Not All Reprogrammed Insulin-Producing Cells Are the Same
Authors: Mingming Gao, Dexi Liu
Journal: Molecular Therapy (2018)

Different strategies have been explored in recent years for diabetes treatment, including islet transplantation, which has been proven effective, but its applications are limited by a lack of sufficient islet suppliers. A newer approach, as described by Wang et al.1Wang Y. Dorrell C. Naugler W.E. Heskett M. Spellman P. Li B. Galivo F. Haft A. Wakefield L. Grompe M. Long-term correction of diabetes in mice by in vivo reprogramming of ancreatic ducts.Mol Ther. 2018; 26 (this issue): 1327-1342Abstract Full Text Full Text PDF Scopus (19) Google Scholar in this issue of Molecular Therapy, is to reprogram cells into insulin-producing β-like cells. The study was designed to systematically compare the reprogramming capacity of cells within the endodermal lineage (hepatocytes, hepatic ductal cells, and pancreatic ductal cells). Using adenoviral vector-mediated delivery of Pdx1, Ngn3, and Mafa (PNM) in mice, the authors demonstrate that pancreatic ductal cells are the preferred target for reprogramming and are superior to hepatocytes, hepatic ductal cells, acinar cells, and gall bladder epithelial cells. Diabetes is one of the earliest identified human diseases caused by loss or dysfunction of β cells in the pancreas. An estimated 30 million Americans, or 9.4% of the US population, suffer from this disease. Another 84 million are pre-diabetic, a condition which, if not treated, often leads to diabetes in 5 years.2CDC. (2017). New CDC report: More than 100 million Americans have diabetes or prediabetes. https://www.cdc.gov/media/releases/2017/p0718-diabetes-report.html.Google Scholar There is currently no cure for diabetes. Life-long frequent injections of insulin are a common practice for most diabetic patients to minimize the risk of diabetes-related comorbidities. Following an intravenous injection of adenoviral vectors carrying PNM genes (Ad-PNM) into mice, Wang and colleagues1Wang Y. Dorrell C. Naugler W.E. Heskett M. Spellman P. Li B. Galivo F. Haft A. Wakefield L. Grompe M. Long-term correction of diabetes in mice by in vivo reprogramming of ancreatic ducts.Mol Ther. 2018; 26 (this issue): 1327-1342Abstract Full Text Full Text PDF Scopus (19) Google Scholar first confirmed that hepatocytes are the predominant type of cell that becomes insulin+. However, despite producing insulin, Ad-PNM-transduced hepatocytes did not express Nkx6.1, a gene critical to β cell identity and function, and they exhibited a low level of Tmem27 mRNA, an important gene for exocytosis of insulin granules. In addition, insulin+ hepatocytes were unable to shut down insulin secretion when the blood glucose level was low, resulting in hypoglycemia in animals 3–5 days after PNM gene transfer. Furthermore, the number of insulin+ hepatocytes in the liver decreased significantly over time. A decrease in transgene expression, accompanied by a decline of the copy number of adenoviral vector genomes, suggests that Ad-PNM-transduced hepatocytes die off with time. This conclusion is consistent with the observation that Ad-PNM-treated RIP-DTR diabetic mice reverted to a hyperglycemic status 3 weeks after Ad-PNM injection. The decrease of transduced hepatocytes was associated with an increase in the F4/80+ monocyte/macrophage population in the liver and hepatocyte regeneration. These results clearly demonstrate that liver hepatocytes may not be the most appropriate type of cell to be reprogrammed by PNM for treatment of diabetes. A similar approach was used to evaluate the properties of hepatic and pancreatic ductal cells, employing retrograde intra-bile duct injection of Ad-PNM. Transgene (Ngn3) and insulin gene overexpression was detected in hepatic ductal cells using fluorescence-activated cell sorting (FACS)-sorted cells. However, unlike their non-reprogrammed neighboring intrahepatic ductal cells, a majority of insulin+ cells stained negative for the ductal epithelial cell transcription factor Sox9 (Sox9−/insulin+ intrahepatic duct cells, 88.2% ± 13.5%). No Nkx6.1 expression (β cell signature gene) was detected in any insulin+ intrahepatic ductal cells in the 8-week period of the experiments, suggesting incomplete reprogramming of these cells into β-like cells. Aside from the liver hepatic ductal cells, very few insulin+ cells (0.08 ± 0.07%) were detected in the gallbladder epithelium, suggesting differences in these cells’ response to PNM-mediated reprogramming. Compared to hepatic ductal cells, the reprogramming potential of pancreatic ductal cells was significantly greater. Following retrograde intra-bile duct injection, the authors showed that Ad-PNM-transduced pancreatic duct cells express not only β cell identity genes such as Nkx6.1, NeuroD1, and Nkx2.2, but also genes involved in β cell function (Tmem27, Slc30a8). At the same time, the pancreatic duct insulin+ cells stained negative for expression of glucagon (GcG) and somatostatin (Sst), the markers for α and δ cell types, respectively, suggesting that the transduced pancreatic duct cells were β cell-specific. Endothelial properties of insulin+ cells in pancreatic ducts were also observed using the endothelial cell surface marker CD31. Moreover, Ad-PNM transduction did not compromise exocrine pancreatic function, as judged by plasma amylase activity 7 and 14 days after Ad-PNM treatment. The insulin+ pancreatic ductal cells appear to possess a more complete β-like phenotype. To evaluate whether intra-ductal injection of Ad-PNM is effective in reverting hyperglycemia, both genetically (RIP-DTR strain) and chemically induced (intra-peritoneal streptozotocin [STZ] injection) diabetic mice were used. Intra-bile duct treatment normalized blood glucose level in 7 days in 35.3% (6/17) of RIP-DTR animals. Another 41.2% (7/17) of the animals showed a transient response, defined by at least one normal blood level during a 7-day period. The remaining 23.5% (4/17) had no response. In STZ-induced diabetic animals, 41.7% (5/12) were rescued by Ad-PNM, but the other 58.3% had either a transient response (16.7%, 2/12) or no response (41.7%, 5/12). Total pancreatic C-peptide content was measured in Ad-PNM-treated diabetic animals induced by STZ and was approximately 50-fold higher in the rescued diabetic animals than in untreated control animals. The rescued diabetic animals showed good glucose response in the glucose tolerance test. In addition, the C-peptide level in rescued animals appeared higher than that of animals with partial or no response to Ad-PNM treatment. These results clearly demonstrate the efficacy of PNM-mediated reprogramming of ductal cells in modulating blood glucose. Additional research should certainly improve the response rate. This is the first comprehensive study of how different cell types respond to the same set of reprogramming factors in vivo. The results clearly demonstrate that hepatocytes, intrahepatic ductal cells, and pancreatic ductal cells can all give rise to insulin+ cells under the influence of PNM overexpression, but pancreatic duct-derived insulin+ cells assume greater β-like characteristics and functions compared with insulin+ cells derived from hepatocytes and intrahepatic duct cells. The results of this study offer the opportunity for further exploration. First, while it is clear that different cell types respond to the same set of reprogramming factors differently, it might be reasonable to hypothesize that different sets of reprogramming factors will lead cells to different fates. If so, it would be reasonable to predict that one type of cell could be converted to another by providing the cells with the appropriate set of reprogramming factors and replacing the functions of disease-causing cells with the reprogrammed cells. Cell replacement therapy via cell reprogramming holds great potential to become the ultimate strategy for the treatment of different, if not all, types of human disease. The achievement of a β cell phenotype in pancreatic ductal cells and a positive response in both genetically and chemically induced diabetic animal models would suggest the applicability of the PNM system to the treatment of diabetes. The data appear to favor such a direction, although a long-term safety study is needed to ensure there are no negative consequences of reprogramming these pancreatic ductal cells. As far as the vectors are concerned, adenoviral vectors may not be ideal for gene delivery due to the transient nature of their transgene expression.3Bouard D. Alazard-Dany D. Cosset F.L. Viral vectors: from virology to transgene expression.Br. J. Pharmacol. 2009; 157: 153-165Crossref PubMed Scopus (240) Google Scholar Adeno-associated viral vectors might be a better choice due to their relatively low immunogenicity, long-term transgene expression, and capacity to target pancreatic ductal cells by intravenous administration, which is a much easier procedure to perform than retrograde intra-bile duct injection. AAV-6 has been shown to be able to target the pancreatic duct in vivo.4Guo P. Xiao X. El-Gohary Y. Criscimanna A. Prasadan K. Rymer C. Shiota C. Wiersch J. Gaffar I. Esni F. Gittes G.K. Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts.Lab. Invest. 2013; 93: 1241-1253Crossref PubMed Scopus (17) Google Scholar In summary, substantial efforts have been devoted to reprogram non-β cells into β-like cells for the purpose of treating diabetes. Wang et al.1Wang Y. Dorrell C. Naugler W.E. Heskett M. Spellman P. Li B. Galivo F. Haft A. Wakefield L. Grompe M. Long-term correction of diabetes in mice by in vivo reprogramming of ancreatic ducts.Mol Ther. 2018; 26 (this issue): 1327-1342Abstract Full Text Full Text PDF Scopus (19) Google Scholar performed an elegant study, showing that pancreatic ducts were superior to other types of cells, including hepatocytes and intrahepatic ducts, as targets for in vivo reprogramming. Importantly, the induced insulin+ cells resembled β cells and generated a sustained glucose-lowering effect in diabetic mice. With additional optimization and safety examinations, this approach should be further evaluated in the clinic for the treatment of diabetes. Long-Term Correction of Diabetes in Mice by In Vivo Reprogramming of Pancreatic DuctsWang et al.Molecular TherapyFebruary 21, 2018In Briefβ cell regeneration has been of great interest in diabetes research. Wang et al. describe an intra-ductal injection approach to reprogram pancreatic ducts into insulin-producing β cells and rescue diabetes in mouse models. This work provides important insights into the mechanism of reprogramming and offers a promising therapeutic strategy for type 1 diabetes. Full-Text PDF Open Archive