Differentiation Of Neural Crest Cells Research Articles

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.

Transcriptomic analysis reveals mitochondrial dysfunction in the pathogenesis of Nager syndrome in sf3b4-depleted zebrafish

Nager syndrome (NS) is a rare acrofacial dysostosis caused by heterozygous loss-of-function variants in the splicing factor 3B subunit 4 (SF3B4). The main clinical features of patients with NS are characterized by facial-mandibular and preaxial limb malformations. The migration and specification of neural crest cells are crucial for craniofacial development, and mitochondrial fitness appears to play a role in such processes. Here, by analyzing our previously published transcriptome dataset, we aim to investigate the potential involvement of mitochondrial components in the pathogenesis of craniofacial malformations, especially in sf3b4 mutant zebrafish. We identified that oxidative phosphorylation (OXPHOS) defects and overproduction of reactive oxygen species (ROS) due to decreased antioxidants defense activity, which leads to oxidative damage and mitochondrial dysfunction. Furthermore, our results highlight that fish lacking sf3b4 gene, primarily display defects in mitochondrial complex I. Altogether, our findings suggest that mitochondrial dysfunction may contribute to the development of the craniofacial anomalies observed in sf3b4-depleted zebrafish.

Assessment of SOX10 expression in 437 canine neoplasms of different embryologic origins.

Several members of the SRY-related HMG-box (SOX) protein family are implicated in tumorigenesis, metastasis, and regulation of the tumor microenvironment. SOX10, which is involved in neural crest cell migration and differentiation, has long been recognized a sensitive and specific immunohistochemical (IHC) marker in the diagnosis of melanoma in humans. However, expression of SOX10 in other tumor types has infrequently been evaluated in humans until recently and has not been thoroughly investigated in the dog. Our aim was to characterize the expression of SOX10 in canine neoplasms to objectively assess its value as a diagnostic IHC marker. Immunohistochemistry for SOX10 was performed on 437 archived, formalin-fixed paraffin-embedded tissues from representative canine neoplasms of ectodermal (15 tumor types), mesodermal (13 tumor types), endodermal (8 tumor types), and mixed/unknown (7 tumor types) embryologic origin. Oral and cutaneous tumors of melanocytic origin were used as positive controls. Intense SOX10 immunolabeling was observed in most tumors of ectodermal origin, including consistent expression in mammary carcinomas, and gliomas. Embryonal and hair follicle neoplasms inconsistently exhibited strong nuclear immunolabeling. Oral fibrosarcomas and undifferentiated oral sarcomas both inconsistently exhibited moderate to strong nuclear immunolabeling. Neoplasms of mesodermal and endodermal origin lacked immunolabeling. Salivary carcinomas, representing an unknown/mixed embryologic origin, were strongly labeled. SOX10 expression is not limited to melanomas, but is expressed by canine tumors of diverse tissues and embryologic derivation. Importantly, expression of SOX10 by a subset of oral sarcomas impairs its value as a marker for spindle cell oral melanomas.

The emerging role of ectodermal neural cortex 1 in cancer

Ectodermal neural cortex 1 (ENC1) is a protein that plays a crucial role in the regulation of various cellular processes such as cell proliferation, differentiation, and apoptosis. Numerous studies have shown that ENC1 is overexpressed in various types of cancers, including breast, lung, pancreatic, and colorectal cancer, and its upregulation is correlated with a poorer prognosis. In addition to its role in cancer growth and spreading, ENC1 has also been linked to neuronal process development and neural crest cell differentiation. In this review, we provide an overview of the current knowledge on the relationship between ENC1 and cancer. We discuss the molecular mechanisms by which ENC1 contributes to tumorigenesis, including its involvement in multiple oncogenic signaling pathways. We also summarize the potential of targeting ENC1 for cancer therapy, as its inhibition has been shown to significantly reduce cancer cell invasion, growth, and metastasis. Finally, we highlight the remaining gaps in our understanding of ENC1’s role in cancer and propose potential directions for future research.

Hyaluronic Acid Induction Promotes the Differentiation of Human Neural Crest-like Cells into Periodontal Ligament Stem-like Cells.

Periodontal ligament (PDL) stem-like cells (PDLSCs) are promising for regeneration of the periodontium because they demonstrate multipotency, high proliferative capacity, and the potential to regenerate bone, cementum, and PDL tissue. However, the transplantation of autologous PDLSCs is restricted by limited availability. Since PDLSCs are derived from neural crest cells (NCs) and NCs persist in adult PDL tissue, we devised to promote the regeneration of the periodontium by activating NCs to differentiate into PDLSCs. SK-N-SH cells, a neuroblastoma cell line that reportedly has NC-like features, seeded on the extracellular matrix of PDL cells for 2 weeks, resulted in the significant upregulation of PDL marker expression. SK-N-SH cell-derived PDLSCs (SK-PDLSCs) presented phenotypic characteristics comparable to induced pluripotent stem cell (iPSC)-derived PDLSCs (iPDLSCs). The expression levels of various hyaluronic acid (HA)-related genes were upregulated in iPDLSCs and SK-PDLSCs compared with iPSC-derived NCs and SK-N-SH cells, respectively. The knockdown of CD44 in SK-N-SH cells significantly inhibited their ability to differentiate into SK-PDLSCs, while low-molecular HA (LMWHA) induction enhanced SK-PDLSC differentiation. Our findings suggest that SK-N-SH cells could be applied as a new model to induce the differentiation of NCs into PDLSCs and that the LMWHA-CD44 relationship is important for the differentiation of NCs into PDLSCs.

CSIG-03. SHH PATHWAY ACTIVATION IN DEDIFFERENTIATION DURING TUMOUR PROGRESSION IN MPNST

Abstract BACKGROUND Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. PURPOSE & HYPOTHESIS To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway. METHODS Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation. RESULTS Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation. CONCLUSIONS The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.

Head dysgenesis and disruption of cranial neural crest stem cells behaviour by 4-octylphenol in fire-bellied toad Bombina orientalis embryos

Alkylphenolic endocrine disruptors (Eds) have been known to affect development of the descendants of multipotent neural crest cells (NCCs) in amphibian embryos. To unravel the mechanism of head dysgenesis induced by alkylphenols in amphibians, the effect of 4-octylphenol (OP) on the differentiation of cranial NCCs in developing embryos and tadpoles, ex vivo NC explant, and isolated NCCs was examined in fire-bellied toad Bombina orientalis with 0, 1, 2, 5, 10, 25 and 50 μM concentrations. Following OP treatment, head cartilages were frequently absent together with the decreased col2a1 mRNA level in tadpoles. While the lipid hydroperoxide (LPO), endoplasmic reticulum stress (ERS), apoptosis, and DNA fragmentation were significantly increased in stage 22 neulurae and heads of stage 45 tadpoles. In stage 22 neulurae, OP decreased sox9 mRNA, the master transcription factor for chondrogenic differentiation and increased undifferentiated NCC markers. The ectopic NCCs were found in endoderm while mesodermal SOX10(+) cells were decreased. In cranial NCCs isolated from stage 22 embryos, OP treatment decreased cellular survival and increased apoptosis, epithelial-mesenchymal transition (EMT) and cell migration. In chondrogenic induced cranial NC explants, OP treatment decreased SOX9(+) chondrocytes and cartilage development. Together, OP potentiated oxidative damage, apoptosis, EMT, and ectopic migration of NCCs. Considering that tissue differentiation requires stem cells to activate the molecular mechanism of differentiation at the correct location during embryonic development, these changes caused by OP may inhibit sox9-dependent chondrogenic differentiation of cranial NCCs, leading to head dysgenesis in B. orientalis embryos. Therefore, developing multipotent NCCs could be an important target of OP, provides new direction for the estimation of the risk of EDs exposure in human and wildlife animals.

Depletion of Neural Crest-Derived Cells Leads to Plasma Noradrenaline Decrease and Alters T Cell Development.

The differentiation of neural crest (NC) cells into various cell lineages contributes to the formation of many organs, including the thymus. In this study, we explored the role of NC cells in thymic T cell development. In double-transgenic mice expressing NC-specific Cre and the Cre-driven diphtheria toxin receptor, plasma noradrenaline and adrenaline levels were significantly reduced, as were thymic T cell progenitors, when NC-derived cells were ablated with short-term administration of diphtheria toxin. Additionally, yellow fluorescent protein+ NC-derived mesenchymal cells, perivascular cells, and tyrosine hydroxylase+ sympathetic nerves in the thymus significantly decreased. Furthermore, i.p. administration of 6-hydroxydopamine, a known neurotoxin for noradrenergic neurons, resulted in a significant decrease in thymic tyrosine hydroxylase+ nerves, a phenotype similar to that of depleted NC-derived cells, whereas administration of a noradrenaline precursor for ablating NC-derived cells or sympathetic nerves rarely rescued this phenotype. To clarify the role of NC-derived cells in the adult thymus, we transplanted thymus into the renal capsules of wild-type mice and observed abnormal T cell development in lethally irradiated thymus with ablation of NC-derived cells or sympathetic nerves, suggesting that NC-derived cells inside and outside of the thymus contribute to T cell development. In particular, the ablation of NC-derived mesenchymal cells in the thymus decreases the number of thymocytes and T cell progenitors. Overall, ablation of NC-derived cells, including sympathetic nerves, in the thymus leads to abnormal T cell development in part by lowering plasma noradrenalin levels. This study reveals that NC-derived cells including mesenchymal cells and sympathetic nerves within thymus regulate T cell development.

Biomedical engineering approaches for the delivery of JAGGED1 as a potential tissue regenerative therapy.

JAG1 is a ligand that activates the NOTCH signaling pathway which plays a crucial role in determining cell fate behavior through cell-to-cell signaling. JAG1-NOTCH signaling is required for mesenchymal stem cell (MSC) differentiation into cardiomyocytes and cranial neural crest (CNC) cells differentiation into osteoblasts, making it a regenerative candidate for clinical therapy to treat craniofacial bone loss and myocardial infarction. However, delivery of soluble JAG1 has been found to inhibit NOTCH signaling due to the requirement of JAG1 presentation in a bound form. For JAG1-NOTCH signaling to occur, JAG1 must be immobilized within a scaffold and the correct orientation between the NOTCH receptor and JAG1 must be achieved. The lack of clinically translatable JAG1 delivery methods has driven the exploration of alternative immobilization approaches. This review discusses the role of JAG1 in disease, the clinical role of JAG1 as a treatment, and summarizes current approaches for JAG1 delivery. An in-depth review was conducted on literature that used both in vivo and in vitro delivery models and observed the canonical versus non-canonical NOTCH pathway activated by JAG1. Studies were then compared and evaluated based on delivery success, functional outcomes, and translatability. Delivering JAG1 to harness its ability to control cell fate has the potential to serve as a therapeutic for many diseases.

Hirschsprung's Disease - Review of Clinical Features, Diagnosis and Treatment

Introduction and purpose: Hirschsprung's disease, also known as congenital aganglionic megacolon, is a rare congenital disorder that affects the large intestine. Due to the absence of ganglion cells, the affected segment of the colon becomes narrow and unable to relax. The disease is present from birth but may not always be immediately apparent. In this paper, we will attempt to present the current state of knowledge regarding the diagnosis and treatment of Hirschsprung's disease, based on the analysis of literature available on the PubMed platform.
 Description of the state of knowledge: Hirschsprung's disease (HSCR), also known as congenital aganglionosis of the colon, involves abnormal migration, proliferation, and differentiation of neural crest cells, leading to the absence of autonomic nerve ganglia within the colon. HSCR is associated with mutations in several genes, with RET, GDNF, EDNRB and SOX10 being identified as the main causes of the disease. Mutations in the RET gene are associated with the hereditary form of Hirschsprung's disease. Symptoms of HSCR appear in newborns and may include bilious vomiting, diarrhea associated with enterocolitis, failure to pass meconium within the first 24 hours of life, impaired peristalsis, jaundice, feeding difficulties, and progressive abdominal distension.
 Summary: Diagnosis is typically made based on clinical presentation, imaging studies, and biopsy. Treatment usually involves surgery to remove the affected segment of the colon and reconnect the healthy portions. Although postoperative complications are relatively common, long-term studies suggest that the majority of children with Hirschsprung's disease function well in society.

Identification of key pathways and genes involved in psoriasis and vitiligo using bioinformatics analysis

Objective: Psoriasis and vitiligo are common immune-related skin disorders. Patients are often clinically diagnosed with both diseases. However, whether psoriasis and vitiligo share common genetic factors and aberrant signal pathways that predispose patients to both diseases remains unclear. This study was performed to clarify these factors using bioinformatics analysis. Methods: Publicly available gene expression profiles for GSE109248 and GSE53552 in psoriasis and GSE75819 and GSE65127 in vitiligo from lesional and non-lesional skin tissues were downloaded from the Gene Expression Omnibus database and analyzed to identify differentially expressed genes (DEGs). Gene Ontology terms analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein–protein interaction analysis was also performed to elucidate the molecular mechanisms. Results: Nine overlapping DEGs were found between psoriasis and vitiligo. They are AKR1B10, CRABP1, FOXC1, GPM6B, KIT, MLPH, SOX10, TAGLN, and TUBB2B, respectively. Except for the upregulation of AKR1B10, others are downregulated. Pathway enrichment analyses revealed that these overlapping DEGs were mainly enriched in melanocyte differentiation; exocrine system development; mesenchymal cell development; stem cell differentiation and development; and neural crest cell differentiation, development, and migration. RT-qPCR was used to verify the expression of the DEGs in lesions compared to adjacent non-lesional tissues from three patients with psoriasis combined with vitiligo. Research confirmed that there were statistically significant differences among AKR1B10, FOXC1, KIT, MLPH and SOX10 in lesions compared to adjacent non-lesional tissues (P<0.05), which was consistent with the bioinformatical results. Conclusion: In this study, we detected potential genes and their associated enriched pathways to help understand the molecular mechanisms underlying the simultaneous occurrence of psoriasis and vitiligo.

SHH PATHWAY ACTIVATION IN DEDIFFERENTIATION DURING TUMOR PROGRESSION IN MPNST

Abstract Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. Purpose & Hypothesis: To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway. METHODS: Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation. RESULTS: Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation. CONCLUSIONS: The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.

BMP signaling during craniofacial development: new insights into pathological mechanisms leading to craniofacial anomalies.

Cranial neural crest cells (NCCs) are the origin of the anterior part of the face and the head. Cranial NCCs are multipotent cells giving rise to bones, cartilage, adipose-tissues in the face, and neural cells, melanocytes, and others. The behavior of cranial NCCs (proliferation, cell death, migration, differentiation, and cell fate specification) are well regulated by several signaling pathways; abnormalities in their behavior are often reported as causative reasons for craniofacial anomalies (CFAs), which occur in 1 in 100 newborns in the United States. Understanding the pathological mechanisms of CFAs would facilitate strategies for identifying, preventing, and treating CFAs. Bone morphogenetic protein (BMP) signaling plays a pleiotropic role in many cellular processes during embryonic development. We and others have reported that abnormalities in BMP signaling in cranial NCCs develop CFAs in mice. Abnormal levels of BMP signaling cause miscorrelation with other signaling pathways such as Wnt signaling and FGF signaling, which mutations in the signaling pathways are known to develop CFAs in mice and humans. Recent Genome-Wide Association Studies and exome sequencing demonstrated that some patients with CFAs presented single nucleotide polymorphisms (SNPs), missense mutations, and duplication of genes related to BMP signaling activities, suggesting that defects in abnormal BMP signaling in human embryos develop CFAs. There are still a few cases of BMP-related patients with CFAs. One speculation is that human embryos with mutations in coding regions of BMP-related genes undergo embryonic lethality before developing the craniofacial region as well as mice development; however, no reports are available that show embryonic lethality caused by BMP mutations in humans. In this review, we will summarize the recent advances in the understanding of BMP signaling during craniofacial development in mice and describe how we can translate the knowledge from the transgenic mice to CFAs in humans.

Transcriptomic analysis reveals the role of SIX1 in mouse cranial neural crest patterning and bone development.

Genetic variants of the transcription factor SIX1 and its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is characterized by craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. In this work, we expand our knowledge of the Six1 gene regulatory network by using a Six1-null mouse line to assess gene expression profiles of E10.5 mandibular arches, which give rise to the neural crest (NC)-derived middle ear ossicles and lower jaw, via bulk RNA sequencing. Our transcriptomic analysis led to the identification of 808 differentially expressed genes that are related to translation, NC cell differentiation, osteogenesis, and chondrogenesis including components of the WNT signaling pathway. As WNT signaling is a known contributor to bone development, we demonstrated that SIX1 is required for expression of the WNT antagonist Frzb in the mandibular arch, and determined that SIX1 expression results in repression of WNT signaling. Our results clarify the mechanisms by which SIX1 regulates the development of NC-derived craniofacial elements that are altered in SIX1-associated disorders. In addition, this work identifies novel genes that could be causative to this birth defect and establishes a link between SIX1 and WNT signaling during patterning of NC cells.

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. Although the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests that an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome, detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity. Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species, suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by using Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore longstanding questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Vwa1 Knockout in Zebrafish Causes Abnormal Craniofacial Chondrogenesis by Regulating FGF Pathway.

Hemifacial microsomia (HFM), a rare disorder of first- and second-pharyngeal arch development, has been linked to a point mutation in VWA1 (von Willebrand factor A domain containing 1), encoding the protein WARP in a five-generation pedigree. However, how the VWA1 mutation relates to the pathogenesis of HFM is largely unknown. Here, we sought to elucidate the effects of the VWA1 mutation at the molecular level by generating a vwa1-knockout zebrafish line using CRISPR/Cas9. Mutants and crispants showed cartilage dysmorphologies, including hypoplastic Meckel's cartilage and palatoquadrate cartilage, malformed ceratohyal with widened angle, and deformed or absent ceratobranchial cartilages. Chondrocytes exhibited a smaller size and aspect ratio and were aligned irregularly. In situ hybridization and RT-qPCR showed a decrease in barx1 and col2a1a expression, indicating abnormal cranial neural crest cell (CNCC) condensation and differentiation. CNCC proliferation and survival were also impaired in the mutants. Expression of FGF pathway components, including fgf8a, fgfr1, fgfr2, fgfr3, fgfr4, and runx2a, was decreased, implying a role for VWA1 in regulating FGF signaling. Our results demonstrate that VWA1 is essential for zebrafish chondrogenesis through effects on condensation, differentiation, proliferation, and apoptosis of CNCCs, and likely impacts chondrogenesis through regulation of the FGF pathway.

Exposure to valproic acid (VPA) reproduces hdac1 loss of function phenotypes in zebrafish.

Histone deacetylases are enzymes that remove acetyl groups from histone tails and are understood to act as repressors of transcriptional activity. Hdac1 has been previously shown to function in eye, pectoral fin, heart, liver, and pharyngeal skeletal development. We show that high doses of Valproic Acid (VPA) reproduce the hdac1 phenotype. We identify tbx5 genes as potential targets of Hdac1 in eye, pectoral fin, and heart development. Using timed exposures, we show that skeletal structures in the pharyngeal arches are impacted by VPA between 24-36 hours post-fertilization, indicating a role for Hdac1 during post-migration patterning, differentiation, or proliferation of cranial neural crest cells.

Increased enteric neural crest cell differentiation after transplantation into aganglionic mouse gut.

In recent years, many studies have made considerable progress in the development of stem cell-based therapies for Hirschsprung's disease (HD). However, the question of whether enteric neural crest-derived cells (ENCCs) that are transplanted into the aganglionic gut can migrate, proliferate, and differentiate in a normal manner remains unanswered. Thus, we designed this study to compare the behavior of ENCCs transplanted into the aganglionic gut of endothelin receptor B knockout (Ednrb-KO) mice versus wild-type (WT) mice. ENCCs were isolated from the fetal guts of Sox10 transgenic mice, in which ENCCs were labeled with an enhanced green fluorescent protein, Venus, on an embryonic day 18.5 (E18.5). Neurospheres were generated and transplanted into the aganglionic region of either Ednrb-KO mice gut, or WT mice gut that had not yet been colonized, on E12.5. Time-lapse imaging of the transplanted ENCCs was performed after 24, 48, and 72h of culture. Neuronal differentiation was evaluated using whole-mount immunohistochemistry. Sox10-positive ENCCs were seen to successfully migrate into the myenteric region of the aganglionic gut following transplantation in both the Ednrb-KO and WT mice. The ratio of Tuj1-positive/Sox10-positive cells was significantly increased after 72h of culture compared to 24h in the Ednrb-KO mice, which suggests that the transplanted ENCCs differentiated over time. In addition, at the 72h timepoint, neuronal differentiation of transplanted ENCC in the aganglionic gut of Ednrb-KO mice was significantly increased compared to that of WT mice. The results of our study demonstrated that transplanted ENCCs migrated into the myenteric region of the aganglionic recipient gut in mice. The increased neuronal differentiation of transplanted ENCC in Endrb-KO mice gut suggests that the microenvironment of this region affects ENCC behavior following transplantation. Further research to explore the characteristics of this microenvironment will improve the potential of developing cell therapy to treat HD patients.

Differentiation of enteric neural crest cells transplanted from SOX10-Venus mouse embryonic stem cells into the gut of the endothelin receptor B null mouse model.

Failure of enteric neural crest-derived cells (ENCCs) to correctly colonize the embryonic gut results in Hirschsprung's disease (HD). Embryonic stem cells (ESCs) have the potential to differentiate into all tissue-specific cells and lineages, including ENCCs. We investigated the cellular differentiation of ESCs from Sox10-Venus+ mice into both control and endothelin receptor-B knockout (Ednrb KO) mouse gut to assess each region. We established ESCs from Sox10-Venus + mice. These cells were cultured for 2days, then selected and co-cultured with either a dissociated control or Sox10-Venus - Ednrb KO mouse gut (both small intestine and colon) on embryonic day (E) 13.5. Four days later, cells were immunolabeled for Tuj1 and visualized using confocal microscopy. Confocal microscopy revealed that transplanted Sox10-Venu + cells from ESCs migrated extensively within the host gut. Moreover, Tuj1-positive neurites were detected in the transplanted ESCs. Tuj1 expression was significantly decreased in aganglionic HD colon compared to controls (p < 0.05) and the HD small intestine (p < 0.05). This study demonstrated that an appropriate host environment is crucial for normal and complete colonization of the gut. Further investigations are required to confirm whether modifying this environment can improve the results of this model.

Development and In Vitro Differentiation of Schwann Cells

Schwann cells are glial cells of the peripheral nervous system. They exist in several subtypes and perform a variety of functions in nerves. Their derivation and culture in vitro are interesting for applications ranging from disease modeling to tissue engineering. Since primary human Schwann cells are challenging to obtain in large quantities, in vitro differentiation from other cell types presents an alternative. Here, we first review the current knowledge on the developmental signaling mechanisms that determine neural crest and Schwann cell differentiation in vivo. Next, an overview of studies on the in vitro differentiation of Schwann cells from multipotent stem cell sources is provided. The molecules frequently used in those protocols and their involvement in the relevant signaling pathways are put into context and discussed. Focusing on hiPSC- and hESC-based studies, different protocols are described and compared, regarding cell sources, differentiation methods, characterization of cells, and protocol efficiency. A brief insight into developments regarding the culture and differentiation of Schwann cells in 3D is given. In summary, this contribution provides an overview of the current resources and methods for the differentiation of Schwann cells, it supports the comparison and refinement of protocols and aids the choice of suitable methods for specific applications.