptozotocin-Induced Diabetes in Nude Mice

et al. 2013

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“…C-peptide is again a very useful marker of cell activity in exploring systems and culture conditions. It has particular importance as an independent indicator, because the source of insulin under the conditions of cell culture is not always unequivocal [39]. Besides several studies with human or murine embryonic stem cells, mesenchymal stem cells were investigated, for example in [40].…”

Section: The Fifth Decade 2007mentioning

confidence: 99%

History and Diagnostic Significance of C‐Peptide

Brandenburg

2008

Journal of Diabetes Research

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Starting with the epoch-making discovery of proinsulin, C-peptide has played an important interdisciplinary role, both as part of the single-chain precursor molecule and as an individual entity. In the pioneering years, fundamental systematic experiments unravelled new biochemical mechanisms and chemical structures. After the first detection of C-peptide in human serum, it quickly became a most useful independent indicator of insulin biosynthesis and secretion, finding application in a rapidly growing number of clinical investigations. A prerequisite was the development of specific immuno assays for proinsulin and C-peptide. Further milestones were: the chemical synthesis of several C-peptides and the accomplishments in the synthesis of proinsulin; the detection of preproinsulin with its bearings on understanding protein biosynthesis; the pioneering role of insulin, proinsulin, C-peptide, and mini-C-peptides in the development of recombinant DNA technology; and the discovery of the enzymes for the endoproteolytic processing of proinsulin into insulin and C-peptide, completing the pathway of biosynthesis. Today, C-peptide continues to serve as a special diagnostic tool in Diabetology and related fields. Thus, its passive role is well established. Evidence for its active role in physiology and pathophysiology is more recent and is subject of the following contributions.

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“…Therefore the differentiating medium should contain glucose. Thus, several investigators suggested that the cells may have the ability to absorb glucose from the medium which enhances the cells to release insulin [34]. Accordingly, to avoid the argument of insulin origin, intracellular immunofluorescence staining was positive in Muse cells for insulin, c-peptide and the co-expression of insulin and c-peptide by the same cell emphasized the capacity of proinsulin synthesis.…”

Section: Discussionmentioning

confidence: 99%

In vitro differentiation of human multilineage differentiating stress-enduring (Muse) cells into insulin producing cells

Fouad

Gabr

Abdel-Hady

et al. 2018

Journal of Genetic Engineering and Biotechnology

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Mesenchymal stem cells (MSCs) is a heterogeneous population. Muse cells is a rare pluripotent subpopulation within MSCs. This study aims to evaluate the pulirpotency and the ability of Muse cells to generate insulin producing cells (IPCs) after in vitro differentiation protocol compared to the non-Muse cells. Muse cells were isolated by FACSAria III cell sorter from adipose-derived MSCs and were evaluated for its pluripotency. Following in vitro differentiation, IPCs derived from Muse and non-Muse cells were evaluated for insulin production. Muse cells comprised 3.2 ± 0.7% of MSCs, approximately 82% of Muse cells were positive for anti stage-specific embryonic antigen-3 (SSEA-3). Pluripotent markers were highly expressed in Muse versus non-Muse cells. The percentage of generated IPCs by flow cytometric analysis was higher in Muse cells. Under confocal microscopy, Muse cells expressed insulin and c-peptide while it was undetected in non-Muse cells. Our results introduced Muse cells as a new adult pluripotent subpopulation, which is capable to produce higher number of functional IPCs.

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Detecting de novo insulin synthesis in embryonic stem cell-derived populations

Cited by 5 publications

References 24 publications

Insulin-Producing Cells from Adult Human Bone Marrow Mesenchymal Stem Cells Control Streptozotocin-Induced Diabetes in Nude Mice

Insulin-Producing Cells from Adult Human Bone Marrow Mesenchymal Stem Cells Control Streptozotocin-Induced Diabetes in Nude Mice

History and Diagnostic Significance of C‐Peptide

In vitro differentiation of human multilineage differentiating stress-enduring (Muse) cells into insulin producing cells

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