Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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.

Regeneration of the testis from pluripotent stem cells is a real challenge, reflecting the complexity of the interaction of germ cells and somatic cells. Here we report the generation of testicular somatic cell-like cells (TesLCs) including Sertoli cell-like cells (SCLCs) from mouse embryonic stem cells (ESCs) in xeno-free culture. We find that Nr5a1/SF1 is critical for interaction between SCLCs and PGCLCs. Intriguingly, co-culture of TesLCs with epiblast-like cells (EpiLCs), rather than PGCLCs, results in self-organised aggregates, or testicular organoids. In the organoid, EpiLCs differentiate into PGCLCs or gonocyte-like cells that are enclosed within a seminiferous tubule-like structure composed of SCLCs. Furthermore, conditioned medium prepared from TesLCs has a robust inducible activity to differentiate EpiLCs into PGCLCs. Our results demonstrate conditions for in vitro reconstitution of a testicular environment from ESCs and provide further insights into the generation of sperm entirely in xeno-free culture.

Abstract A nanotechnology-based sealing fluid was developed to solve compromised integrity in pathways too small, smaller than 120 μm, for conventional methods such as cement squeezes. Well integrity and environmental stewardship are at the forefront of our industry's relation with the public as oil and gas fields continue to encroach toward urban centers and the places we live and work. This push towards improved well integrity and a growing number of mature wells requires new and novel technologies and materials to achieve our goals. The nanotechnology-based sealing fluid is capable of penetrating small gaps, as small as 20 μm, and seals through a reaction from either set cement or brine in the leak path. Candidate wells were selected based on very low injectivity rates that conventional remediation techniques could not tackle. Six candidate wells were selected, and of them, two were selected to cure a leaking gas microannulus causing sustained casing pressure and four were selected to cure a pinhole leak in the casing to pass the Texas Railroad Commission H-5 pressure test. For the pinhole leak, the nanosealant was placed across the leak point, and pressure was applied at surface continually until leakoff was minimized. The leaking gas microannulus was squeezed from surface until the leakoff was eliminated. The nanotechnology-based sealing fluid was successful in each case. For the leaking microannulus wells, hesitation squeeze schedules were applied, and both leaks were sealed with a projected penetration greater than 500 ft. These wells were then tested with light detection and ranging (LIDAR) to ensure no gas leaks were occurring at surface after the treatment. For the candidates selected with casing leaks, all passed the regulatory Texas Railroad Commission H-5 pressure test and were put back into service. Three of the wells resulted in a final pressure drop of 0 psi/min based on the hesitation squeezes, and the other one well resulted in a pressure bleedoff reduction more than 25 times the original bleedoff rate. The activation mechanism based on contact with annular materials is a significant breakthrough in squeeze operations because it removes all complexity from the fluid design, which historically required extensive laboratory testing. It also removes time-based boundaries on placement, and ultimately, it eliminates or reduces the drillout time compared to conventional or resin applications. Conventional remedial placement techniques can be used with the sealant, thus further simplifying the job execution. This combination of simplified execution techniques and the lack of necessary laboratory testing and well condition input allows for a quick fit-for-purpose implementation as problems arise, which saves both time and money.

Type 2 diabetes is a widespread metabolic disorder that is on the rise worldwide. Type 2 diabetes is a significant economic burden and increases the frequency of other diseases, such as hypertension and stroke. Reduced secretion of the hormone insulin by pancreatic beta cells, and resistance of the body to insulin contribute to the development of the disease. Insulin secretion can be influenced by circulating mediators, such as hormones, many of which act though G protein‐coupled receptors (GPCR). GPCRs are the largest receptor family in the human genome and are the targets for >30% of approved drugs. GPR75, an orphan GPCR, has been proposed to regulate insulin secretion, thus we aimed to determine its expression in pancreatic beta cells and changes with type 2 diabetes. Using real‐time PCR we found GPR75 is expressed in primary human pancreatic beta cells and the pancreas from control C57Bl/6 mice. Immunofluorescence staining of MIN‐6 cells (a pancreatic beta cell line) showed GPR75 expression on the cell membrane and punctate intracellular staining (indicative of constitutive activity). We found GPR75 to be upregulated in diabetic mice, either with a spontaneous knock‐out of the leptin gene or in LDL receptor knock‐out mice. Our data show expression of GPR75 in pancreatic beta cells and increased expression with diabetes. Uncovering the function of GPR75 in pancreatic beta cells may advance the understanding of type 2 diabetes and even uncover novel treatments for the disease.Support or Funding InformationWellcome Trust

Type 2 diabetes (T2DM) is associated with pancreatic islet dysfunction. Loss of beta-cell identity has been implicated via dedifferentiation or conversion to other pancreatic endocrine cell types. How these transitions contribute to the onset and progression of T2DM in vivo is unknown. The aims of this study were to determine the degree of epithelial-to-mesenchymal transition occurring in alpha and beta cells in vivo and to relate this to diabetes-associated (patho) physiological conditions. The proportion of islet cells expressing the mesenchymal marker vimentin was determined by immunohistochemistry and quantitative morphometry in specimens of pancreas from human donors with T2DM (n = 28) and without diabetes (ND, n = 38) and in non-human primates at different stages of the diabetic syndrome: normoglycaemic (ND, n = 4), obese, hyperinsulinaemic (HI, n = 4) and hyperglycaemic (DM, n = 8). Vimentin co-localised more frequently with glucagon (alpha-cells) than with insulin (beta-cells) in the human ND group (1.43% total alpha-cells, 0.98% total beta-cells, median; P < 0.05); these proportions were higher in T2DM than ND (median 4.53% alpha-, 2.53% beta-cells; P < 0.05). Vimentin-positive beta-cells were not apoptotic, had reduced expression of Nkx6.1 and Pdx1, and were not associated with islet amyloidosis or with bihormonal expression (insulin + glucagon). In non-human primates, vimentin-positive beta-cell proportion was larger in the diabetic than the ND group (6.85 vs 0.50%, medians respectively, P < 0.05), but was similar in ND and HI groups. In conclusion, islet cell expression of vimentin indicates a degree of plasticity and dedifferentiation with potential loss of cellular identity in diabetes. This could contribute to alpha-and beta-cell dysfunction in T2DM.

Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.

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.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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.

Abstract Drilling fluid removal by spacer fluids is an important step in ensuring proper cementing and therefore adequate zonal isolation, especially when non-aqueous fluids (NAF) are used. In addition to the fluid mechanics aspect of displacement, a tensioactive package containing surfactant(s) and solvent(s) is typically added to the spacer base fluid to clean all surfaces, including the casing. However, current surfactants and solvents on the market have limited application in terms of temperature and type of drilling fluids. API and ISO standards recommend practices for evaluating the suitability of a spacer. The viscosity of NAF, spacer, and cement mixtures at various fixed ratios determines their rheological compatibility. The reverse-emulsion test or spacer-surfactant screening test (SSST) determines the percentage of spacer for inverting the NAF emulsion. Other non-standardized tests such as bottle tests or grid tests determine the efficiency of a surfactant/solvent aqueous solution to remove NAF from metallic or glass surfaces. But all these tests suffer from lack of reproducibility and limited automation. Improved alternative laboratory procedures have already been proposed. They include measuring rheology during SSST, which clearly shows the positive impact of early emulsion inversion on viscosity of NAF/spacer mixtures. In addition, proper preparation of metal surfaces clearly improves repeatability of the cleaning test, which now is performed with weighted spacer under temperature and pressure. Using these improved experimental methods, we performed more than 3,000 different tests on more than 200 tensioactive blends. Statistical analysis helped in selecting the optimum chemistry as a function of the conditions (type of base oil, salinity, and temperature). This allowed developing both guidelines and a tensioactive package comprising a limited number of chemicals (surfactants and solvents), from which the field user would select the ones to combine for the application. Only a few confirmation tests would be necessary at the location for planning a given cement operation. An advantage of developing a package of chemicals is ease of implementation at field level since it requires having only a few chemicals in inventory to cover all situations and limited local laboratory testing. The new methods have been successfully applied in several wells in which different types of nonaqueous fluids were used at various temperatures.

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.