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.

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.

Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis.

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

Cutting Edge: Low-Affinity TCRs Support Regulatory T Cell Function in Autoimmunity.

Regulatory T cells (Tregs) use a distinct TCR repertoire and are more self-reactive compared with conventional T cells. However, the extent to which TCR affinity regulates the function of self-reactive Tregs is largely unknown. In this study, we used a two-TCR model to assess the role of TCR affinity in Treg function during autoimmunity. We observed that high- and low-affinity Tregs were recruited to the pancreas and contributed to protection from autoimmune diabetes. Interestingly, high-affinity cells preferentially upregulated the TCR-dependent Treg functional mediators IL-10, TIGIT, GITR, and CTLA4, whereas low-affinity cells displayed increased transcripts for Areg and Ebi3, suggesting distinct functional profiles. The results of this study suggest mechanistically distinct and potentially nonredundant roles for high- and low-affinity Tregs in controlling autoimmunity.

Gut bacteria-derived succinate induces enteric nervous system regeneration.

Enteric neurons control gut physiology by regulating peristalsis, nutrient absorption, and secretion 1 . Disruptions in microbial communities caused by antibiotics or enteric infections result in the loss of enteric neurons and long-term motility disorders 2-5 . However, the signals and underlying mechanisms of this microbiota-neuron communication are unknown. We studied the effects of microbiota on the recovery of the enteric nervous system after microbial dysbiosis caused by antibiotics. We found that both enteric neurons and glia are lost after antibiotic exposure, but recover when the pre-treatment microbiota is restored. Using murine gnotobiotic models and fecal metabolomics, we identified neurogenic bacterial species and their derived metabolite succinate as sufficient to rescue enteric neurons and glia. Unbiased single-nuclei RNA-seq analysis uncovered a novel neural precursor-like population marked by the expression of the neuronal gene Nav2. Genetic fate-mapping showed that Plp1+ enteric glia differentiate into neurons following antibiotic exposure. In contrast, Nav2+ neurons expand upon succinate treatment and indicate an alternative mode of neuronal regeneration under recovery conditions. Our findings highlight specific microbial species, metabolites, and the underlying cellular mechanisms involved in neuronal regeneration, with potential therapeutic implications for peripheral neuropathies.

Pervasive environmental chemicals impair oligodendrocyte development.

Exposure to environmental chemicals can impair neurodevelopment, and oligodendrocytes may be particularly vulnerable, as their development extends from gestation into adulthood. However, few environmental chemicals have been assessed for potential risks to oligodendrocytes. Here, using a high-throughput developmental screen in cultured cells, we identified environmental chemicals in two classes that disrupt oligodendrocyte development through distinct mechanisms. Quaternary compounds, ubiquitous in disinfecting agents and personal care products, were potently and selectively cytotoxic to developing oligodendrocytes, whereas organophosphate flame retardants, commonly found in household items such as furniture and electronics, prematurely arrested oligodendrocyte maturation. Chemicals from each class impaired oligodendrocyte development postnatally in mice and in a human 3D organoid model of prenatal cortical development. Analysis of epidemiological data showed that adverse neurodevelopmental outcomes were associated with childhood exposure to the top organophosphate flame retardant identified by our screen. This work identifies toxicological vulnerabilities for oligodendrocyte development and highlights the need for deeper scrutiny of these compounds' impacts on human health.

Enteric glial hub cells coordinate intestinal motility.

Enteric glia are the predominant cell type in the enteric nervous system yet their identities and roles in gastrointestinal function are not well classified. Using our optimized single nucleus RNA-sequencing method, we identified distinct molecular classes of enteric glia and defined their morphological and spatial diversity. Our findings revealed a functionally specialized biosensor subtype of enteric glia that we call "hub cells." Deletion of the mechanosensory ion channel PIEZO2 from adult enteric glial hub cells, but not other subtypes of enteric glia, led to defects in intestinal motility and gastric emptying in mice. These results provide insight into the multifaceted functions of different enteric glial cell subtypes in gut health and emphasize that therapies targeting enteric glia could advance the treatment of gastrointestinal diseases.

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.

Non-canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function.

Mammalian cells respond to insufficient oxygen through transcriptional regulators called hypoxia-inducible factors (HIFs). Although transiently protective, prolonged HIF activity drives distinct pathological responses in different tissues. Using a model of chronic HIF1a accumulation in pluripotent-stem-cell-derived oligodendrocyte progenitors (OPCs), we demonstrate that HIF1a activates non-canonical targets to impair generation of oligodendrocytes from OPCs. HIF1a activated a unique set of genes in OPCs through interaction with the OPC-specific transcription factor OLIG2. Non-canonical targets, including Ascl2 and Dlx3, were sufficient to block differentiation through suppression of the oligodendrocyte regulator Sox10. Chemical screening revealed that inhibition of MEK/ERK signaling overcame the HIF1a-mediated block in oligodendrocyte generation by restoring Sox10 expression without affecting canonical HIF1a activity. MEK/ERK inhibition also drove oligodendrocyte formation in hypoxic regions of human oligocortical spheroids. This work defines mechanisms by which HIF1a impairs oligodendrocyte formation and establishes that cell-type-specific HIF1a targets perturb cell function in response to low oxygen.

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.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology. Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

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.

Endocrine lineage biases arise in temporally distinct endocrine progenitors during pancreatic morphogenesis.

Decoding the molecular composition of individual Ngn3 + endocrine progenitors (EPs) during pancreatic morphogenesis could provide insight into the mechanisms regulating hormonal cell fate. Here, we identify population markers and extensive cellular diversity including four EP subtypes reflecting EP maturation using high-resolution single-cell RNA-sequencing of the e14.5 and e16.5 mouse pancreas. While e14.5 and e16.5 EPs are constantly born and share select genes, these EPs are overall transcriptionally distinct concomitant with changes in the underlying epithelium. As a consequence, e16.5 EPs are not the same as e14.5 EPs: e16.5 EPs have a higher propensity to form beta cells. Analysis of e14.5 and e16.5 EP chromatin states reveals temporal shifts, with enrichment of beta cell motifs in accessible regions at later stages. Finally, we provide transcriptional maps outlining the route progenitors take as they make cell fate decisions, which can be applied to advance the in vitro generation of beta cells.

High self-reactivity drives T-bet and potentiates Treg function in tissue-specific autoimmunity.

T cell receptor (TCR) affinity is a critical factor of Treg lineage commitment, but whether self-reactivity is a determining factor in peripheral Treg function remains unknown. Here, we report that a high degree of self-reactivity is crucial for tissue-specific Treg function in autoimmunity. Based on high expression of CD5, we identified a subset of self-reactive Tregs expressing elevated levels of T-bet, GITR, CTLA-4, and ICOS, which imparted significant protection from autoimmune diabetes. We observed that T-bet expression in Tregs, necessary for control of Th1 autoimmunity, could be induced in an IFNγ-independent fashion and, unlike in conventional T cells (Tconv), was strongly correlated with the strength of TCR signaling. The level of CD5 similarly identified human Tregs with an increased functional profile, suggesting that CD5hi Tregs may constitute an efficacious subpopulation appropriate for use in adoptive Treg therapies for treatment of inflammatory conditions. Overall, this work establishes an instrumental role of high TCR self-reactivity in driving Treg function.

Organotypic pancreatoids with native mesenchyme develop Insulin producing endocrine cells.

Replacement of lost beta cells in patients with diabetes has the potential to alleviate them of their disease, yet current protocols to make beta cells are inadequate for therapy. In vitro screens can reveal the signals necessary for endocrine maturation to improve beta cell production, however the complexities of in vivo development that lead to beta cell formation are lost in two-dimensional systems. Here, we create three-dimensional organotypic pancreatic cultures, named pancreatoids, composed of embryonic day 10.5 murine epithelial progenitors and native mesenchyme. These progenitors assemble in scaffold-free, floating conditions and, with the inclusion of native mesenchyme, develop into pancreatoids expressing markers of different pancreatic lineages including endocrine-like cells. Treatment of pancreatoids with (-)-Indolactam-V or phorbol 12-myristate 13-acetate, two protein kinase C activators, leads to altered morphology which otherwise would be overlooked in two-dimensional systems. Protein kinase C activation also led to fewer Insulin+ cells, decreased Ins1 and Ins2 mRNA levels, and increased Pdx1 and Hes1 mRNA levels with a high number of DBA+ cells. Thus, organotypic pancreatoids provide a useful tool for developmental studies, and can further be used for disease modeling, small molecules and genetic screens, or applied to human pluripotent stem cell differentiation for beta-like cell formation.

Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases.

Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies.

Alternative splicing regulates vesicular trafficking genes in cardiomyocytes during postnatal heart development.

During postnatal development the heart undergoes a rapid and dramatic transition to adult function through transcriptional and post-transcriptional mechanisms, including alternative splicing (AS). Here we perform deep RNA-sequencing on RNA from cardiomyocytes and cardiac fibroblasts to conduct a high-resolution analysis of transcriptome changes during postnatal mouse heart development. We reveal extensive changes in gene expression and AS that occur primarily between postnatal days 1 and 28. Cardiomyocytes and cardiac fibroblasts show reciprocal regulation of gene expression reflecting differences in proliferative capacity, cell adhesion functions and mitochondrial metabolism. We further demonstrate that AS plays a role in vesicular trafficking and membrane organization. These AS transitions are enriched among targets of two RNA-binding proteins, Celf1 and Mbnl1, which undergo developmentally regulated changes in expression. Vesicular trafficking genes affected by AS during normal development (when Celf1 is downregulated) show a reversion to neonatal splicing patterns after Celf1 re-expression in adults. Short-term Celf1 induction in adult animals results in disrupted transverse tubule organization and calcium handling. These results identify potential roles for AS in multiple aspects of postnatal heart maturation, including vesicular trafficking and intracellular membrane dynamics.