Birger Petersson

The renal subcapsular site offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen

SummaryIn order to investigate the importance of the transplantation site for the replication of grafted islet cells, we implanted syngeneic mouse pancreatic islets intrasplenically, intraportally and subcapsularly in the kidney. Fourteen days later the alloxan-diabetic mice were killed after an injection of tritiated thymidine, and the graft-bearing organs fixed and autoradiography. The highest labelling indices were recorded for subcapsularly grafted islets, followed by intraportal and intrasplenic islets in that order. In separate experiments some islet-containing kidney sections were immune stained for insulin before the autoradiographic process. The labelling index of the insulin-positive cells was as high as in the entire islet cell population of the sections from the same mice stained with haematoxylin only. This indicates that the B cells of the islets replicate as often as the other islet cell types. The present data also suggest that the renal subcapsular space offers better growth conditions for transplanted islet cells than the liver or spleen.

Tissue Culture of Human Fetal Pancreas: Development and Function of B-Cells In Vitro and Transplantation of Explants to Nude Mice

The present study evaluates the development and function of human fetal B-cells in vitro with a view to using such cells in future attempts for transplantation of human fetal pancreas to diabetic patients. A method previously described in our laboratory for preparing islets in vitro from the fetal rat pancreas has been applied and modified for use with human fetal pancreas. Pancreatic glands of different gestational ages were obtained from 37 consecutive prostaglandin-induced abortions. After a mild collagenase treatment, the partially disintegrated tissue was maintained in culture for 7 days in tissue culture medium RPM1 1640 plus 20% fetal calf serum to permit cell attachment and outgrowth of endocrine cells. In 17 of the 37 consecutively cultured fetal pancreatic glands, islet-like cell clusters were formed. The 20 remaining glands were lost because of either bacterial contamination or lack of viability already before dissection had occurred. Sections of the newly formed cell clusters revealed well-preserved pancreatic cells showing frequent mitotic figures. The tissue exhibited a high rate of (pro)insulin biosynthesis and a modest insulin response to secretory stimuli, suggesting that the mechanism of glucose regulation by the fetal B-cells is not yet fully developed. Electron micrographs showed a large number of granule-containing cells, some of which were identified as B-cells. In nine cases, harvested cell clusters were implanted beneath the kidney capsule of nude mice. When these animals were killed after 2 mo, seven mice showed a considerable growth of the grafts with numerous islet-like structures containing insulin- and glucagon-positive cells. A high replicatory activity of the grafted cells was further supported by the finding of numerous labeled nuclei in autoradiographs of the graft-bearing kidneys. Since no immunocytochemical staining was carried out, the cell replicatory rate is representative for the whole grafted cell population rather than for the endocrine cells. It isconcluded that this improved culture technique provides a suitable system for the study of growthand differentiation of human fetal pancreas and may facilitate the preservation of pancreatic B-cells intended for transplantation.

Somatostatin in the pancreas, stomach and hypothalamus of the diabetic Chinese hamster

SummaryThe inhibitory effects of somatostatin on the release of insulin and glucagon, as well as its localization to the A1-cells (D-cells) of the pancreatic islets, suggest a role of this peptide in carbohydrate metabolism. In the present study we have measured the percentage islet volume, the total weight of the A1-cells and the somatostatin concentration in the pancreas of normal and spontaneously diabetic Chinese hamsters. In addition, the concentration of somatostatin in the stomach and hypothalamus as well as the insulin and glucagon content of the pancreas were evaluated. The percentage islet volume in the normal hamsters was 0.66±0.12, which was in marked excess of that in the diabetic group, 0.38±0.04. Similarly, the total weight of the A1-cells in the controls, 0.17±0.02 mg, was significantly larger than that in the diabetic animals, 0.12±0.02 mg. In agreement with these findings there was also a decreased pancreatic concentration of insulin and somatostatin, whereas the glucagon concentration was in the normal range. Also the stomach of the diabetic hamsters showed a decreased concentration of somatostatin. In the hypothalamus the total content of somatostatin appeared similar in the two groups of animals, but when expressed per mg wet weight this value was also decreased in the diabetic hamsters. These observations strongly suggest that, in the diabetic Chinese hamster, apart from the well-known B-cell deficiency there exists also a decreased functional activity of the somatostatin-producing cells.

Some Phylogenetical Aspects on the Occurrence of Somatostatin in the Gastro-Entero-Pancreatic Endocrine System

Rodioimmunoassayable somatostatin (SRIF) was found in acid ethanol extracts from various parts of the gastro-entero-pancreatic (GEP) endocrine system in reptiles, amphibians, teleost bony fish, cartilaginous fish, and jawless fish, as well as in a deuterostomian invertebrate, the tunicate, Ciona intestinalis. The cellular sites could, as a rule, be easily visualized light-microscopically by the peroxidase-anti-peroxidase (PAP) immunocytochemical procedure, using guinea-pig and rabbit antisera against synthetic SRIF. The standard Hellerström-Hellman technique, used to detect argyrophi SRIF-storing D cells, failed to visualize the SRIF cells in teh GEP endocrine system of the tumicate and of the jaw-less fish. Moreover, the results comfirmed the previous description that this technique only exceptionally (and sometimes only after further modifications) gave positive results when applied to the GEP endocrine system of bony fish, amphibians, and reptiles. In cartilaginous fish, however, it worked adequately and confirmed the radio-immunological and immunocytochemical observations. In the mucosa of the alimentary tract and in the parenchyma of its associated glands of one echinoderm and two pelecypod molluscs and one crustacean arthropod no sgns of the occurrence of SRIF-storing cells were observed using the three correlated procedures. In several of these tissues, signs of the occurrence of insulin-producing cells had perviously been observed. Thus, SRIF seems to appear at a later evolutionary stage than insulin. The principal islets (Brockmann corpusles) of the marine teleost fish, Cottus scorpius, had the highest concentrations of radioimmunoassayable SRIF of all the GEP organs and tissues investigated, viz. about 200 ng/mg wet weight. Nevertheless, it was only 1/5 of the actual insulin content.

Some characteristics of the two types of A-cells in the islets of Langerhans of guinea-pigs

SummaryTwo types of A cells were identified in the islets of Langerhans of guinea-pigs. The silver-positive A1 cells were found to correspond to the “C cells” previously described by some authors in this species. The most conspicuous feature of the A2 cells was their strongly luminescent granulation in dark field illumination and intense staining with ponceaufuchsin or phloxine. The nuclei of the A1 cells were smaller than those of the A2 cells, while no significant differences were found for their nuclear eccentricities. The previous hypothesis, that the A2 cells are the source of glucagon, obtained support from the finding that this type of A cell was distinctly tryptophane positive. A great susceptibility to cobalt is probably another characteristic property for the A2 cells in the guinea-pig.

Tissue Culture of Human Fetal Pancreas; Effects of Nicotinamide on Insulin Production and Formation of Isletlike Cell Clusters

Human fetal pancreas (HFP) is a potential source of β-cells for transplantation to insulin-dependent diabetic patients. We have previously described a method for tissue culture of HFP that results in the in vitro development of isletlike cell clusters (ICCs) containing a minority of insulin-positive cells. Recently we found that nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, induces an increased islet cell DNA replication both in vivo and in vitro. In this study, this culture technique was used to evaluate the effects of addition of 10 mM nicotinamide on HFP expiants cultured in RPMI-1640 medium plus 10% human serum. ICCs developed in 11 of 19 consecutive cultures with nicotinamide increased the yield of ICCs by 40%. Also, the insulin content of ICCs increased ∼50% with nicotinamide supplementation, although measurements of DNA indicated an unchanged number of cells in each ICC. Neither the rates of insulin release in response to 16.7 mM glucose plus 5 mM theophylline nor the (pro)insulin or total protein biosynthesis rates were affected by nicotinamide addition. The combined results of this study suggest that nicotinamide is useful for stimulating the formation of ICCs from HFP.

Failure of Successful Intrasplenic Transplantation of Islets from Lean Mice to Cure Obese.Hyperglycaemic Mice, Despite Islet Growth

SummaryImplantation of allogeneic pancreatic islets encapsulated in Millipore diffusion chambers has been reported to normalize the obese-hyperglycaemic syndrome in mice. In the present study, both young and adult ob/ob mice remained hyperglycaemic and gained weight after intrasplenic implantation of 500 isogeneic islets isolated from lean mice. Such islets normalized the elevated blood-glucose of alloxan-diabetic lean mice. Morphometric analysis of the intrasplenically implanted islets showed that the mean islet volume in the ob/ob mice was five times larger than that of the lean, non-diabetic mice. Immunocytochemical staining of the spleens showed an increased proportion of B-cells in the enlarged, intrasplenic islets in the ob/ob mice. Moreover, autoradiographical examination of these islets demonstrated the presence of several labelled cells. These results suggest that the growth of the implanted “lean” islets is due to extrapancreatic factors which stimulate islet cell replication in the obese-hyperglycaemic mouse.

Phase-contrast microscopy of fresh and cultured pancreatic islet cells of guinea-pigs

SummaryIsolated cells were prepared from microdissected pancreatic islets of guinea-pigs. Phase-contrast microscopy of the fresh islet cells suspended in a balanced salt solution displayed a number of cellular details, including cytoplasmic secretion granules. There was morphologic evidence of the survival of islet cells in monolayer cultures for up to 3 weeks. A moderate proliferation of cells occurred during the first 2 weeks after explantation. Different types of islet cells could not be distinguished in the phase-contrast microscope.

Culture and Function in Vitro

Human fetal pancreas, obtained at prostaglandin-in-duced legal abortions, was maintained in organ culture for 6–14 days in medium TCM 199 (5.5 mM glucose) or RPMI 1640 (11.1 mM glucose) supplemented with 20% calf serum and antibiotics. Light microscopic examination indicated that the cultured explants were composed of fibrous tissue and well-preserved ducts and islets, whereas acinar cells had disappeared. The hormone content (insulin, C-peptide, glucagon, somatostatin) per milligram wet pancreatic weight increased 10–35 times during the culture period. In static incubations of cultured explants there was a poor insulin response to glucose (16.7 mM). By contrast, insulin release was markedly stimulated in the presence of both glucose (16.7 mM) and theophylline (10 mM). The maximal rate of insulin release in response to glucose plus theophylline was observed after 11 days of culture. When pancreatic explants were incubated in a perifusion system after 7 days in culture, 25% of the fetuses showed a low and monophasic insulin response to glucose alone (15 mM), whereas the remaining ones failed to respond. When perifusions were performed with glucose (15 mM) plus theophylline (5 mM), the insulin response was high in about 25% of the fetuses, intermediate in 40%, and low in 35%. Neither in the static incubations nor in the perifusions was there any clear correlation between the rates of insulin release and the tissue culture medium used. It is concluded that human fetal B cells may retain viability in organ culture for more than a week. The fetal pancreatic material prepared and stored by this means may be used in attempts to cure human diabetes by transplantation.

Transplantation of Fetal Pancreatic Microfragments via the Portal Vein to a Diabetic Patient

Human fetal pancreas preserved in culture was used as a donor organ in a 45-yr-old man with diabetes of 14-yr duration complicated by severe retinopathy and nephropathy. Renal failure had been successfully treated by a cadaveric renal transplant 2 yr earlier. Six fetal pancreases, obtained within 30 min of delivery after prostaglandin-induced abortion at 14–20 wk of gestation, were minced and placed in tissue culture for 3 h at the earliest and 15 days at the longest duration. The cultures were harvested 2–3 h before transplantation. Approximately 3 ml of tissue was infused into a right portal vein branch. Azathioprine was continued at 2 mg/kg and prednisolone increased from 10 mg to 100 mg/day on the day of transplantation and gradually reduced to 25 mg/day. Only two doses of antilymphocytic globulin were given because of a severe reaction. During the 40 days since transplantation, insulin requirements have not changed, but C-peptide has appeared in the urine, suggesting function of the transplanted tissue.