Non-invasive quantification of stem cell-derived islet graft size and composition.

AIMS/HYPOTHESIS: Stem cell-derived islets (SC-islets) are being used as cell replacement therapy for insulin-dependent diabetes. Non-invasive long-term monitoring methods for SC-islet grafts, which are needed to detect misguided differentiation in vivo and to optimise their therapeutic effectiveness, are lacking. Positron emission tomography (PET) has been used to monitor transplanted primary islets. We therefore aimed to apply PET as a non-invasive monitoring method for SC-islet grafts. METHODS: We implanted different doses of human SC-islets, SC-islets derived using an older protocol or a state-of-the-art protocol and SC-islets genetically rendered hyper- or hypoactive into mouse calf muscle to yield different kinds of grafts. We followed the grafts with PET using two tracers, glucagon-like peptide 1 receptor-binding [18F]F-dibenzocyclooctyne-exendin-4 ([18F]exendin) and the dopamine precursor 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA), for 5 months, followed by histological assessment of graft size and composition. Additionally, we implanted a kidney subcapsular cohort with different SC-islet doses to assess the connection between C-peptide and stem cell-derived beta cell (SC-beta cell) mass. RESULTS: Small but pure and large but impure grafts were derived from SC-islets. PET imaging allowed detection of SC-islet grafts even <1 mm3 in size, [18F]exendin having a better detection rate than [18F]FDOPA (69% vs 44%, <1 mm3; 96% vs 85%, >1 mm3). Graft volume quantified with [18F]exendin (r2=0.91) and [18F]FDOPA (r2=0.86) strongly correlated with actual graft volume. [18F]exendin PET delineated large cystic structures and its uptake correlated with graft SC-beta cell proportion (r2=0.68). The performance of neither tracer was affected by SC-islet graft hyper- or hypoactivity. C-peptide measurements under fasted or glucose-stimulated conditions did not correlate with SC-islet graft volume or SC-beta cell mass, with C-peptide under hypoglycaemia having a weak correlation with SC-beta cell mass (r2=0.52). CONCLUSIONS/INTERPRETATION: [18F]exendin and [18F]FDOPA PET enable non-invasive assessment of SC-islet graft size and aspects of graft composition. These methods could be leveraged for optimising SC-islet cell replacement therapy in diabetes.

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function.

AIMS/HYPOTHESIS: Regulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown. METHODS: To understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice. RESULTS: Stem cell models of the homozygous variant RFX6-/- predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell-Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/- SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes. CONCLUSIONS/INTERPRETATION: Our allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes. DATA AVAILABILITY: Ultra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley ( https://data.mendeley.com/datasets/g75drr3mgw/2 ).

Aberrant metabolite trafficking and fuel sensitivity in human pluripotent stem cell-derived islets.

Pancreatic islets regulate blood glucose homeostasis through the controlled release of insulin; however, current metabolic models of glucose-sensitive insulin secretion are incomplete. A comprehensive understanding of islet metabolism is integral to studies of endocrine cell development as well as diabetic islet dysfunction. Human pluripotent stem cell-derived islets (SC-islets) are a developmentally relevant model of human islet function that have great potential in providing a cure for type 1 diabetes. Using multiple 13C-labeled metabolic fuels, we demonstrate that SC-islets show numerous divergent patterns of metabolite trafficking in proposed insulin release pathways compared with primary human islets but are still reliant on mitochondrial aerobic metabolism to derive function. Furthermore, reductive tricarboxylic acid cycle activity and glycolytic metabolite cycling occur in SC-islets, suggesting that non-canonical coupling factors are also present. In aggregate, we show that many facets of SC-islet metabolism overlap with those of primary islets, albeit with a retained immature signature.

The type 1 diabetes gene TYK2 regulates ß-cell development and its responses to interferon-α.

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic ß-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Ι interferon (IFN-Ι) mediated intracellular signalling. To study the role of TYK2 in ß-cell development and response to IFNα, we generated TYK2 knockout human iPSCs and directed them into the pancreatic endocrine lineage. Here we show that loss of TYK2 compromises the emergence of endocrine precursors by regulating KRAS expression, while mature stem cell-islets (SC-islets) function is not affected. In the SC-islets, the loss or inhibition of TYK2 prevents IFNα-induced antigen processing and presentation, including MHC Class Ι and Class ΙΙ expression, enhancing their survival against CD8+ T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in ß-cell development and support TYK2 inhibition in adult ß-cells as a potent therapeutic target to halt T1D progression.

Differentiating functional human islet-like aggregates from pluripotent stem cells.

We present here a robust and reliable protocol by which to differentiate pancreatic islet-like aggregates (SC-islets) from human pluripotent stem cells. The 7-stage protocol mimics developmental patterning factors that induce endocrine lineage formation and spans monolayer, microwell, and aggregate suspension culture. The SC-islets demonstrate dynamic glucose-sensitive insulin secretion and an endocrine cell composition similar to those of primary human islets. SC-islets generated using this optimized protocol are suitable for in vitro modeling of islet cell pathophysiology and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Balboa et al. (2022).

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Stem Cell Based Models in Congenital Hyperinsulinism - Perspective on Practicalities and Possibilities.

Congenital hyperinsulinism (CHI) is a severe inherited neonatal disorder characterized by inappropriate insulin secretion caused by genetic defects of the pancreatic beta cells. Several open questions remain in CHI research, such as the optimal treatment for the most common type of CHI, caused by mutations in the genes encoding ATP-sensitive potassium channels, and the molecular mechanisms of newly identified CHI genes. Answering these questions requires robust preclinical models, particularly since primary patient material is extremely scarce and accurate animal models are not available. In this short review, we explain why pluripotent stem cell derived islets present an attractive solution to these issues and outline the current progress in stem-cell based modeling of CHI. Stem cell derived islets enable the study of molecular mechanisms of CHI and the discovery of novel antihypoglycemic drugs, while also providing a valuable model to study the biology of variable functional states of beta cells.

Loss of MANF Causes Childhood-Onset Syndromic Diabetes Due to Increased Endoplasmic Reticulum Stress.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-resident protein that plays a crucial role in attenuating ER stress responses. Although MANF is indispensable for the survival and function of mouse ß-cells, its precise role in human ß-cell development and function is unknown. In this study, we show that lack of MANF in humans results in diabetes due to increased ER stress, leading to impaired ß-cell function. We identified two patients from different families with childhood diabetes and a neurodevelopmental disorder associated with homozygous loss-of-function mutations in the MANF gene. To study the role of MANF in human ß-cell development and function, we knocked out the MANF gene in human embryonic stem cells and differentiated them into pancreatic endocrine cells. Loss of MANF induced mild ER stress and impaired insulin-processing capacity of ß-cells in vitro. Upon implantation to immunocompromised mice, the MANF knockout grafts presented elevated ER stress and functional failure, particularly in recipients with diabetes. By describing a new form of monogenic neurodevelopmental diabetes syndrome caused by disturbed ER function, we highlight the importance of adequate ER stress regulation for proper human ß-cell function and demonstrate the crucial role of MANF in this process.

SUR1-mutant iPS cell-derived islets recapitulate the pathophysiology of congenital hyperinsulinism.

AIMS/HYPOTHESIS: Congenital hyperinsulinism caused by mutations in the KATP-channel-encoding genes (KATPHI) is a potentially life-threatening disorder of the pancreatic beta cells. No optimal medical treatment is available for patients with diazoxide-unresponsive diffuse KATPHI. Therefore, we aimed to create a model of KATPHI using patient induced pluripotent stem cell (iPSC)-derived islets. METHODS: We derived iPSCs from a patient carrying a homozygous ABCC8V187D mutation, which inactivates the sulfonylurea receptor 1 (SUR1) subunit of the KATP-channel. CRISPR-Cas9 mutation-corrected iPSCs were used as controls. Both were differentiated to stem cell-derived islet-like clusters (SC-islets) and implanted into NOD-SCID gamma mice. RESULTS: SUR1-mutant and -corrected iPSC lines both differentiated towards the endocrine lineage, but SUR1-mutant stem cells generated 32% more beta-like cells (SC-beta cells) (64.6% vs 49.0%, p = 0.02) and 26% fewer alpha-like cells (16.1% vs 21.8% p = 0.01). SUR1-mutant SC-beta cells were 61% more proliferative (1.23% vs 0.76%, p = 0.006), and this phenotype could be induced in SUR1-corrected cells with pharmacological KATP-channel inactivation. The SUR1-mutant SC-islets secreted 3.2-fold more insulin in low glucose conditions (0.0174% vs 0.0054%/min, p = 0.0021) and did not respond to KATP-channel-acting drugs in vitro. Mice carrying grafts of SUR1-mutant SC-islets presented with 38% lower fasting blood glucose (4.8 vs 7.7 mmol/l, p = 0.009) and their grafts failed to efficiently shut down insulin secretion during induced hypoglycaemia. Explanted SUR1-mutant grafts displayed an increase in SC-beta cell proportion and SC-beta cell nucleomegaly, which was independent of proliferation. CONCLUSIONS/INTERPRETATION: We have created a model recapitulating the known pathophysiology of KATPHI both in vitro and in vivo. We have also identified a novel role for KATP-channel activity during human islet development. This model will enable further studies for the improved understanding and clinical management of KATPHI without the need for primary patient tissue.

YIPF5 mutations cause neonatal diabetes and microcephaly through endoplasmic reticulum stress.

Neonatal diabetes is caused by single gene mutations reducing pancreatic ß cell number or impairing ß cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in ß cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human ß cell models (YIPF5 silencing in EndoC-ßH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects ß cells. Loss of YIPF5 function in stem cell-derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and ß cell failure. Partial YIPF5 silencing in EndoC-ßH1 cells and a patient mutation in stem cells increased the ß cell sensitivity to ER stress-induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in ß cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes.

Epidemiology of two human protoparvoviruses, bufavirus and tusavirus.

Two human parvoviruses were recently discovered by metagenomics in Africa, bufavirus (BuV) in 2012 and tusavirus (TuV) in 2014. These viruses have been studied exclusively by PCR in stool and detected only in patients with diarrhoea, although at low prevalence. Three genotypes of BuV have been identified. We detected, by in-house EIA, BuV1-3 IgG antibodies in 7/228 children (3.1%) and 10/180 adults (5.6%), whereas TuV IgG was found in one child (0.4%). All children and 91% of the adults were Finnish, yet interestingly 3/6 adults of Indian origin were BuV-IgG positive. By competition EIA, no cross-reactivity between the BuVs was detected, indicating that the BuV genotypes represent distinct serotypes. Furthermore, we analysed by BuV qPCR stool and nasal swab samples from 955 children with gastroenteritis, respiratory illness, or both, and found BuV DNA in three stools (0.3%) and for the first time in a nasal swab (0.1%). This is the first study documenting the presence of BuV and TuV antibodies in humans. Although the seroprevalences of both viruses were low in Finland, our results indicate that BuV infections might be widespread in Asia. The BuV-specific humoral immune responses appeared to be strong and long-lasting, pointing to systemic infection in humans.