Insulin-Secreting Pituitary GH3 Cells: A Potential β-Cell Surrogate for Diabetes Cell Therapy

Abstract

In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 x 10(6) InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic betaTC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines, and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced betaTC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential beta-cell surrogate because they are more resistant than pancreatic beta cells to different apoptotic insults and might therefore be particularly suitable for encapsulation.

Reduced Expression of the Cytokine Transducer gp130 Inhibits Hormone Secretion, Cell Growth, and Tumor Development of Pituitary Lactosomatotrophic GH3 Cells

“…Although it is still uncertain how TtT/GF cells regulate MtT/S's cell behavior, it is likely that the microenvironment determined by cellular interactions and extracellular matrix modifications would contribute to support their tumor development. In contrast, GH3 cells have innate capacity to develop tumors in nude mice (41)(42)(43). They respond to angiogenic and growth factors and do not need the presence of other pituitary cells (i.e.…”

Insulin gene transfer enhances the function of human islet grafts

“…However, due to the extreme complexity of beta-cell physiology, it is quite difficult to induce nonbeta cells to produce and secrete appropriate amounts of insulin in response to changes in blood glucose, that adequately mimic the native beta cell. Some progress has been made by using a glucose controllable promoter or by transferring multiple genes into surrogate cells [11,12,13]. Nevertheless, with the current knowledge and technology, it is not likely that genetically engineered perfectly insulin-regulating "pseudo beta-cells" will be available for large-scale clinical use in the near future.…”

Novel Culture Technique Involving an Histone Deacetylase Inhibitor Reduces the Marginal Islet Mass to Correct Streptozotocin-Induced Diabetes

“…Various parameters affecting islet quality and quantity such as cold ischemia time, organ storage solution, and collagenase incubation time have been tested to optimize islet yield (19,26,37). Second, several surrogate β-cells have been developed (6,15,28). Third, pancreatic progenitor cells have been directed toward islet-like clusters (3,42).…”