Progenitor and bihormonal cells in neuroinsular complexes of the developing human pancreas

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Abstract

BACKGROUND: The development of effective treatments for diabetes mellitus directly depends on a comprehensive understanding of the mechanisms underlying endocrine cell differentiation and pancreatic islet morphogenesis. Experimental studies have demonstrated that the nervous system plays an important role in regulating these processes. In the developing human pancreas, neural structures closely interact with hormone-containing cells, forming neuroinsular complexes, as well as with epithelial cells, suggesting a potential involvement of the nervous system in pancreatic islet development. To support this hypothesis, data on interactions between neural structures and cells exhibiting a progenitor immunophenotype are essential; however, such data are currently lacking in the scientific sources.

AIM: To investigate the distribution of cells expressing the transcription factor PDX1 and bihormonal cells within neuroinsular complexes during human pancreatic development.

METHODS: The study was performed on autopsy specimens of the pancreas obtained from 15 human fetuses (gestational age of 9–25 weeks). Multiple immunofluorescence staining and confocal microscopy were used. To assess the cellular composition of neuroinsular complexes, various combinations of antibodies were applied: against endocrine cell hormones (insulin, glucagon, somatostatin), against the transcription factor PDX1 (pancreatic and duodenal homeobox 1), and against the neural marker neuron-specific β3-tubulin.

RESULTS: Triple immunofluorescence staining with antibodies against insulin, PDX1, and β3-tubulin revealed cells with a progenitor immunophenotype characteristic of ductal epithelial cells within neuroinsular complexes: insulin−/PDX1+/β3-tubulin− and insulin−/PDX1+/β3-tubulin+ cells. Starting from the 9th week of gestation, such cells were detected in isolated ganglia, and from the 14th week onward, in ganglia integrated with pancreatic islets within neuroinsular complexes. In specimens stained with insulin/glucagon/β3-tubulin and insulin/somatostatin/β3-tubulin antibody combinations, bihormonal insulin+/glucagon+ and insulin+/somatostatin+ cells were identified within neuroinsular complexes.

CONCLUSION: The present study demonstrates spatial integration of neural structures with cells exhibiting a progenitor immunophenotype and with bihormonal cells in the developing human pancreas. These bihormonal cells are regarded by most authors as endocrine cells at a stage of differentiation and maturation. Such integration may be necessary for the regulation of β-cell mass and maturation, as well as for modulation of pancreatic islet morphogenesis, as previously shown in experimental studies in animal models and cell cultures.

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About the authors

Yuliya S. Krivova

Petrovsky National Research Centre of Surgery

Author for correspondence.
Email: homulkina@rambler.ru
ORCID iD: 0000-0001-9692-3616
SPIN-code: 4653-9661

Cand. Sci. (Biology)

Russian Federation, Moscow

Alexandra E. Proshchina

Petrovsky National Research Centre of Surgery

Email: proshchina@yandex.ru
ORCID iD: 0000-0002-0515-8275
SPIN-code: 8899-5104

Dr. Sci. (Biology), Assistant Professor

Russian Federation, Moscow

Olga S. Godovalova

Petrovsky National Research Centre of Surgery

Email: godovalova.brain@gmail.com
ORCID iD: 0000-0002-9285-7241
SPIN-code: 8770-0481

Cand. Sci. (Biology)

Russian Federation, Moscow

Sergey V. Saveliev

Petrovsky National Research Centre of Surgery

Email: embrains@hotmail.com
ORCID iD: 0000-0002-1447-7198
SPIN-code: 2079-6351

Dr. Sci. (Biology), Professor

Russian Federation, Moscow

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2. Fig. 1. Optical section images of the fetal pancreas: a–d, at 9 weeks of gestation; e–h, at 15–16 weeks of development; i–l, at 20–21 weeks of development; m–p, at 21–22 weeks of development. Arrows indicate insulin−/PDX1+/β3-tubulin− cells; short arrows indicate insulin−/PDX1+/β3-tubulin+ cells; green arrows indicate single β-cells. a–d, i–l, separately located ganglia; c–h, ganglia forming neuroinsular complexes with mantle-type pancreatic islets; m–p, ganglia forming neuroinsular complexes with bipolar-type pancreatic islets. Triple immunofluorescent staining with antibodies: b, f, j, n, against insulin (green); c, g, k, o, against the transcription factor PDX1 (purple); d, h, l, p, against β3-tubulin (white). a, e, i, m, merged images. Nuclei stained with 4',6-diamidino-2-phenylindole (blue). 1, pancreatic duct; 2, nerve ganglion; 3, pancreatic islet. Scale bar: 20 μm.

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3. Fig. 2. Optical section images of the fetal pancreas. Bihormonal cells: a–d, i–l, at 16 weeks of gestation; e–h, at 18 weeks of development; m–p, at 21–22 weeks of development. Arrows indicate insulin+/glucagon+/β3-tubulin− cells within neuroinsular complexes; short arrows indicate insulin+/glucagon+/β3-tubulin+ cells within neuroinsular complexes. Triple immunofluorescent staining with antibodies: b, f, j, n, against insulin (green); c, g, k, o, against glucagon (red); d, h, l, p, against β3-tubulin (white). a, e, i, m, merged images. Nuclei stained with 4',6-diamidino-2-phenylindole (blue). a–d, insulin+/glucagon+ cells in a ganglion integrated with a pancreatic islet; e–h, an insulin+/glucagon+ cell in a cluster of endocrine cells integrated with a bundle of nerve fibers; i–l, a single insulin+/glucagon+ cell in a nerve fiber extending from a ganglion; m–p, insulin+/glucagon+ cells in a separately located ganglion. 1, nerve ganglion; 2, pancreatic islet; 3, nerve fibers. Scale bar: 20 μm.

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4. Fig. 3. Optical section images of the fetal pancreas. Ddistribution of bihormonal cells in neuroinsular complexes: a–d, at 15–16 weeks of gestation; e–h, at 19 weeks of development; i–l, at 16 weeks of development; m–p, at 25 weeks of development. Arrows indicate insulin+/somatostatin+/β3-tubulin− cells; short arrows indicate insulin+/somatostatin+/β3-tubulin+ cells. Triple immunofluorescent staining with antibodies: b, f, j, n, against insulin (green); c, g, k, o, against somatostatin (red); d, h, l, p, against β3-tubulin (white). a, e, i, m, merged images. Nuclei stained with 4',6-diamidino-2-phenylindole (blue). a–d, insulin+/somatostatin+ cells in complexes formed by neural structures integrated with pancreatic islets; e–h, insulin+/somatostatin+ cells in complexes with neural structures integrated with clusters of endocrine cells; i–l, monohormonal somatostatin+ cells in complexes composed of individual hormone-containing cells integrated with neurons; m–p, monohormonal somatostatin+ cells in complexes composed of individual hormone-containing cells integrated with nerve fibers. 1, nerve ganglion; 2, pancreatic islet; 3, nerve fibers. Scale bar: 20 μm.

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