iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment.

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

Modulatory role of RNA helicases in MBNL-dependent alternative splicing regulation.

Muscleblind-like splicing regulators (MBNLs) activate or repress the inclusion of alternative splicing (AS) events, enabling the developmental transition of fetal mRNA splicing isoforms to their adult forms. Herein, we sought to elaborate the mechanism by which MBNLs mediate AS related to biological processes. We evaluated the functional role of DEAD-box (DDX) RNA helicases, DDX5 and DDX17 in MBNL-dependent AS regulation. Whole-transcriptome analysis and validation approaches revealed a handful of MBNLs-dependent AS events to be affected by DDX5 and DDX17 in mostly an opposite manner. The opposite expression patterns of these two groups of factors during muscle development and coordination of fetal-to-adult splicing transition indicate the importance of these proteins at early stages of development. The identified pathways of how the helicases modulate MBNL splicing activity include DDX5 and DDX17-dependent changes in the ratio of MBNL splicing isoforms and most likely changes in accessibility of MBNL-binding sites. Another pathway involves the mode of action of the helicases independent of MBNL activity. These findings lead to a deeper understanding of the network of interdependencies between RNA-binding proteins and constitute a valuable element in the discussion on developmental homeostasis and pathological states in which the studied protein factors play a significant role.

Deep into the niche: Deciphering local endoderm-microenvironment interactions in development, homeostasis, and disease of pancreas and intestine.

Unraveling molecular and functional heterogeneity of niche cells within the developing endoderm could resolve mechanisms of tissue formation and maturation. Here, we discuss current unknowns in molecular mechanisms underlying key developmental events in pancreatic islet and intestinal epithelial formation. Recent breakthroughs in single-cell and spatial transcriptomics, paralleled with functional studies in vitro, reveal that specialized mesenchymal subtypes drive the formation and maturation of pancreatic endocrine cells and islets via local interactions with epithelium, neurons, and microvessels. Analogous to this, distinct intestinal niche cells regulate both epithelial development and homeostasis throughout life. We propose how this knowledge can be used to progress research in the human context using pluripotent stem cell-derived multilineage organoids. Overall, understanding the interactions between the multitude of microenvironmental cells and how they drive tissue development and function could help us make more therapeutically relevant in vitro models.

FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks.

The natural product jasplakinolide is widely used to stabilize F-actin. Based on extensive structure-activity relationship studies, we have developed a new generation of photoswitchable jasplakinolides that feature rationally designed red-shifted azobenzene photoswitches. Our lead compound, nOJ, can be activated with longer wavelengths in the visible range (e.g. 440-475 nm) and rapidly returns to its inactive state through thermal relaxation. nOJ enables the reversible control of F-actin dynamics, as shown through live-cell imaging, cell migration, and cell proliferation assays. Short, local irradiation with blue light resulted in highly localized and reversible actin aggregation with subcellular precision. Our optical tool can be useful in diverse fields to study actin dynamics with excellent spatiotemporal resolution.

SARS-CoV-2 infects an in vitro model of the human developing pancreas through endocytosis.

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic ß-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences.

Diabetes and SARS-CoV-2-Is There a Mutual Connection?

SARS-CoV-2, a newly emerged virus described for the first time in late 2019, affects multiple organs in humans, including the pancreas. Here, we present the bilateral link between the pathophysiology of diabetes and COVID-19, with diabetes being COVID-19 comorbidity, and a complication of SARS-CoV-2 infection. Analysis of clinical data indicates that patients with chronic conditions like diabetes are at increased risk of severe COVID-19, hospitalization, ICU admission, and death compared to the healthy subjects. Further, we show that SARS-CoV-2 infection might be also associated with the development of new-onset diabetes and diabetic ketoacidosis. We then discuss the options for studying SARS-CoV-2 infection in pancreatic settings, including the use of human pluripotent stem cell-derived pancreatic organoids. Further, we review the presence of SARS-CoV-2 receptors in different pancreatic cell types and the infection efficiency based on pancreatic sections from COVID-19 patients and primary human islet in vitro studies. Finally, we discuss the impact of SARS-CoV-2 infection on human pancreatic cell homeostasis, focusing on ß-cells.

SMER28 Attenuates PI3K/mTOR Signaling by Direct Inhibition of PI3K p110 Delta.

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here, we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and, consequently, growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction in phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ and to a lesser extent p110γ. The biological relevance of our observations was underscored by SMER28 interfering with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor tyrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited. Lastly, in B cell lymphoma cells, which predominantly depend on tonic signaling through PI3Kδ, apoptosis upon SMER28 treatment is profound in comparison to non-hematopoietic cells. This indicates SMER28 as a possible drug candidate for the treatment of diseases that derive from aberrant PI3Kδ activity.

A dormant T-cell population with autoimmune potential exhibits low self-reactivity and infiltrates islets in type 1 diabetes.

The contribution of low-affinity T cells to autoimmunity in the context of polyclonal T-cell responses is understudied due to the limitations in their capture by tetrameric reagents and low level of activation in response to antigenic stimulation. As a result, low-affinity T cells are often disregarded as nonantigen-specific cells irrelevant to the immune response. Our study aimed to assess how the level of self-antigen reactivity shapes T-cell lineage and effector responses in the context of spontaneous tissue-specific autoimmunity observed in NOD mice. Using multicolor flow cytometry in combination with Nur77GFP reporter of TCR signaling, we identified a dormant population of T cells that infiltrated the pancreatic islets of prediabetic NOD mice, which exhibited reduced levels of self-tissue reactivity based on expression of CD5 and Nur77GFP . We showed that these CD5low T cells had a unique TCR repertoire and exhibited low activation and minimal effector function; however, induced rapid diabetes upon transfer. The CD4+ CD5low T-cell population displayed transcriptional signature of central memory T cells, consistent with the ability to acquire effector function post-transfer. Transcriptional profile of CD5low T cells was similar to T cells expressing a low-affinity TCR, indicating TCR affinity to be an important factor in shaping CD5low T-cell phenotype and function at the tissue site. Overall, our study suggests that autoimmune tissue can maintain a reservoir of undifferentiated central memory-like autoreactive T cells with pathogenic effector potential that might be an important source for effector T cells during long-term chronic autoimmunity.

Human pancreatic microenvironment promotes ß-cell differentiation via non-canonical WNT5A/JNK and BMP signaling.

In vitro derivation of pancreatic ß-cells from human pluripotent stem cells holds promise as diabetes treatment. Despite recent progress, efforts to generate physiologically competent ß-cells are still hindered by incomplete understanding of the microenvironment's role in ß-cell development and maturation. Here, we analyze the human mesenchymal and endothelial primary cells from weeks 9-20 fetal pancreas and identify a time point-specific microenvironment that permits ß-cell differentiation. Further, we uncover unique factors that guide in vitro development of endocrine progenitors, with WNT5A markedly improving human ß-cell differentiation. WNT5A initially acts through the non-canonical (JNK/c-JUN) WNT signaling and cooperates with Gremlin1 to inhibit the BMP pathway during ß-cell maturation. Interestingly, we also identify the endothelial-derived Endocan as a SST+ cell promoting factor. Overall, our study shows that the pancreatic microenvironment-derived factors can mimic in vivo conditions in an in vitro system to generate bona fide ß-cells for translational applications.

Pancreatic Differentiation of Stem Cells Reveals Pathogenesis of a Syndrome of Ketosis-Prone Diabetes.

Genetic analysis of an adult patient with an unusual course of ketosis-prone diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the 5'-untranslated region (UTR) of PDX1, a ß-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm (DE) stage. Metabolomics analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the DE to the pancreatic progenitor (PP) stage, and RNA sequencing showed that defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR/Cas9 manipulation of the PDX1 variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the PDX1 5'-UTR, leading to decreased PDX1 expression at the early PP stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical ß-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset ß-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multiomics analysis of patient-specific stem cells for clinical discovery.

Endocrine Pancreas Development and Dysfunction Through the Lens of Single-Cell RNA-Sequencing.

A chronic inability to maintain blood glucose homeostasis leads to diabetes, which can damage multiple organs. The pancreatic islets regulate blood glucose levels through the coordinated action of islet cell-secreted hormones, with the insulin released by ß-cells playing a crucial role in this process. Diabetes is caused by insufficient insulin secretion due to ß-cell loss, or a pancreatic dysfunction. The restoration of a functional ß-cell mass might, therefore, offer a cure. To this end, major efforts are underway to generate human ß-cells de novo, in vitro, or in vivo. The efficient generation of functional ß-cells requires a comprehensive knowledge of pancreas development, including the mechanisms driving cell fate decisions or endocrine cell maturation. Rapid progress in single-cell RNA sequencing (scRNA-Seq) technologies has brought a new dimension to pancreas development research. These methods can capture the transcriptomes of thousands of individual cells, including rare cell types, subtypes, and transient states. With such massive datasets, it is possible to infer the developmental trajectories of cell transitions and gene regulatory pathways. Here, we summarize recent advances in our understanding of endocrine pancreas development and function from scRNA-Seq studies on developing and adult pancreas and human endocrine differentiation models. We also discuss recent scRNA-Seq findings for the pathological pancreas in diabetes, and their implications for better treatment.

A human liver chimeric mouse model for non-alcoholic fatty liver disease.

BACKGROUND & AIMS: The accumulation of neutral lipids within hepatocytes underlies non-alcoholic fatty liver disease (NAFLD), which affects a quarter of the world's population and is associated with hepatitis, cirrhosis, and hepatocellular carcinoma. Despite insights gained from both human and animal studies, our understanding of NAFLD pathogenesis remains limited. To better study the molecular changes driving the condition we aimed to generate a humanised NAFLD mouse model. METHODS: We generated TIRF (transgene-free Il2rg -/-/Rag2 -/-/Fah -/-) mice, populated their livers with human hepatocytes, and fed them a Western-type diet for 12 weeks. RESULTS: Within the same chimeric liver, human hepatocytes developed pronounced steatosis whereas murine hepatocytes remained normal. Unbiased metabolomics and lipidomics revealed signatures of clinical NAFLD. Transcriptomic analyses showed that molecular responses diverged sharply between murine and human hepatocytes, demonstrating stark species differences in liver function. Regulatory network analysis indicated close agreement between our model and clinical NAFLD with respect to transcriptional control of cholesterol biosynthesis. CONCLUSIONS: These NAFLD xenograft mice reveal an unexpected degree of evolutionary divergence in food metabolism and offer a physiologically relevant, experimentally tractable model for studying the pathogenic changes invoked by steatosis. LAY SUMMARY: Fatty liver disease is an emerging health problem, and as there are no good experimental animal models, our understanding of the condition is poor. We here describe a novel humanised mouse system and compare it with clinical data. The results reveal that the human cells in the mouse liver develop fatty liver disease upon a Western-style fatty diet, whereas the mouse cells appear normal. The molecular signature (expression profiles) of the human cells are distinct from the mouse cells and metabolic analysis of the humanised livers mimic the ones observed in humans with fatty liver. This novel humanised mouse system can be used to study human fatty liver disease.

Mitogen Synergy: An Emerging Route to Boosting Human Beta Cell Proliferation.

Decreased number and function of beta cells are a key aspect of diabetes mellitus (diabetes), a disease that remains an onerous global health problem. Means of restoring beta cell mass are urgently being sought as a potential cure for diabetes. Several strategies, such as de novo beta cell derivation via pluripotent stem cell differentiation or mature somatic cell transdifferentiation, have yielded promising results. Beta cell expansion is another promising strategy, rendered challenging by the very low proliferative capacity of beta cells. Many effective mitogens have been identified in rodents, but the vast majority do not have similar mitogenic effects in human beta cells. Extensive research has led to the identification of several human beta cell mitogens, but their efficacy and specificity remain insufficient. An approach based on the simultaneous application of several mitogens has recently emerged and can yield human beta cell proliferation rates of up to 8%. Here, we discuss recent advances in restoration of the beta cell population, focusing on mitogen synergy, and the contribution of RNA-sequencing (RNA-seq) to accelerating the elucidation of signaling pathways in proliferating beta cells and the discovery of novel mitogens. Together, these approaches have taken beta cell research up a level, bringing us closer to a cure for diabetes.

Human NK cell deficiency as a result of biallelic mutations in MCM10.

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Optical Manipulation of F-Actin with Photoswitchable Small Molecules.

Cell-permeable photoswitchable small molecules, termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resolution that light affords. Optojasps can be expected to find applications in diverse areas of cell biological research. They may also provide a template for photopharmacology targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.

Two drugs converged in a pancreatic ß cell.

Combining a DYRK1A inhibitor and GLP-1 receptor agonist boosts human pancreatic ß cell proliferation and glucose homeostasis in vivo (Ackeifi et al., this issue).

A Critical Insulin TCR Contact Residue Selects High-Affinity and Pathogenic Insulin-Specific T Cells.

Type 1 diabetes is an autoimmune-mediated disease that culminates in the targeted destruction of insulin-producing ß-cells. CD4 responses in NOD mice are dominated by insulin epitope B:9-23 (InsB9-23) specificity, and mutation of the key T-cell receptor (TCR) contact residue within the epitope prevents diabetes development. However, it is not clear how insulin self-antigen controls the selection of autoimmune and regulatory T cells (Tregs). Here we demonstrate that mutation of insulin epitope results in escape of highly pathogenic T cells. We observe an increase in antigen reactivity, clonality, and pathogenicity of insulin-specific T cells that develop in the absence of cognate antigen. Using a single TCR system, we demonstrate that Treg development is greatly diminished in mice with the Y16A mutant epitope. Collectively, these results suggest that the tyrosine residue at position 16 is necessary to constrain TCR reactivity for InsB9-23 by both limiting the development of pathogenic T cells and supporting the selection of Tregs.

Pancreatic Cell Fate Determination Relies on Notch Ligand Trafficking by NFIA.

Notch is activated globally in pancreatic progenitors; however, for progenitors to differentiate into endocrine cells, they must escape Notch activation to express Neurogenin-3. Here, we find that the transcription factor nuclear factor I/A (NFIA) promotes endocrine development by regulating Notch ligand Dll1 trafficking. Pancreatic deletion of NFIA leads to cell fate defects, with increased duct and decreased endocrine formation, while ectopic expression promotes endocrine formation in mice and human pancreatic progenitors. NFIA-deficient mice exhibit dysregulation of trafficking-related genes including increased expression of Mib1, which acts to target Dll1 for endocytosis. We find that NFIA binds to the Mib1 promoter, with loss of NFIA leading to an increase in Dll1 internalization and enhanced Notch activation with rescue of the cell fate defects after Mib1 knockdown. This study reveals NFIA as a pro-endocrine factor in the pancreas, acting to repress Mib1, inhibit Dll1 endocytosis and thus promote escape from Notch activation.