Neural Crest Research Articles

The Neuroblastoma Microenvironment, Heterogeneity and Immunotherapeutic Approaches.

Neuroblastoma is a peripheral nervous system tumor that almost exclusively occurs in young children. Although intensified treatment modalities have led to increased patient survival, the prognosis for patients with high-risk disease is still around 50%, signifying neuroblastoma as a leading cause of cancer-related deaths in children. Neuroblastoma is an embryonal tumor and is shaped by its origin from cells within the neural crest. Hence, neuroblastoma usually presents with a low mutational burden and is, in the majority of cases, driven by epigenetically deregulated transcription networks. The recent development of Omic techniques has given us detailed knowledge of neuroblastoma evolution, heterogeneity, and plasticity, as well as intra- and intercellular molecular communication networks within the neuroblastoma microenvironment. Here, we discuss the potential of these recent discoveries with emphasis on new treatment modalities, including immunotherapies which hold promise for better future treatment regimens.

A bioinformatics approach to reveal common genes and molecular pathways shared by cutaneous melanoma and uveal melanoma

Background Melanomas are highly aggressive in nature known for metastasis and death. Melanocytes that gave rise to melanomas are neural crest progenitor cells. Our research was primarily concerned with uveal melanoma (UM) and cutaneous melanoma (CM), respectively. Although they both have the same melanocytic origin, the biology of their respective is different.Aim The aim of our study was to recognize the common differentially expressed genes (DEGs) between UM and CM.MethodologyThe gene expression profile was downloaded from the GEO and analyzed by GEO2R to recognize DEGs. By applying DAVID, GO, and KEGG, pathway enrichment analysis was performed. PPI of these DEGs was analyzed using STRING and visualized by Cytoscape and MCODE. Further, we utilized HPA and GEPIA to obtain Kaplan–Meier graph for survival analysis in order to assess the prognostic value of hub genes.Results We examined the UM and CM datasets and discovered three common upregulated and eight downregulated DEGs based on computational analysis. HMGCS1 and ELOVL5 were shown to be enriched in a variety of altered molecular pathways and pathways in cancer. Overexpression of HMGCS1 and ELOVL5 was linked to a poor prognosis in CM.Conclusion Computational evaluation found that HMGCS1 and ELOVL5 were upregulated in both melanomas. Enrichment analysis showed that these genes are involved in cancer metabolism pathway and associated with poor prognosis in CM. However, the molecular study of these genes in UM is limited. Therefore, a better understanding of the cancer metabolism pathways should be carried to pave the way for clinical benefits.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Evaluating neural crest cell migration in a Col4a1 mutant mouse model of ocular anterior segment dysgenesis

The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome – a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.

Piezo1 and Piezo2 collectively regulate jawbone development.

Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.

Internal Carotid Artery:Development and Anomalies

This chapter outlines the cerebrovascular developmental anatomy with emphasis on the internal carotid artery(ICA), which is important for optimal endovascular treatment of cerebrovascular system disorders. Gene expression, neural crest cells, and pharyngeal arches play key roles in the embryonic development of the carotid arteries. Evolutionary inheritance in vertebrates contributes to the formation, regression, and segmental structure of these arteries. We present examples of current mutations with regard to their segmental nature; ICA mutations are discussed primarily with regard to their developmental origin from the first to third pharyngeal arches and the role of the ductus caroticus. This comprehensive review highlights the importance of understanding the developmental and anatomical nuances of the ICA to aid with accurate diagnosis and management of vascular anomalies in the clinical setting. We have focused on the complexity associated with ICA mutations, particularly those associated with the third pharyngeal arch and the need for a solid foundation of developmental and anatomical knowledge to accurately identify and interpret their significance in the adult phenotype.

Pathological Eye Growth in Myopia: a Limited Review on Endocrine Physiology for Future Research

Purpose: Myopia or short-sightedness: first manifests in school-children (early-onset), and also in young adults (late-onset). Pathological myopia can lead to stretching of the eyeball to the point of blindness from a detached retina. This title presents a preliminary understanding of endocrine, juxtacrine, and paracrine factors: hoping to stimulate further research and enable early therapeutic intervention. Background: The optical stimulus for eye growth is a blurred image on the retina that results from under-focused accommodation of the internal crystalline lens. Neural signals and biochemical messaging from the retina and vascular choroid provide local impetus to alter collagen structure in the outer white sclera of the eye. In persons that develop highly myopic refractive error, additional factors from blood and tissue chemistry, might influence intra-ocular pressure (IOP), posited to influence eyeball elongation. New research shows that GABA and dopamine are involved, but other factors remain unexplored. Eye Physiology: Human sclera has developmental origination same as any other connective tissue derived from the neural crest and mesoderm. Bundles of collagen fibers exist in the white opaque sclera and are more stringently oriented in the dome of the clear, transparent cornea. “Deformation compliance” of the sclera is governed by complexity of the collagen architecture, and “reduced scleral rigidity” is a known factor for myopic elongation, but developmental physiology is not well understood. Muscular contraction is required for sustained eye focusing to objects located at normal working distances. Short-term contractions can reduce IOP but increased muscle density from sustained near accommodation can promote progression of myopia. This implicates nitric oxide and calcium channels, and a role for associated biochemical and nutritional agonists and antagonists. Endocrine Influences: In his 1954 lecture to the Royal College of Surgeons, Dr. Howard C. Naffziger pointed to all forms of “ocular protrusion,” not just “thyroid exophthalmos,” as being physiologically related. A 2016 review paper on autonomic nervous physiology, aside from sympathetic (adrenergic) and parasympathic (collinergic) control, also implicates Substance P (SP), Vasoactive Intestinal Peptide (VIP), and Phospholipase C (PC). These topics are further described in limited detail. SP is a neuropeptide of the tachykinin family, is released from the terminals of sensory nerves, and regulates emotion (hypothalamus, amygdala, and the periaqueductal gray). It is also a vasodilator dependent on release of nitric oxide. Experimental eye physiology and clinical research pertaining to these topics appears warranted. VIP regulates cardiac contractions, relaxes smooth muscle of the trachea, and affects prolactin secretion by the pituitary gland. Cardiac contractions lead to pulsations of eye pressure about three times in two seconds, and pulmonary effects on IOP occur once every five seconds. Research into these mechanisms requires cross-disciplinary collaborations. PC is a membrane-associated enzyme that cleaves dietary phospholipids and has downstream effects on calcium channels. Binding to blood platelets, of agonists such as thrombin, epinephrine, or collagen: activates PC to release arachidonic acid from cleavage of phosphatidylinositol and phosphatidyl choline. Behavioral effects include visual attention-span and memory: both of which also concern education of children, and effectiveness of adults at work. Conclusions: The author is pleased to report growing interest and evidence for endocrine influences upon the outer coat of the eye (cornea and sclera). A 2022 study on “keratoconus” from Texas and Oklahoma provides in-vitro as well as in-vivo data, suggesting the existence of what they call, the Hypothalamic-Pituitary-Adrenal-Corneal axis. Effective communication across university disciplines and clinic doctors appears necessary for further progress. Funding: The author received no funds from Federal or State granting mechanisms, and no wages from corporate employers. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melanocyte stem cells in the skin: Origin, biological characteristics, homeostatic maintenance and therapeutic potential.

Melanocyte stem cells (MSCs), melanocyte lineage-specific skin stem cells derived from the neural crest, are observed in the mammalian hair follicle, the epidermis or the sweat gland. MSCs differentiate into mature melanin-producing melanocytes, which confer skin and hair pigmentation and uphold vital skin functions. In controlling and coordinating the homeostasis, repair and regeneration of skin tissue, MSCs play a vital role. Decreased numbers or impaired functions of MSCs are closely associated with the development and therapy of many skin conditions, such as hair graying, vitiligo, wound healing and melanoma. With the advancement of stem cell technology, the relevant features of MSCs have been further elaborated. In this review, we provide an exhaustive overview of cutaneous MSCs and highlight the latest advances in MSC research. A better understanding of the biological characteristics and micro-environmental regulatory mechanisms of MSCs will help to improve clinical applications in regenerative medicine, skin pigmentation disorders and cancer therapy. KEY POINTS: This review provides a concise summary of the origin, biological characteristics, homeostatic maintenance and therapeutic potential of cutaneous MSCs. The role and potential application value of MSCs in skin pigmentation disorders are discussed. The significance of single-cell RNA sequencing, CRISPR-Cas9 technology and practical models in MSCs research is highlighted.

Creating 3D constructs with cranial neural crest-derived cell lines using a bio-3D printer

ObjectivesThe development of bio-three-dimensional (bio-3D) printers has led to significant advances in regenerative medicine. Three-dimensional constructs, including spheroids, are maintained by extracellular matrix proteins secreted by cells so that the cells can be cultured in conditions closer to the physiological environment. This study aimed to create a useful 3D construct as a model of the dentin-pulp complex. MethodsWe examined the expression patterns of extracellular matrix proteins and cell proliferation areas in a 3D construct created using O9-1 cells derived from cranial neural crest cells of mice. The 3D construct was created by sticking the spheroid cultures onto a needle array using a bio-3D printer. ResultsCell proliferation areas along with characteristic expression of tenascin C and DMP1 were evaluated. The expression of tenascin C and DMP1 was significantly enhanced in the spheroids compared to that in two-dimensional cultures. Moreover, cell proliferation regions and tenascin C expression were confirmed in the outer layer of spheroids in the embryonic stem cell medium, with insignificant DMP1 expression being observed. Interestingly, in a 3D construct cultured in calcification-induction medium, DMP1 expression was promoted, and DMP1-positive cells existed in the outermost layer without overlapping with tenascin C expression. ConclusionsThe extracellular matrix proteins, tenascin C and DMP1, were expressed in a polarized manner in spheroids and 3D constructs, similar to the findings in the dental papilla. Therefore, these 3D constructs show potential as artificial models for studying odontogenesis.

The hiPSC-derived corneal endothelial progenitor-like cell recovers the rabbit model of corneal endothelial dystrophy

IntroductionCorneal endothelial dysfunction results in cornea opacity, damaging sightedness, and affecting quality of life. A corneal transplant is the current effective intervention. Due to the scarcity of donated cornea, such an unmet medical need requires a novel therapeutic modality. ObjectivesCustomizing patients' corneal endothelial progenitor cells with proliferative activity and lineage restriction properties shall offer sufficient therapeutic cells for corneal endothelial dystrophy. MethodsThe customized induced human corneal endothelial progenitor-like cell (iHCEPLC) was obtained through cell fate conversions starting from PBMC (peripheral blood mononuclear cell), hiPSC (human induced pluripotent stem cell), and hNCC (human neural crest cell), while it finally reached the iHCEPLC state via a series of induction. Several molecular diagnoses were applied to depict its progenitor state, including RNAseq, FlowCytometer, immunostainings, and rtPCR. Significantly, it can be induced to gain differentiation maturity through contact inhibition. In addition, a BAK-mediated rabbit model of corneal endothelial dystrophy was established in the present study to test the therapeutic effectiveness of the iHCEPLC. ResultsAfter inducing cell fate conversion, the specific HCEC markers were detected by rtPCR and immunostaining in iHCEPLC. Further, RNAseq was applied to distinguish its progenitor-like cell fate from primary human corneal endothelial cells (HECE). FlowCytometry profiled the heterogeneity subpopulation, consistently displaying a subtle difference from primary HCEC. A terminal differentiation can be induced in iHCEPLC, addressing its progenitor-like fate. iHCEPLC can restore the BAK-based rabbit model of corneal endothelial dystrophy. Immunohistochemistry verified that such acuity restoration of the BAK-treated cornea is due to the introduced iHCEPLC, and such therapeutic effectiveness is observed in the long term. ConclusionHere, we demonstrated that customized iHCEPLC has long-term therapeutic efficacy. As a progenitor cell, our iHCEPLC has a restricted cell lineage nature and can proliferate in vitro, supporting sufficient therapeutic candidate cells. Due to the immune-privileged nature of the cornea, our iHCEPLC proves the principle of therapeutical feasibility in both autogenic and allogeneic modalities.

COMPOSITE PHEOCHROMOCYTOMA (WITH ASSOCIATED GANGLIONEUROMA) AS A CAUSE OF SECONDARY ENDOCRINOLOGICAL HYPERTENSION

Objective: To illustrate and understand a rare variant of pheochromocytoma, its clinical implications and prognosis, based on a clinical case of secondary hypertension. Design and method: Review of the patient's clinical record and the relevant literature. Results: Composite paraganglioma or pheochromocytoma is a rare tumour (<100 reported cases) composed of paraganglioma or pheochromocytoma plus another neurogenic tumour, most often (∼75%) ganglioneuroma, a typically benign tumour derived from the sympathogonia of the neural crest. It can be associated with neurofibromatosis type I, von Hippel-Lindau disease, or MEN2. Our patient is a 64-year-old woman with uncontrolled hypertension, without a history of hypertensive crises. During follow-up for breast adenocarcinoma, a CT scan revealed bilateral adrenal nodules consistent with metastases. PET-CT showed increased metabolic uptake suggesting malignancy in the right adrenal. Plasma metanephrines were very high and MIBG scintigraphy was consistent with a right pheochromocytoma. Following doxazosin blockade, robotic adrenalectomy was performed, and a month later plasma metanephrines and blood pressure were normal. Pathology revealed a single mass of 4x3x2 cm labelled as composite pheochromocytoma (30%) - ganglioneuroma (705). There was no vascular invasion or necrosis, S-100 staining was preserved, and mitotic activity was minimal. In addition to the PASS scale for prognosis of malignancy in pheochromocytoma, there is a specific COPPS scale for composite pheochromocytoma, based on necrosis, loss of S100, loss of SDHB, vascular invasion, and size >7 cm (5/2/1/1/1 points, respectively), with low risk if <3; our patient scores 0-1 (pending SDHB). The patient continues oncological follow-up for breast pathology, but her secondary hypertension is clinically resolved and the risk of pheochromocytoma-ganglioneuroma metastases is apparently low. There are no clinical signs of neurofibromatosis type 1, von Hippel-Lindau, or MEN2. Conclusions: Composite pheochromocytoma is a rare cause of secondary hypertension, often indistinguishable clinically from simple pheochromocytoma, except occasionally due to the larger lesion size. Diagnosis is typically confirmed through histopathological examination. In the most common association (with ganglioneuroma), additional malignancy implications are rare, unlike with other neurogenic tumours such as neuroblastoma or ganglioneuroblastoma. Due to insufficient current knowledge of its natural history, close monitoring is required.

Rapamycin and/or valproic acid pre-treatment protects epidermal neural crest stem cells from oxygen glucose deprivation-induced oxidative stress through NRF2 pathway

Rapamycin and/or valproic acid pre-treatment protects epidermal neural crest stem cells from oxygen glucose deprivation-induced oxidative stress through NRF2 pathway

A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

The development of brain pericytes requires expression of the transcription factor nkx3.1 in intermediate precursors.

Brain pericytes are one of the critical cell types that regulate endothelial barrier function and activity, thus ensuring adequate blood flow to the brain. The genetic pathways guiding undifferentiated cells into mature pericytes are not well understood. We show here that pericyte precursor populations from both neural crest and head mesoderm of zebrafish express the transcription factor nkx3.1 develop into brain pericytes. We identify the gene signature of these precursors and show that an nkx3.1-, foxf2a-, and cxcl12b-expressing pericyte precursor population is present around the basilar artery prior to artery formation and pericyte recruitment. The precursors later spread throughout the brain and differentiate to express canonical pericyte markers. Cxcl12b-Cxcr4 signaling is required for pericyte attachment and differentiation. Further, both nkx3.1 and cxcl12b are necessary and sufficient in regulating pericyte number as loss inhibits and gain increases pericyte number. Through genetic experiments, we have defined a precursor population for brain pericytes and identified genes critical for their differentiation.

Diphthamide deficiency promotes association of eEF2 with p53 to induce p21 expression and neural crest defects

Diphthamide is a modified histidine residue unique for eukaryotic translation elongation factor 2 (eEF2), a key ribosomal protein. Loss of this evolutionarily conserved modification causes developmental defects through unknown mechanisms. In a patient with compound heterozygous mutations in Diphthamide Biosynthesis 1 (DPH1) and impaired eEF2 diphthamide modification, we observe multiple defects in neural crest (NC)-derived tissues. Knockin mice harboring the patient’s mutations and Xenopus embryos with Dph1 depleted also display NC defects, which can be attributed to reduced proliferation in the neuroepithelium. DPH1 depletion facilitates dissociation of eEF2 from ribosomes and association with p53 to promote transcription of the cell cycle inhibitor p21, resulting in inhibited proliferation. Knockout of one p21 allele rescues the NC phenotypes in the knockin mice carrying the patient’s mutations. These findings uncover an unexpected role for eEF2 as a transcriptional coactivator for p53 to induce p21 expression and NC defects, which is regulated by diphthamide modification.

Functional and Genetic Analyses Unveil the Implication of CDC27 in Hemifacial Microsomia.

Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27-/- mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.

Adult hair follicle stem cells differentiate into neuronal cells in explanted rat intestinal tissue.

Hair follicle stem cells (HFSCs) are adult stem cells located in the outer root sheath of the follicle bulge with high neural plasticity, which promise a potential for the stem cell therapy for neurological diseases. Hirschsprung's disease (HD) is characterized by the absence of ganglia in the distant bowel. In this study, we elucidated the capacity of HFSCs to differentiate into neuronal cells in the aganglionic colon from embryonic rat. HFSCs were isolated from adult Sprague-Dawley (SD) rats and formed spheres that could be passaged. The cultured HFSCs expressed neural crest stem cells (NCSCs) markers such as SOX10, CD34, and nestin, which indicated their neural crest lineage. Subsequent differentiation assays demonstrated that these cells could give rise to neural progeny that expressed neuronal or glial markers. The aganglionic colon from the embryonic intestine was applied as in vitro explant to test the capacity of proliferation and differentiation of HFSCs. The HFSCs expressing GFP or RFP integrated in intestinal explants and maintained proliferative capacity. Moreover, the HFSCs differentiated into Tuj1- or S100β-positive cells in the cultured intestinal explants. The results proposed that the HFSCs might be an alternative source of neural stem cells for the HD therapy.

GFRA4 improves the neurogenic potential of enteric neural crest stem cells via hedgehog pathway.

Hirschsprung disease (HSCR) is a congenital intestinal disease characterised by functional obstruction of the colon. Herein, we investigated the role and mechanism of the gene GFRA4 in HSCR. GFRA4 expression in the ganglionic and aganglionic segment tissues in patients with HSCR and healthy colon tissues were detected using qRT-PCR, western blot, and immunohistochemistry. Cell proliferation, cycle distribution, apoptosis, changes in mitochondrial membrane potential, and differentiation were assessed in mouse enteric neural crest stem cells (ENCSCs) using the CCK-8 assay, EdU staining, flow cytometry, JC-1 probe, and immunofluorescence, respectively. GSEA analysis was performed to screen the signaling pathways regulated by GFRA4. GFRA4 was downregulated in aganglionic segment tissues compared to control and ganglionic segment tissues. GFRA4 overexpression promoted proliferation and differentiation, and inhibited apoptosis in ENCSCs, while GFRA4 down-regulation had the opposite result. GFRA4 activated the hedgehog pathway. GFRA4 overexpression enhanced the expression of key factors of the hedgehog pathway, including SMO, SHH, and GLI1. However, GFRA4 down-regulation reduced their expression. An antagonist of hedgehog pathway, cyclopamine, attenuated the effect of GFRA4 overexpression on proliferation, differentiation, and apoptosis of ENCSCs. GFRA4 promotes proliferation and differentiation but inhibits apoptosis of ENCSCs via the hedgehog pathway in HSCR. This study confirms that GFRA4 improves the proliferation and differentiation of ENCSCs via modulation of the hedgehog pathway. This study for the first time revealed the role and the mechanism of the action of GFRA4 in HSCR, which indicates that GFRA4 may play a role in the pathological development of HSCR. Our findings may lay the foundation for further investigation of the mechanisms underlying HSCR development and into targets of HSCR treatment.

Patient-reported burden associated with pheochromocytoma/paraganglioma diagnosis.

Pheochromocytoma and paragangliomas (PPGLs) originate from the chromaffin cells of the adrenal medulla or neural crest progenitors outside the adrenal gland, respectively. The estimated annual incidence of PPGL is between 2.0 and 8.0/million adults. Minimal data exist on the impact of PPGL from the patient's perspective. Therefore, a survey was adapted from a previously published study on gastroenteropancreatic neuroendocrine tumors to explore the voice of patients with PPGL and learn ways to improve clinical care while understanding the current gaps to direct future research. A self-reported online survey was available to patients with PPGL and those with genetic predisposition even without PPGL from June to July 2022. Survey questions captured sociodemographic and clinical characteristics, the diagnostic workup, treatment and monitoring, quality and access to care, and financial impact. Here, we report the most relevant findings on patient experience of disease burden following diagnosis. A total of 270 people responded, the majority of whom were from the USA (79%), Caucasian (88%), and female (81%). The results of this survey highlight the burden of disease on a patient's daily life, resulting in moderate to severe financial distress, increased travel time to specialized facilities resulting in loss of work and wages, and significant delays in care. Respondents reported being unheard and unacknowledged. With a median time to diagnosis just over 2 years, the physical, mental, and emotional toll are substantial. Increasing access to PPGL specialists and centers could lead to faster diagnoses and better management, which may reduce the burden on both patients and healthcare centers.

GFP-labeled Schwann cell-like cells derived from hair follicle epidermal neural crest stem cells promote the acellular nerve allografts to repair facial nerve defects in rats

BackgroundAcellular nerve allografts (ANAs) have been successfully applied to bridge facial nerve defects, and transplantation of stem cells may enhance the regenerative results. Up to now, application of hair follicle epidermal neural crest stem cell-derived Schwann cell-like cells (EPI–NCSC–SCLCs) combined with ANAs for bridging facial nerve defects has not been reported. MethodsThe effect of ANAs laden with green fluorescent protein (GFP)-labeled EPI–NCSC–SCLCs (ANA + cells) on bridging rat facial nerve trunk defects (5-mm-long) was detected by functional and morphological examination, as compared with autografts and ANAs, respectively. Results(1) EPI–NCSC–SCLCs had good compatibility with ANAs in vitro. (2) In the ANA + cells group, the GFP signals were observed by in vivo imaging system for small animals within 8 weeks, and GFP-labeled EPI–NCSC–SCLCs were detected in the tissue slices at 16 weeks postoperatively. (3) The facial symmetry at rest after surgery in the ANA + cells group was better than that in the ANA group (p < 0.05), and similar to that in the autograft group (p > 0.05). The initial recovery time of vibrissal and eyelid movement in the ANA group was 2 weeks later than that in the other two groups. (4) The myelinated fibers, myelin sheath thickness and diameter of the axons of the buccal branches in the ANA group were significantly worse than those in the other two groups (P < 0.05), and the results in the ANA + cells group were similar to those in the autograft group (p > 0.05). ConclusionsEPI–NCSC–SCLCs could promote functional and morphological recovery of rat facial nerve defects, and GFP labeling could track the transplanted EPI–NCSC–SCLCs in vivo for a certain period of time. These may provide a novel choice for clinical treatment of peripheral nerve defects.