Muhammad T. Tabiin

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic β cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose–response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for β cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic β cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic β cells and become a therapy for the treatment of type I diabetes.

Comparison of size, viability, and function of fetal pig islet-like cell clusters after digestion using collagenase or liberase.

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 ± 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 ± 2 vs. 31 ± 2 min). The size of ICCs was similar for both collagenase (83 ± 0.5 μm) and Liberase (79 ± 0.4 μm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at 1 month after transplantation of 1500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

LOWERING OF BLOOD GLUCOSE TO NONDIABETIC LEVELS IN A HYPERGLYCEMIC PIG BY ALLOGRAFTING OF FETAL PIG ISLETLIKE CELL CLUSTERS1

Background. Fetal pig isletlike cell clusters (ICCs) will differentiate when grafted into the thymus gland of outbred immunosuppressed nondiabetic pigs for up to 3 months. Whether these cells will survive for a similar period in a diabetic recipient and will mature with secretion of insulin to ameliorate the hyperglycemia is unknown. Methods. Between 40,000 and 125,000 ICCs (7,000 to 11,400 ICCs/kg) were injected into the thymus gland of five juvenile pigs immunosuppressed with cyclosporine and deoxyspergualin, and the animals were subsequently made diabetic by the injection of streptozotocin. Insulin was administered subcutaneously, with one pig dying from hypoglycemia. The animal with the least number of ICCs transplanted was killed 81 days later, and the graft was analyzed histologically. Blood glucose levels and porcine C-peptide in the remaining animals were monitored for a median of 101 days. Results. Histological analysis of the graft showed numerous epithelial cell clusters; the percentage of cells that contained insulin, glucagon, somatostatin, and pancreatic polypeptide were 61%, 64%, 25%, and 18%, respectively. Some cells contained more than one hormone. Porcine C-peptide was detected from 21 days after induction of diabetes but not before. In the pig receiving the most ICCs, blood glucose levels were lowered to nondiabetic levels 109 days after transplantation. Plasma C-peptide levels in response to glucagon in this pig steadily increased after grafting; peak levels were 0, 0.21, 0.45, and 0.52 ng/ml at 4, 21, 49, and 80 days after induction of diabetes compared to 0.09 ng/ml in control diabetic pigs. The secretion of C-peptide in response to oral and intravenous glucose and arginine also was greater than in untransplanted diabetic pigs, the pattern of secretion being consistent with developing fetal &bgr; cells as the source of the C-peptide. Pancreatic insulin content was 0.1 mU/mg, 4% of that in nondiabetic pigs, and the number of &bgr; cells per islet was 3 to 6 compared to 90 in nondiabetic controls. Conclusions. ICCs will differentiate and function for up to 111 days when transplanted into outbred immunosuppressed pigs rendered diabetic. Blood glucose levels can be lowered to nondiabetic levels when sufficient numbers of ICCs are grafted.

Role of pancreatic polypeptide as a market of transplanted insulin-producing fetal pig cells.

Transplantation of insulin-producing fetal pancreatic tissue into diabetic recipients has been shown to normalize blood glucose levels after several months. This time period is required for the growth and maturation of the fetal tissue so insulin levels cannot be used as a marker of graft function while the β-cell is immature. Therefore, we have examined the use of another pancreatic endocrine hormone, pancreatic polypeptide (PP), to monitor graft function. The cell that produces this hormone has been shown to be the first mature endocrine cell in the fetal pancreas. Fetal pig pancreatic tissue, both in the form of 1 mm3 explants and islet-like cell clusters (ICCs), was transplanted into immunodeficient SCID mice and the levels of PP and insulin were measured in plasma and in the graft for up to 12 weeks. PP was detected in the untransplanted explants (0.58 pmol/mg) and ICCs (0.06 pmol/ICC) and the PP to insulin ratio was 2.7% and 5.8%, respectively. PP (but not porcine C-peptide, a marker of insulin secretion) was detectable in the plasma of SCID mice from 4 days to 3 weeks after transplantation, but not thereafter. The highest values were obtained at 4 days to 1 week. In the grafted tissue PP and insulin were present at all time points and the ratio of PP to insulin was 59%, 87%, 75%, 56%, 7%, 8%, and 7% at 4 days, 1, 2, 3, 6, 9, and 12 weeks, respectively. The decline in PP levels 3 weeks after transplantation was associated with β-cell development in the graft. PP was also secreted by fetal pig pancreatic explants transplanted into diabetic NOD/SCID mice, with plasma levels measurable in the first week after the tissue was grafted. In immunocompetent BALB/c mice transplanted with the tissue, PP was detectable in plasma for 2 days after transplantation but not at 4 days, when cellular rejection commenced, or thereafter. We conclude that plasma PP levels can be used as a marker of the viability of fetal porcine pancreatic tissue in the first 3 weeks after it is transplanted into mice. These findings may have relevance to fetal pancreatic tissue transplanted into humans if suitable techniques can be developed to separate pig from human PP.