In VitroProliferation of Porcine Pancreatic Islet Cells forβ-Cell Therapy Applications

β-Cell replacement through transplantation is the only curative treatment to establish a long-term stable euglycemia in diabetic patients. Owing to the shortage of donor tissue, attempts are being made to develop alternative sources of insulin-secreting cells. Stem cells differentiation and reprograming as well as isolating pancreatic progenitors from different sources are some examples; however, no approach has yet yielded a clinically relevant solution. Dissociated islet cells that are cultured in cell numbers byin vitroproliferation provide a promising platform for redifferentiation towardsβ-cells phenotype. In this study, we cultured islet-derived cellsin vitroand examined the expression ofβ-cell genes during the proliferation. Islets were isolated from porcine pancreases and enzymatically digested to dissociate the component cells. The cells proliferated well in tissue culture plates and were subcultured for no more than 5 passages. Only 10% of insulin expression, as measured by PCR, was preserved in each passage. High glucose media enhanced insulin expression by about 4–18 fold, suggesting a glucose-dependent effect in the proliferated islet-derived cells. The islet-derived cells also expressed other pancreatic genes such as Pdx1, NeuroD, glucagon, and somatostatin. Taken together, these results indicate that pancreatic islet-derived cells, proliferatedin vitro, retained the expression capacity for key pancreatic genes, thus suggesting that the cells may be redifferentiated into insulin-secretingβ-like cells.

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Co-localization of nestin and insulin and expression of islet cell markers in long-term human pancreatic nestin-positive cell cultures

Journal of Endocrinology ◽

10.1677/joe.1.05703 ◽

2004 ◽

Vol 183 (3) ◽

pp. 455-467 ◽

Cited By ~ 26

Author(s):

Silvya Stuchi Maria-Engler ◽

Maria Lúcia C Corrêa-Giannella ◽

Letícia Labriola ◽

Karin Krogh ◽

Christian Colin ◽

...

Keyword(s):

Cell Cultures ◽

Islet Cell ◽

Islet Cells ◽

Human Islet ◽

Β Cells ◽

Short Term ◽

Cell Markers ◽

Insulin Expression

Strategies to differentiate progenitor cells into β cells in vitro have been considered as an alternative to increase β cell availability prior to transplantation. It has recently been suggested that nestin-positive cells could be multipotential stem cells capable of expressing endocrine markers upon specific stimulation; however, this issue still remains controversial. Here, we characterized short- and long-term islet cell cultures derived from three different human islet preparations, with respect to expression of nestin and islet cell markers, using confocal microscopy and semi-quantitative RT-PCR. The number of nestin-positive cells was found to be strikingly high in long-term cultures. In addition, a large proportion (49.7%) of these nestin-positive cells, present in long-term culture, are shown to be proliferative, as judged by BrdU incorporation. The proportion of insulin-positive cells was found to be high in short-term (up to 28 days) cultures and declined thereafter, when cells were maintained in the presence of 10% serum, concomitantly with the decrease in insulin and PDX-1 expression. Interestingly, insulin and nestin co-expression was observed as a rare event in a small proportion of cells present in freshly isolated human islets as well as in purified islet cells cultured in vitro for long periods of time. In addition, upon long-term subculturing of nestin-positive cells in 10% serum, we observed reappearance of insulin expression at the mRNA level; when these cultures were shifted to 1% serum for a month, expression of insulin, glucagon and somatostatin was also detected, indicating that manipulating the culture conditions can be used to modulate the nestin-positive cell’s fate. Attempts to induce cell differentiation by plating nestin-positive cells onto Matrigel revealed that these cells tend to aggregate to form islet-like clusters, but this is not sufficient to increase insulin expression upon short-term culture. Our data corroborate previous findings indicating that, at least in vitro, nestin-positive cells may undergo the early stages of differentiation to an islet cell phenotype and that long-term cultures of nestin-positive human islet cells may be considered as a potential source of precursor cells to generate fully differentiated/ functional β cells.

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Generation of Islet-Like Hormone-Producing Cells In Vitro from Adult Human Pancreas

Cell Transplantation ◽

10.3727/000000005783982602 ◽

2005 ◽

Vol 14 (10) ◽

pp. 735-748 ◽

Cited By ~ 9

Author(s):

Fouad Atouf ◽

Yong Choi ◽

Michael J. Fowler ◽

Greg Poffenberger ◽

Jan Vobecky ◽

...

Keyword(s):

Pancreatic Islet ◽

Fluorescent Protein ◽

De Novo ◽

Islet Cells ◽

In Vitro Release ◽

Diabetic Patients ◽

Human Pancreas ◽

Cell Clusters ◽

Adult Human

Transplantation of pancreatic islets can provide long-lasting insulin independence for diabetic patients, but the current islet supply is limited. Here we describe a new in vitro system that utilizes adult human pancreatic islet-enriched fractions to generate hormone-producing cells over 3–4 weeks of culture. By labeling proliferating cells with a retrovirus-expressing green fluorescent protein, we show that in this system hormone-producing cells are generated de novo. These hormone-producing cells aggregate to form islet-like cell clusters. The cell clusters, when tested in vitro, release insulin in response to glucose and other secretagogues. After transplantation into immunodeficient, nondiabetic mice, the islet-like cell clusters survive and release human insulin. We propose that this system will be useful as an experimental tool for investigating mechanisms for generating new islet cells from the postnatal pancreas, and for designing strategies to generate physiologically competent pancreatic islet cells ex vivo.

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Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes

Diabetes ◽

10.2337/diabetes.45.9.1197 ◽

1996 ◽

Vol 45 (9) ◽

pp. 1197-1203 ◽

Cited By ~ 13

Author(s):

J. Saldeen ◽

D. T. Curiel ◽

D. L. Eizirik ◽

A. Andersson ◽

E. Strandell ◽

...

Keyword(s):

Gene Transfer ◽

Pancreatic Islet ◽

Islet Cells ◽

Pancreatic Islet Cells ◽

Dna Complexes ◽

Human Pancreatic Islet ◽

Efficient Gene

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Dietary fatty acids modify insulin secretion of rat pancreatic islet cells in vitro

Journal of Endocrinological Investigation ◽

10.1007/bf03344034 ◽

2002 ◽

Vol 25 (5) ◽

pp. 436-441 ◽

Cited By ~ 4

Author(s):

F. J. Tinahones ◽

A. Pareja ◽

F. J. Soriguer ◽

J. M. Gómez-Zumaquero ◽

F. Cardona ◽

...

Keyword(s):

Fatty Acids ◽

Insulin Secretion ◽

Pancreatic Islet ◽

Islet Cells ◽

Dietary Fatty Acids ◽

Pancreatic Islet Cells ◽

Rat Pancreatic Islet

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Pancreatic Islet Cells Antibodies in Diabetic Patients Submitted to Pancreas Transplantation

Transplantation Proceedings ◽

10.1016/j.transproceed.2006.08.167 ◽

2006 ◽

Vol 38 (9) ◽

pp. 3031-3032 ◽

Cited By ~ 3

Author(s):

J. Repka ◽

J. Nicoluzzi ◽

R. Belila ◽

M. Antunes ◽

P. Caron

Keyword(s):

Pancreatic Islet ◽

Pancreas Transplantation ◽

Islet Cells ◽

Diabetic Patients ◽

Pancreatic Islet Cells

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Characterization of insulin glycation in insulin-secreting cells maintained in tissue culture

Journal of Endocrinology ◽

10.1677/joe.0.1520059 ◽

1997 ◽

Vol 152 (1) ◽

pp. 59-67 ◽

Cited By ~ 31

Author(s):

Y H A Abdel-Wahab ◽

F P M O'Harte ◽

C R Barnett ◽

P R Flatt

Keyword(s):

Tissue Culture ◽

Secretory Activity ◽

Protein Glycation ◽

Subsequent Exposure ◽

Insulin Secreting Cells ◽

Per Se ◽

Stimulation Of

Abstract Characteristics of cellular insulin glycation were examined in the pancreatic B-cell line, BRIN-BD11. The extent of insulin glycation increased stepwise during 72 h of culture at 5·6–33·3 mmol/l glucose, attaining levels up to 27%. Glycation of insulin at 33·3 mmol/l glucose was rapid, reaching maximal values within 2 h, and not readily reversible during 2 to 24 h of subsequent exposure to 5·6 mmol/l glucose. Glycated insulin was readily secreted by BRIN-BD11 cells upon active stimulation with glucose and other secretagogues. Cellular insulin glycation was decreased by 66–80% by inhibitors of protein glycation, vitamin C, aminoguanidine or acetylsalicylic acid. Modulation of insulin-secretory activity of BRIN-BD11 cells by co-culture at high glucose with diazoxide, l-alanine or glibenclamide indicated that long-term stimulation of secretion was associated with a decrease in the extent of insulin glycation. Glycation of insulin in vitro was substantially less extensive than in BRIN-BD11 cells, although glucose-6-phosphate and glyceraldehyde-3-phosphate were 1·4- to 2·0-fold more reactive than glucose per se. These observations indicate that insulin is readily glycated and secreted from insulin-secreting cells under hyperglycaemic conditions in culture. Journal of Endocrinology (1997) 152, 59–67

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