Target Cells for Stem Cell Factor in the Adult Islets of Langerhans, Simultaneously Synthesizing Glucagon and Insulin
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C-kit—a stem cell receptor, or CD117, is one of the markers of human pancreatic endocrinocyte progenitor cells. Its important role in the proliferation and differentiation of pancreatic endocrine cells during human prenatal development has already been proven. However, the presence and role of c-kit-positive cells in adult pancreatic islets remains unclear. The purpose of our study was to establish the presence of c-kit-positive cells in the adult pancreatic islets and to assess whether they retain the ability to proliferate during a lifetime. We studied pancreatic autopsies of adults aged 50 to 70 years, which were obtained in pathology departments of Kazan hospitals. Samples of the pancreas were embedded in paraffin using standard techniques. Paraffin sections of the pancreas were stained immunohistochemically with commercial antibodies against c-kit, insulin, glucagon, and Ki-67. Few c-kit-positive cells simultaneously secreting insulin or glucagon were detected in all samples studied. These data suggest that such cells are in a state of differentiation into endocrinocytes, but no proliferation of c-kit-positive cells was observed.
KeywordsC-kit CD117 Adult pancreatic islets Pancreatic progenitor cells
Diabetes mellitus and a number of other metabolic disorders, along with cardiovascular diseases, tumors, and injuries, are the main causes of death in developed countries. In addition, diabetes is a social and economic problem, since it leads to early disability of the population due to complications it causes. Recently, the incidence of diabetes mellitus has steadily increased . Type I diabetes is a chronic disease caused by damage of β-cells of the islets of Langerhans that produce insulin, which leads to decreased blood insulin levels. To date, substitution therapy, which is a constant introduction of exogenous insulin or insulin-like drugs, is one of the main methods of treatment for type I diabetes. However, this therapy does not always prevent the development of diabetes complications resulting in disability.
The development of new cell-based technologies and other methods of regenerative medicine aimed to restore the population of β-cells can be a very promising solution. However, cellular sources of the islets development and regeneration are still under a question.
C-kit—a stem cell receptor, or CD117, is one of the markers of human pancreatic endocrinocyte precursor cells. To date, its role in the prenatal development of the human pancreatic islets has already been proven [2, 3, 4, 5]. However, the presence and role of c-kit-positive cells in adult pancreatic islets remains still unclear. Nowadays, there are two opposing points of view in the literature. Part of researchers suggest that normal adult pancreatic islets have no c-kit-positive cells , others detect rare c-kit-positive cells in the adult rat pancreatic islets . Another issue is the proliferative potential of these cells in adults, since it is known, that the number of proliferating c-kit-positive cells in the islets decreases during gestation . It is essential to clarify the presence and proliferative potential of c-kit-positive cells in the adult pancreas to develop the criteria for donor selection for transplantation purposes.
Considering the above, our study was aimed to establish the presence of c-kit-positive cells in the adult pancreatic islets and to evaluate their ability to proliferate.
2 Materials and Methods
Autopsies of the pancreas of adults at the age of 50 to 70 years (a total of eight samples), who died from acute or chronic pathologies not associated with pancreatic disorders, were obtained in pathology departments of Kazan hospitals. The research was approved by the Kazan (Volga region) Federal University local ethics committee.
Samples of the pancreas were embedded in paraffin using standard techniques. Paraffin sections of the pancreas were stained immunohistochemically with commercial antibodies against c-kit (1:200, mouse monoclonal T595, “Novocastra,” UK), insulin (1:75, mouse monoclonal 2D11-H5, “Novocastra,” UK), glucagon (1:50, rabbit polyclonal, “DAKO,” DK), and Ki-67 (1:200, rabbit polyclonal SP6 “Abcam,” UK).
Double immunohistochemical staining to detect c-kit+/glucagon+, c-kit+/insulin+, glucagon+/insulin+, and c-kit+/Ki-67+ islet cells was performed; combinations of different AP- and HRP-conjugated detection systems were used for mouse and rabbit primary antibodies (LSAB AP with alkaline phosphatase (Dako, DK), Novolink with peroxidase (“Novocastra,” UK), HRP-conjugated CSA Rabbit Link (Dako, DK), HRP-conjugated CSA (Dako, DK), and CSA with alkaline phosphatase (Dako, DK)). AEC for detection of peroxidase activity (red), and BCIP/NBT substrate system (Dako, DK) for detection of alkaline phosphatase activity (blue) were used.
Then, histological sections were studied under a microscope (Axio Imager, Z2, Zeiss, Germany), followed by image acquisition (AxioCam HRc, Zeiss, Germany). Images were processed with the ZEN pro 2012 software.
There was no statistically significant age-dependent difference in the number of c-kit-positive cells.
Double staining for c-kit and insulin or glucagon revealed single c-kit-positive cells expressing insulin (Fig. 1a) and glucagon (Fig. 1b). We also found single cells that simultaneously synthesized insulin and glucagon (Fig. 1c).
Double staining for c-kit and Ki-67 revealed no double stained cells in any sample (Fig. 1d). As previously, c-kit-positive cells were located in the islets, and Ki-67-positive cells were found both in the islets and in acini of the pancreas.
The detection of c-kit-positive cells in rat pancreatic islets by Gong et al.  and the absence of these cells in human pancreatic islets according to the results obtained by Amsterdam et al.  raised a question whether c-kit-positive cells in the islets is a specific feature the of rat pancreas.
Our results proved the presence of c-kit-positive cells in the adult pancreatic islets. We found out that in humans there can be different numbers of these cells in the islets.
Since we detected c-kit-positive cells secreting insulin and glucagon, it can be assumed that these are differentiating endocrinocyte precursor cells, as the presence of c-kit indicates proliferation and differentiation processes, and the secretion of hormones—the onset of differentiation into α- or β-endocrinocytes .
When studying the prenatal development of the islets we and another group of researchers previously established a similar pattern of expression [2, 3, 4, 5], which may indicate the same sequence of differentiation throughout a lifetime.
The presence of cells, simultaneously synthesizing insulin and glucagon, indicates the phenotypic plasticity of developing endocrinocytes, and also suggests that the same cells can be C-kit-positive. In addition, these data correlate with other studies of c-kit-positive cells in a human prenatal period [2, 3, 4, 5]. The first proliferating c-kit-positive cells were detected on the 8th week of gestation, insulin, and glucagon appeared later . Moreover, our group has shown that c-kit-positive cells first begin to synthesize glucagon, and later on they also secrete insulin. We have made a conclusion that c-kit-positive cells are common precursors of α- and β-cells [4, 5]. Interestingly, in prenatal ontogenesis, cells begin to synthesize hormones after their proliferative activity decreases which becomes significant after 19–21 weeks of gestation . Obviously, the proliferative activity of c-kit-positive cells continues to decrease with age. Since there were no dividing c-kit-positive cells in the islets, we can state that the proliferation capacity of these cells is over, at least after the age of 50 years. However, it cannot be ruled out that at a younger age (samples of patients younger than 50 years were not available for the study); these cells retain the ability to proliferate.
Our results first showed the population of c-kit-positive cells, secreting insulin and glucagon in the adult pancreas. This indicates the continuing differentiation of c-kit-positive cells into α- and β-cells of the islets along the same pathway as in the prenatal period [2, 3, 4, 5].
The results obtained are valuable not only for fundamental, but also for practical medicine. The presence of c-kit-positive cells in normal adult pancreatic islets suggests that disorders can occur in these c-kit-positive progenitor cells, and the cells themselves can be a source of the development of pancreatic tumors expressing c-kit . Therefore, the detection of these cells in a patient’s biopsy requires additional diagnostics, as their presence can indicate both the physiological differentiation of c-kit-positive cells into α- and β-cells of the islets and the pathological process of neoplasm formation.
C-kit-positive cells in the adult pancreatic islets can also be of interest for the development of new treatment approaches to type I diabetes. As C-kit is a transmembrane receptor, it is the most attractive and promising marker for purposes of possible isolation of these cells for their subsequent culture and transplantation. Since c-kit-positive cells remain in the pancreas for at least 70 years, not only a child, but also an adult can be their donor. However, the lack of proliferation activity of these cells in the islets of people older than 50 years indicates a greater expediency of isolating these cells from younger donors.
The work is performed according to the Russian Government Program of Competitive Growth of the Kazan Federal University and a subsidy allocated to the Kazan Federal University for the state assignment in the sphere of scientific activities and financial support of the Russian president grant № МК-3632.2011.7.
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