Maturation of beta cells : lessons from in vivo and in vitro models

The ability to maintain normoglycaemia, through glucose-sensitive insulin release, is a key aspect of postnatal beta cell function. However, terminally differentiated beta cell identity does not necessarily imply functional maturity. Beta cell maturation is therefore a continuation of beta cell development, albeit a process that occurs postnatally in mammals. Although many important features have been identified in the study of beta cell maturation, as of yet no unified mechanistic model of beta cell functional maturity exists. Here, we review recent findings about the underlying mechanisms of beta cell functional maturation. These findings include systemic hormonal and nutritional triggers that operate through energy-sensing machinery shifts within beta cells, resulting in primed metabolic states that allow for appropriate glucose trafficking and, ultimately, insulin release. We also draw attention to the expansive synergistic nature of these pathways and emphasise that beta cell maturation is dependent on overlapping regulatory and metabolic networks.Peer reviewe

Protective actions of des-acylated ghrelin on brain injury and blood-brain barrier disruption after stroke in mice

The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, emerging evidence indicates that these peptides may have other functions including neuro- and vaso-protection. Here, we investigated whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin could improve functional and histological endpoints of stroke outcome in mice after transient middle cerebral artery occlusion (tMCAo). We found that des-acylated ghrelin (1 mg/kg) improved neurological and functional performance, reduced infarct and swelling, and decreased apoptosis. In addition, it reduced blood-brain barrier (BBB) disruption in vivo and attenuated the hyper-permeability of mouse cerebral microvascular endothelial cells after oxygen glucose deprivation and reoxygenation (OGD + RO). By contrast, acylated ghrelin (1 mg/kg or 5 mg/kg) had no significant effect on these endpoints of stroke outcome. Next we found that des-acylated ghrelin's vasoprotective actions were associated with increased expression of tight junction proteins (occludin and claudin-5), and decreased cell death. Moreover, it attenuated superoxide production, Nox activity and expression of 3-nitrotyrosine. Collectively, these results demonstrate that post-stroke treatment with des-acylated ghrelin, but not acylated ghrelin, protects against ischaemia/reperfusion-induced brain injury and swelling, and BBB disruption, by reducing oxidative and/or nitrosative damage

Differentiating functional human islet-like aggregates from pluripotent stem cells

Publisher Copyright: © 2022 The Author(s)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).Peer reviewe

The type 1 diabetes gene TYK2 regulates beta-cell development and its responses to interferon-alpha

The TYK2 gene is associated with development of type 1 diabetes. Here the authors show that TYK2 regulates beta-cell development, but at the same time TYK2 inhibition in the islets prevents IFN alpha responses and enhances their survival against CD8(+) T-cell cytotoxicity; representing a potent therapeutic target to halt T1D progression. Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin producing pancreatic beta-cells. One of the genes associated with T1D is TYK2, which encodes a Janus kinase with critical roles in type-Iota interferon (IFN-Iota) mediated intracellular signalling. To study the role of TYK2 in beta-cell development and response to IFN alpha, 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 IFN alpha-induced antigen processing and presentation, including MHC Class Iota and Class Iota Iota expression, enhancing their survival against CD8(+) T-cell cytotoxicity. These results identify an unsuspected role for TYK2 in beta-cell development and support TYK2 inhibition in adult beta-cells as a potent therapeutic target to halt T1D progression.Peer reviewe

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. Pancreatic islets derived from stem cells are benchmarked against primary cells.Peer reviewe

Conserved Genes Act as Modifiers of Invertebrate SMN Loss of Function Defects

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species

Biofabrication of Three-Dimensional Neural Tissue Constructs from Human Induced Pluripotent Stem Cells

The human brain is arguably one of the most complex structures known to humankind. To understand the development, function, and dysfunction of this organ, both historical and modern neurological research methods have intrinsic limitations. Modern functional imaging and electrophysiological techniques obtain important data on the structural and functional aspects of specific brain regions but are unable to accumulate biomolecular information, whereas animal models of brain development allow for complete molecular interrogation, but show intrinsic morphological differences compared to native human brain tissue. In vitro modelling of human brain tissue through the differentiation of induced pluripotent stem cells (iPSCs) offers a completely novel method of studying human brain development. iPSCs can differentiate into any somatic cell derived from the three germ layers, and as such, can form cells of specific neural lineages from multiple brain regions. Furthermore, this differentiation in vitro follows the same principles of in utero brain development, and so can be used to model specific genetically-linked neurodevelopmental pathologies such as the epilepsies. Neural cell differentiation within three-dimensional cell culture scaffolds offers a way of replicating a more in vivo-like microenvironment compared to standard planar culture. Therefore, the generation and optimisation of cytocompatible biomaterials to form these three-dimensional scaffolds will be integral for the future of in vitro neural tissue engineering. This thesis focuses on the combination of both biomaterial engineering and iPSCbased neurological development to assess the synergy of targeted neuronal cell differentiation within three-dimensional hydrogel environments

Scleraxis Is Essential for Tendon Differentiation by Equine Embryonic Stem Cells and in Equine Fetal Tenocytes

The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes

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

Aims/hypothesis Congenital hyperinsulinism caused by mutations in the K-ATP-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 ABCC8(V187D) mutation, which inactivates the sulfonylurea receptor 1 (SUR1) subunit of the K-ATP-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 K-ATP-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 K-ATP-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 K-ATP-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.Peer reviewe