ABSTRACT
Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba2+ crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.
Subject(s)
Alginates/chemistry , Cell Transplantation/methods , Insulin-Secreting Cells/transplantation , Luminescence , Animals , Cell Survival , Cross-Linking Reagents/chemistry , Diabetes Mellitus/therapy , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Insulin/metabolism , Insulin Secretion , Male , Mice , Mice, Inbred BALB C , Pancreas/metabolism , Parkinson Disease/therapy , Time Factors , Transplantation, HeterologousABSTRACT
Histone deacetylase inhibitors (HDACi) were recently identified as having significant clinical potential in reversing ß-cell functional inhibition caused by inflammation, a shared precursor of Type 1 and Type 2 diabetes. However, HDACi are highly complex and little is known of their direct effect on important cell secretion pathways for blood glucose regulation. The aims of the present study were to investigate the effect of HDACi on insulin secretion from ß-cells, GLP-1 secretion from L-cells, and recombinant insulin secretion from engineered L-cells. The ß-cell line ßTC-tet, L-cell line GLUTag, or recombinant insulin-secreting L-cell lines were exposed to Trichostatin A for 24h. Effects on insulin or GLP-1 mRNA, intracellular protein content, processing efficiency, and secretion were measured by real-time PCR, ELISA, and radioimmunoassay. HDACi increased secretion per viable cell in a dose-dependent manner for all cell types. Effects on mRNA levels were variable, but enhanced intracellular polypeptide content and secretion were comparable among cell types. Enhanced recombinant insulin secretion was sustained for seven days in alginate microencapsulated L-cells. HDACi enhances ß- and L-cell secretion fluxes in a way that could significantly improve blood glucose regulation in diabetes patients and holds potential as a novel method for enhancing insulin-secreting non-ß or ß-cell grafts.
Subject(s)
Enteroendocrine Cells/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Insulin-Secreting Cells/drug effects , Secretory Pathway , Animals , Cell Line , Enteroendocrine Cells/metabolism , Glucagon-Like Peptide 1/genetics , Glucagon-Like Peptide 1/metabolism , Insulin/genetics , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Cell-based insulin therapies can potentially improve glycemic regulation in insulin-dependent diabetic patients. Enteroendocrine cells engineered to secrete recombinant insulin have exhibited glycemic efficacy, but have been primarily studied as uncontrollable growth systems in immune incompetent mice. Furthermore, reports suggest that suboptimal insulin secretion remains a barrier to expanded application. METHODS: Genetic and tissue engineering strategies were applied to improve recombinant insulin secretion from intestinal L-cells on both a per-cell and per-graft basis. Transduction of insulin-expressing GLUTag L-cells with lentivirus carrying an additional human insulin gene-enhanced secretion twofold. We infected cells with lentivirus expressing a luciferase reporter gene to track cell survival in vivo. To provide a growth-controlled and immune protective environment without affecting secretory capacity, cells were microencapsulated in barium alginate. Approximately 9×10(7) microencapsulated cells were injected intraperitoneally in immune competent streptozotocin-induced diabetic mice for therapeutic efficacy evaluation. RESULTS: Graft insulin secretion was increased to 16 to 24 mU insulin per day. Transient normoglycemia was achieved in treated mice two days after transplantation, and endogenous insulin was sufficient to sustain body weights of treated mice receiving minimal supplementation. CONCLUSION: Glycemic efficacy of a bioartificial pancreas based on insulin-secreting enteroendocrine cells is insufficient as a standalone therapy, despite enhancement of graft insulin secretion capacity. Supplemental strategies to alleviate secretion limitations should be pursued.
