Porcine pancreas mesenchymal cell characterization and functional differentiation into insulin‑producing cells in vitro

There have been considerable efforts towards understanding the potential of human pancreatic endocrine cells to proliferate and transition into mesenchymal cell populations. Since rodent studies have demonstrated that mouse insulin-producing cells do not proliferate in vitro, a similar possibility has been considered for human islet endocrine cells. Considering the inherent differences in mouse and human pancreatic islets, we decided to assess the potential of human fetal pancreatic insulin-producing cells to proliferate in vitro. We studied the proliferative potential of human fetal pancreatic islet-derived populations from second or third trimester fetal pancreas and characterized the cells that grow out during their expansion. We have used seven different approaches including in situ hybridization and immunostaining, quantitative estimation of multiple gene transcripts in populations as well as in single cells, clonal analysis of islet cells, assessment of heritable marks of active insulin promoter, and thymidine analog-based lineage tracing. Our studies demonstrate that human fetal pancreatic insulin-producing cells proliferate in vitro to generate mesenchymal cell populations. Interestingly, epigenetic modifications that mark open chromatin conformation of insulin promoter regions are retained even after a million fold expansion/proliferation in vitro. These findings demonstrate that hormone-producing cells in pancreatic islets proliferate in vitro and retain epigenetic marks that characterize an active insulin promoter. Such in vitro-derived mesenchymal cells may be of potential use in cell-replacement therapy for diabetes.

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Faculty Opinions recommendation of INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13984975.15442093 ◽

2012 ◽

Author(s):

Gordon Weir

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Insulin Producing Cells

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Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666201229124429 ◽

2020 ◽

Vol 16 ◽

Author(s):

Bruna O. S. Câmara ◽

Bruno M. Bertassoli ◽

Natália M. Ocarino ◽

Rogéria Serakides

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Culture Medium ◽

Adipogenic Differentiation ◽

Medium Composition ◽

Cell Therapies ◽

Insulin Producing Cells ◽

Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

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In Vitro Generated Regulatory T-Cells and Insulin Producing Cells-A Potential Therapeutic Tool For End Stage Renal Failure Diabetic Nephropathy.

Transplantation Journal ◽

10.1097/00007890-201407151-00692 ◽

2014 ◽

Vol 98 ◽

pp. 232

Author(s):

S. Dave ◽

A. Vanikar ◽

H. Trivedi

Keyword(s):

Renal Failure ◽

T Cells ◽

Diabetic Nephropathy ◽

Regulatory T Cells ◽

Therapeutic Tool ◽

End Stage Renal Failure ◽

Insulin Producing Cells ◽

End Stage

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Bioactive Lipid Mediators Regulate Lung Epithelial and Mesenchymal Cell Reparative Processes In Vitro

Annals of the American Thoracic Society ◽

10.1513/annalsats.201606-457mg ◽

2017 ◽

Vol 14 (Supplement_3) ◽

pp. S252-S253

Author(s):

Tara M. Nordgren ◽

Art J. Heires ◽

Kristina L. Bailey ◽

Debra J. Romberger

Keyword(s):

Mesenchymal Cell ◽

Lipid Mediators ◽

Lung Epithelial

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TESTOSTERONE SECRETION BY FOETAL RAT TESTES IN VITRO

Journal of Endocrinology ◽

10.1677/joe.0.0710231 ◽

1976 ◽

Vol 71 (2) ◽

pp. 231-238 ◽

Cited By ~ 67

Author(s):

RÉGINE PICON

Keyword(s):

Cyclic Amp ◽

Synthetic Medium ◽

Functional Differentiation ◽

Dibutyryl Cyclic Amp ◽

Testosterone Secretion ◽

Testosterone Production ◽

Foetal Rat

SUMMARY Testosterone secretion by foetal rat testes (13½–21½ days of gestation) explanted for 3 days in a synthetic medium was measured every 24 h by radioimmunoassay. During the first day of explantation, the foetal testis produced, respectively, 1013 ± 132, 8734 ± 1118, 9179 ± 2185 and 3886 ± 309 (s.e.m.) pg/testis when explanted at 14½, 16½, 18½ and 21½ days respectively. Testosterone production by 13½-day-old testes was not detectable on the first day of culture, but appeared on subsequent days. Daily testosterone secretion increased on the 2nd and 3rd days of culture in 14½-day-old testes and decreased in older stages. These results suggest that the functional differentiation of the testis is independent of stimulatory factors like gonadotrophins. Dibutyryl cyclic AMP was found to stimulate testosterone production significantly from 14½ days of gestation onwards.

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The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

Reproduction ◽

10.1530/rep-09-0518 ◽

2010 ◽

Vol 139 (4) ◽

pp. 759-769 ◽

Cited By ~ 42

Author(s):

F P Yuan ◽

X Li ◽

J Lin ◽

C Schwabe ◽

E E Büllesbach ◽

...

Keyword(s):

Mesenchymal Cell ◽

Postnatal Period ◽

Testosterone Replacement Therapy ◽

Developmental Defect ◽

Mrna Levels ◽

Lh Receptor ◽

Protein Levels ◽

Testis Descent

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT).In vivoandin vitroexperiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development inLhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression ofRxfp2which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase inRxfp2mRNA levels in a time-dependent fashion inLhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediatedRxfp2knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent inLhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

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Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1547-1552.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1547-1552

Author(s):

D Leshkowitz ◽

M D Walker

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Activity ◽

Insulin Gene ◽

Hybrid Cells ◽

Dna Binding Activity ◽

Insulin Producing Cells ◽

Insulin Gene Expression ◽

Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

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