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Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2B-polyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells. Single-cell transcriptomics revealed that expression of PHOX2B+7Ala PARM alters the differentiation trajectories of the hindbrain neurons and hampers the formation of the RTN-like neurons in HBSOs. With the unguided cerebral organoids (HCOs), PHOX2B+7Ala PARM interrupted the patterning of PHOX2B+ neurons with dysregulation of Hedgehog pathway and HOX genes. With complementary use of HBSOs and HCOs with a patient and two mutant induced pluripotent stem cell lines carrying different polyalanine repetition in PHOX2B, we further defined the association between the length of polyalanine repetition and malformation of RTN-respiratory center and demonstrated the potential toxic gain of function of PHOX2B-PARMs, highlighting the uniqueness of these organoid models for disease modeling., Competing Interests: Conflict of interests The authors are filing a patent related to the method of generating HBSOs as described in this manuscript., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis., (© 2023. The Author(s).)

Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. Due to the oligogenic nature of the disease, some HSCR conditions could not be phenocopied in animal models. Building the patient-based disease model using human induced pluripotent stem cells (hPSC) has opened up a new opportunity to untangle the unknowns of the disease. The expanding armamentarium of hPSC-based therapies provides needed new tools for developing cell-replacement therapy for HSCR. Here we summarize the recent studies of hPSC-based models of ENS in 2-D and 3-D culture systems. These studies have highlighted how hPSC-based models complement the population-based genetic screens and bioinformatic approaches for the discovery of new HSCR susceptibility genes and provide a human model for the close-to-physiological functional studies. We will also discuss the potential applications of these hPSC-based models in translational medicines and their advantages and limitations. The use of these hPSC-based models for drug discovery or cell replacement therapy likely leads to new treatment strategies for HSCR in the future. Further improvements in incorporating hPSC-based models with the human-mouse chimera model and organ-on-a-chip system for establishing a better disease model of HSCR and for drug discovery will further propel us to success in the development of an efficacious treatment for HSCR.

Gastrointestinal motility disorders occur frequently in patients with ciliopathy, but the underlying genetic link is unclear. The ciliary protein Kif7 can positively or negatively regulate Hedgehog signaling in different cellular contexts. Mice with neural crest cell (NCC)–specific Kif7 deficiency show a marked reduction of enteric NOS + inhibitory neurons. Malformation of enteric nervous system (ENS) causes growth retardation and gut motility defect in mice. Mechanistically, Kif7 inhibits Gli2 in enteric NCCs (ENCCs), where Gli2 positively regulates the expression of Ezh2 by inhibiting the miR124 -mediated suppression. In developing ENCCs, Ezh2 is a master regulator of 102 core genes underlying ENCC differentiation. Deletion of Gli2 or inhibition of Ezh2 favors the neurogenic lineage differentiation of mouse and human ENCCs and rescues the ENS defects of Kif7 mutants. In summary, Hedgehog signal, via Kif7-Gli-Ezh2, controls the timely expressions of the core genes to mediate the differentiation of ENCCs.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET , RASGEF1A , and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain., (© 2020 Fu et al.; Published by Cold Spring Harbor Laboratory Press.)

Background & Aims: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants., Methods: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay., Results: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10 -7 ). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the β-secretase 2 gene (BACE2) (P = 2.9 × 10 -6 ). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid β precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease., Conclusions: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Stem cells possess the ability of self-renewal and the potency to differentiate into multiple cell lineages. Somatic stem cells are present in adult tissues, but they usually exhibit limited differentiation capacity and life span. On the other hand, somatic cells from adult tissues can be reprogrammed into induced pluripotent stem cells (iPSCs) that retain a full differentiation capacity with unlimited self-renewal ability. Autologous origin of iPSCs makes them an ideal source of cells for regenerative medicine to replenish the missing or damaged cells in the patients. iPSCs nowadays have also been widely used to build human disease models to study pathological mechanisms of the diseases. Hirschsprung disease (HSCR) is a congenital disorder caused by defects in the development of enteric neural crest stem cells. The failures of the ENCCs to proliferate, differentiate, and/or migrate lead to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. The lack of effective treatment for HSCR urges continuous efforts to develop new therapies for this congenital disorder. In this review, we will discuss the potential applications of somatic stem cells and iPSCs for the cell-based therapy of HSCR. We will also highlight the recent advances in stem cell research for the establishment of human HSCR models for the development of novel therapies., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)