Kevin Docherty

This article was written as a contribution to mark the centenary of the first administration of insulin to a human in 1922. Writing from an Aberdeen perspective, an introductory passage will place emphasis on the role of JJR MacLeod, under whose supervision the discovery of insulin by Banting and Best was made. The major thrust of the article, however, will be on the cloning and sequencing of the human insulin gene, and the impact it had on the scientific career of the author. It initiated a journey to find alternative therapies for diabetes that led sequentially though gene therapy, embryonic stem cell-derived islets, and reprogramming. Our experience in these areas will be described, with emphasis on the strengths and weaknesses of each of these approaches.

The shortage of donor material has driven research towards finding a replenishable supply of islets for transplantation. We have previously shown that the exocrine material that results as a by-product of the islet isolation procedure can be transdifferentiated towards functional islet-like structures by overexpression of the pancreatic transcription factors (TFs) Pdx1, MafA, Ngn3 and Pax4 followed by culture in the presence of betacellulin, nicotinamide and exendin-4. These cells secreted insulin in a glucose responsive manner and rescued diabetes in a streptozotocin-induced diabetic mouse model, but expressed 1% of the insulin levels of mature islets. Modification of the culture conditions to include laminin and a low glucose concentration has further enhanced transdifferentiation towards endocrine lineages. A major increase in insulin production was observed upon siRNA knockdown of the α-cell TF ARX at the later stages of the protocol. Specific ELISAs for human proinsulin, insulin and C-peptide and ultrastructural analysis demonstrated that the newly transdifferentiated cells were able to efficiently store (33.5±7.3 pg/μg protein) and process insulin, secreting C-peptide in a regulated glucose-responsive manner, with insulin levels rising to 15% of those found in human islets. ARX knockdown further decreased glucagon expression. Removal of Pax4 abolished regulated glucose-response, supporting its critical role for functionality and maturation of the transdifferentiated cells. This population was monohormonal, comprising 40% C-peptide⁺, 4% glucagon⁺, and + cells. Grafted in diabetic Scid/Bg mice, these cells had an immediate and prolonged (100 days) effect in normalising blood glucose levels and body weights. We have shown for the first time that late inhibition of ARX, along with Pax4 overexpression, is crucial for the transdifferentiation of human exocrine cells towards mature beta-like cells. We estimate that ~3 billion of these cells would have an immediate and therapeutic effect following transplantation in people with type 1 diabetes.

A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome.

Aims/hypothesis Type 1 diabetes complicated by hypoglycaemia is prevalent in socioeconomically deprived populations. Islet transplantation is of proven efficacy in type 1 diabetes complicated by hypoglycaemia, but it is not known if nationally funded programmes reach the socioeconomically deprived. Our aim was to determine: (1) socioeconomic indices in participants referred to our nationally funded programme; and (2) if metabolic outcomes in our transplant recipients were improved. Methods Participants referred (n = 106) and receiving transplants (n = 18; 32 infusions) were examined with respect to socioeconomic status (deprivation category score) and their ability to work and drive. In participants followed for ≥12 months after transplantation, metabolic and anthropometric measurements (n = 14) were recorded pre- and post-transplant (assessed ~1, ~3, ~6 and ~12 months with mixed-meal tolerance tests and 6 day continuous glucose monitoring assessments). Donor data was also examined. Results There was a greater prevalence of socioeconomic deprivation in referred and transplant recipients than the general population (p < 0.05). Of the transplant recipients, 73% were socioeconomically deprived, 88% did not hold a driver’s license and 94% had reduced ability to work (all p < 0.01 vs referred participants). Donors were predominantly obese and included circulatory death donors. At 12 months, 93% of participants who had received transplants had graft function, diminished frequency of hypoglycaemia (10 [4–11] vs 0 [0–2] hypoglycaemic episodes/week), improved awareness of hypoglycaemia (Gold score 7 [5–7] vs 1 [1–2]) and glycaemic control (HbA_1c: 7.9% [7.2–8.5%]; 63 [55–69] mmol/mol vs 7.2% [6.8–7.5%]; 55 [51–58] mmol/mol), diminished glycaemic lability and decreased central adiposity (all p < 0.05). Conclusions/interpretation A nationally funded islet transplant programme reaches the socioeconomically deprived and outcomes are significantly improved in this group.

Cell therapy in the form of human islet transplantation has been a successful form of treatment for patients with type 1 diabetes for over 10 years, but is significantly limited by lack of suitable donor material. A replenishable supply of insulin-producing cells has the potential to address this problem; however to date success has been limited to a few preclinical studies. Two of the most promising strategies include differentiation of embryonic stem cells and induced pluripotent stem cells towards insulin-producing cells and transdifferentiation of acinar or other closely related cell types towards β-cells. Here, we discuss recent progress and challenges that need to be overcome in taking cell therapy to the clinic.

Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.

Aims/hypothesis Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) offer unique opportunities for regenerative medicine and for the study of mammalian development. However, developing methods to differentiate hESCs/hIPSCs into specific cell types following a natural pathway of development remains a major challenge. Methods We used defined culture media to identify signalling pathways controlling the differentiation of hESCs/hIPSCs into pancreatic or hepatic progenitors. This approach avoids the use of feeders, stroma cells or serum, all of which can interfere with experimental outcomes and could preclude future clinical applications. Results This study reveals, for the first time, that activin/TGF-β signalling blocks pancreatic specification induced by retinoic acid while promoting hepatic specification in combination with bone morphogenetic protein and fibroblast growth factor. Using this knowledge, we developed culture systems to differentiate human pluripotent stem cells into near homogenous population of pancreatic and hepatic progenitors displaying functional characteristics specific to their natural counterparts. Finally, functional experiments showed that activin/TGF-β signalling achieves this essential function by controlling the levels of transcription factors necessary for liver and pancreatic development, such as HEX and HLXB9. Conclusion/interpretation Our methods of differentiation provide an advantageous system to model early human endoderm development in vitro, and also represent an important step towards the generation of pancreatic and hepatic cells for clinical applications.