Pancreatic endocrine and exocrine cells are of endodermal origin and differentiate during prenatal development from the epithelial cells of the primitive ducts. A specific program of transcription factor genes expression is subsequently activated and defines the fate of pancreatic progenitors. In our studies we have analyzed the distribution of two transcription factors - PDX1 and Nkx6.1 in the developing human pancreas. The study was performed on pancreatic samples from 22 fetuses (gestational age 10-40 weeks) using double and triple immunohistochemistry with antibodies to Pdx1, Nkx6.1, insulin and glucagon. In the adult human pancreas, the maintenance of β-cell identity is associated with the presence of key transcription factors (in particular Pdx1 and Nkx6.1), and changes in their expression and/or localisation have been described in the islets of type 2 diabetic individuals. In agreement with the literature, we have shown that Pdx1 and Nkx6.1 are present in the fetal pancreas already at 10th g.w. In all investigated pancreatic samples, we have observed two types of β-cells: Pdx1-positive β-cells and population of β-cells with the Pdx1-immunonegative nuclei. The results of reactions with antibodies to Nkx6.1 were similar: β-cells with both Nkx6.1 -positive and negative nuclei were detected in the human fetal pancreas. It can be suggested, that Pdx1 and Nkx6.1 are transiently expressed in the differentiating β-cells, thereafter their expression decreases and further restores in the mature β-cells. On the other hand, differentiation of some β-cells may possibly occur without Pdx1 or Nkx6.1 expression. In addition, Pdx1-positive glucagon-containing cells were observed in the developing human pancreas. Moreover, in contrast to other researchers, we found rare glucagon-containing cells with Nkx6.1-positive nuclei. This activation of Pdx1 and Nkx6.1 possibly indicates a partial transformation of α-cells toward a β-cell phenotype (and vice-versa). Disclosure A. Proshchina: None. Y. Krivova: None. L. Gurevich: None. D. Otlyga: None. S. Saveliev: None. Funding Russian Foundation for Basic Research (18-015-00147)

Tyrosine hydroxylase (TH) - the specific marker of the catecholamine synthesizing cells, has been detected in the pancreatic endocrine, acinar and ductal cells in some mammals. In the developing mouse pancreas, TH+ cells are often contained glucagon and shared some features that are characteristics for differentiated endocrine cells. As it was shown in experimental studies on mice, a non-neural catecholaminergic pathway appears to modulate pancreatic endocrine precursor and insulin producing cell neogenesis. Our aim was to evaluate whether TH+ cells are present in the developing human pancreas. We have analyzed pancreatic autopsies from 34 human fetuses (10-37 gestational weeks (g.w.)) using double immunofluorescent labeling with antibodies to TH and either insulin or glucagon. Rare TH+ cells were found in the pancreas of all fetuses from 10th to 21th g.w., which were not detected later in development. At early stages (10-12 g.w.), such cells were located among the epithelial cells of primitive ducts, often in close proximity to the endocrine cell clusters, or were associated with the TH+ nerve bundles. In the pancreas of fetuses from 14th to 21th g.w., TH+ cells were observed in the nerve bundles, nerve ganglia and in the neuro-inular complexes type 1 (endocrine cells integrated with the neurons and nerve fibers). Some of the TH+ cells simultaneously contained glucagon. However, the majority of the TH+ cells were negative for insulin or glucagon. Thus, we first detected TH+ cells in the human fetal pancreas. These cells may be differentiating endocrine cells, because some of the TH+ cells were located in the ductal epithelium and rarely contained glucagon. Similarly with mice, non-neural catecholamines may be involved in the endocrine cells differentiation during human pancreas development. Disclosure Y. Krivova: None. A. Proshchina: None. D. Otlyga: None. O. Leonova: None. V. Popenko: None. V. Barabanov: None. S. Saveliev: None. Funding Russian Foundation for Basic Research (18-015-00146)

As it was recently shown, vimentin is expressed in some epithelial and endocrine cells of the pancreas during its development, regeneration and in type 2 diabetes (DM2). It is suggested, that activation of vimentin expression may occurs during the neogenesis of β-cells from epithelial progenitors or during dedifferentiation of β-cells and their reprogramming into α-cells. Our aim was to study the distribution of vimentin in the endocrine pancreas in DM2. The study was performed on pancreatic autopsies from 13 persons with DM2 (DM 2 group) and 9 persons without carbohydrate metabolism disorders (control group) using double immunohistochemistry and immunofluoresence with antibodies to vimentin and either insulin or glucagon, morphometric methods and statistical analysis. Vimentin+ cells, some of which simultaneously contain insulin or glucagon, were observed in the pancreatic islets both in DM2 and control groups. In the DM2 group, all the parameters reflecting the amount of vimentin+ cells were significantly higher than in control group (Table 1). In the DM2 group, we also found characteristic changes in the endocrine pancreas - an increase in α-cell density and a decrease in the β- to α-cell ratio (Table 1). We assume that an increase in the amount of vimentin+ cells in the pancreatic islets may reflect alterations in the endocrine cell mass, and vimentin may be used as a marker of endocrine pancreas disorders. Disclosure Y. Krivova: None. A. Proshchina: None. D. Otlyga: None. S. Saveliev: None.

Pancreatic disorders are the most common pathologies in humans worldwide. Detailed information on pancreatic cytoarchitecture, vascularisation, innervation, morphogenesis, and cell differentiation is required for the development of new approaches to the treatment of these diseases. Currently, the majority of studies on pancreas development are performed on experimental animals (mainly rodents). Studies on human pancreatic prenatal development are restricted in number by ethical constraints and some technical difficulties. However, interspecies differences in pancreatic structure and development are considerable. Therefore, data obtained in experiments on animals and cell cultures must be supplemented with information obtained directly from human pancreatic autopsies. In this chapter, we summarise our previous results and the literature data on human pancreatic ontogeny. Special attention has been paid to the endocrine pancreas, which undergoes morphogenetic restructuring during human development. Several forms of structural organisation of the endocrine pancreas (single endocrine cells, small clusters of endocrine cells, mantle, bipolar, and mosaic islets) gradually appear during development. It is important that this restructuring is accompanied by changes in the ratio of pancreatic endocrine cells. The mechanisms of these changes are still unclear. The difficulties in identifying progenitor cells and tracking cell differentiation are the main problems associated with this issue.

The main cause of diabetes is the loss or dysfunction of beta cells that leads to insulin deficiency and hyperglycemia. However, there are some opposite pathologies, in which insulin secretion results in hypoglycemia. Data on the islets morphology in such diseases may be useful for understanding of the pathogenesis of diabetes. Diabetic fetopathy (DF) is a complication of gestational diabetes or preexisting maternal diabetes mellitus, which is characterized by the accelerated fetal growth and neonatal hypoglycemia. These complications mainly develop due to fetal hyperinsulinemia as a compensatory response to the increased delivery of the glucose to the fetus. Congenital hyperinsulinism (CHI) is a severe genetic disease, in which hypoglycemia is caused by excessive insulin secretion. There are two main CHI forms based on histology: diffuse and focal. Pancreatic samples of 4 newborns with DF from mothers with diabetes, 7 infants with diffuse CHI, and 7 infants with focal CHI were compared to 6 control infants without hypoglycemia. The diagnosis of CHI and histological form were established based on increased insulin level during hypoglycemia, presence of pathologic mutations in ABCC8/KCNJ11 genes, and immunohistochemistry results. All pancreatic samples were examined by immunohistochemistry with antibodies against insulin, glucagon, and vimentin. Histological changes such as nuclei enlargement in some beta-cells, islets hypertrophy, and prominent islets vascularization were observed in the endocrine pancreas of infants with DF as well as in patients with CHI. Swelling of the perivascular space was the distinguishing feature, which was revealed only in newborns with DF. In children with CHI, the abundant vascularization in large islets was not accompanied by swelling. We assume that a long-term increase of blood glucose in fetuses with DF leads to vasodilatation, which subsequently causes impaired endothelial permeability and swelling. Disclosure A. Proshchina: None. D. Gubaeva: None. Y. Krivova: None. D. Otlyga: None. M. Melikyan: None.