Neural Crest-like Cells Research Articles

Cell type and regulatory analysis in amphioxus illuminates evolutionary origin of the vertebrate head

To shed light on the enigmatic origin of the vertebrate head, our study employs an integrated approach that combines single-cell transcriptomics, perturbations in signaling pathways, and cis-regulatory analysis in amphioxus. As a representative of a basal lineage within the chordate phylum, amphioxus retains many characteristics thought to have been present in the common chordate ancestor. Through cell type characterization, we identify the presence of prechordal plate-like, pre-migratory, and migratory neural crest-like cell populations in the developing amphioxus embryo. Functional analysis establishes conserved roles of the Nodal and Hedgehog signaling pathways in prechordal plate-like populations, and of the Wnt signaling pathway in neural crest-like populations’ development. Furthermore, our trans-species transgenic experiments highlight similarities in the regulatory environments that drive neural crest-like and prechordal plate-like developmental programs in both vertebrates and amphioxus. Our findings provide evidence that the key features of vertebrate head development can be traced back to the common ancestor of all chordates.

Schwannian and Perineuriomatous Differentiation in a Series of Giant Congenital Melanocytic Nevi.

Close relationship between melanocytes and neural cells is accepted to reflect their common derivation from the neural crest and tumors combining both elements. We present a series of 10 patients with giant congenital melanocytic nevi (CMN) in which a secondary proliferation (11 lesions) with schwannian and/or perineuriomatous differentiation developed in the course of the disease. The age of the patients (4 male and 6 female) at the time of surgery and histological assessment varied from 3 months to 57 years. Histopathologically, the following subgroups were delineated: (1) nodular/tumoriform "neurotization" in CMN, (2) diffuse neurofibroma-like proliferation within CMN, (3) plexiform neurofibroma-like proliferation within CMN, and (4) diffuse perineuriomatous (hybrid schwannomatous-perineuriomatous) differentiation in CMN. We review the pertinent literature, including the role of recently identified Schwann cell precursors which are believed to represent the nerve-associated state of neural crest-like cells that persists into later developmental stages.

Axl as a potential therapeutic target for adamantinomatous craniopharyngiomas: Based on single nucleus RNA-seq and spatial transcriptome profiling

Craniopharyngiomas (CPs), particularly Adamantinomatous Craniopharyngiomas (ACPs), often exhibit a heightened risk of postoperative recurrence and severe complications of the endocrine and hypothalamic function. The primary objective of this study is to investigate potential novel targeted therapies within the microenvironment of ACP tumors. Cancer-Associated Fibroblasts (CAFs) were identified in the craniopharyngioma microenvironment, notably in regions characterized by cholesterol clefts, wet keratin, ghost cells, and fibrous stroma in ACPs. CAFs, alongside ghost cells, basaloid-like epithelium cells and calcifications, were found to secrete PROS1 and GAS6, which can activate AXL receptors on the surface of tumor epithelium cells, promoting immune suppression and tumor progression in ACPs. Additionally, the AXL inhibitor Bemcentinib effectively inhibited the proliferation organoids and enhanced the immunotherapeutic efficacy of Atezolizumab. Furthermore, neural crest-like cells were observed in the glial reactive tissue surrounding finger-like protrusions. Overall, our results revealed that the AXL might be a potentially effective therapeutic target for ACPs.

Effect of Fibrillin-2 on Differentiation into Periodontal Ligament Stem Cell-Like Cells Derived from Human-Induced Pluripotent Stem Cells.

Periodontal tissue regeneration is important for preserving teeth. Periodontal ligament stem cells (PDLSCs) are useful in periodontal tissue regeneration; however, tooth extraction is required to obtain these cells. Therefore, we focused on induced pluripotent stem (iPS) cells and established a method to obtain PDLSC-like cells from iPS cells. Specifically, we first differentiated iPS cells into neural crest-like cells (iNCs). Next, we obtained PDLSC-like cells (iPDLSCs) by culturing iNCs on extracellular matrix (ECM) derived from human primary periodontal ligament cells (HPDLCs). This differentiation method suggested that ECM derived from HPDLCs is important for iPDLSC differentiation. Thus, we aimed to identify the PDLSC-inducing factor present in HPDLC-derived ECM in this study. We first performed comprehensive analyses of HPDLC genes and identified fibrillin-2 (FBN2), an ECM-related factor. Furthermore, to clarify the effect of FBN2 on iPDLSC differentiation, we cultured iNCs using ECM derived from HPDLCs with FBN2 knocked down. As a result, expression of PDL-related markers was reduced in iNCs cultured on ECM derived from HPDLCs transfected with FBN2 siRNA (iNC-siFBN2) compared with iPDLSCs. Furthermore, the expression of CD105 (a mesenchymal stem cell marker), proliferation ability, and multipotency of iNC-siFBN2 were lower compared with iPDLSCs. Next, we cultured iNCs on FBN2 recombinant protein; however, expression of PDL-related markers did not increase compared with iPDLSC. The present results suggest the critical involvement of FBN2 in inducing iPDLSCs from iNCs when in fact it does not promote iPDLSC differantiation. Therefore, we need to elucidate the entire HPDLC-ECMs, responsible for iPDLSCs induction.

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.

Lipid droplets are a metabolic vulnerability in melanoma

Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the melanocytic cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved across human melanoma cell lines and patient tumors. This melanocytic state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon fatty acid oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt cell cycle progression and slow melanoma growth in vivo. Because the melanocytic cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body’s immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.

PAX9 Is Involved in Periodontal Ligament Stem Cell-like Differentiation of Human-Induced Pluripotent Stem Cells by Regulating Extracellular Matrix.

Periodontal ligament stem cells (PDLSCs) play central roles in periodontal ligament (PDL) tissue homeostasis, repair, and regeneration. Previously, we established a protocol to differentiate human-induced pluripotent stem cell-derived neural crest-like cells (iNCs) into PDLSC-like cells (iPDLSCs) using human PDL cell-derived extracellular matrix (ECM). However, it remained unclear what factors principally regulate the differentiation of iNCs into iPDLSCs. In this study, we aimed to identify the transcription factor regulating production of human PDL cell-derived ECM, which is responsible for the generation of iPDLSCs. We cultured iNCs on ECMs of two human PDL cell lines (HPDLC-3S and HPDLC-3U) and of human dermal fibroblasts (HDF). iNCs cultured on HPDLC-3U demonstrated higher iPDLSC-associated gene expression and mesenchymal differentiation capacity than cells cultured on HDF or HPDLC-3S. The transcription factor PAX9 was highly expressed in HPDLC-3U compared with HDF and HPDLC-3S. iNCs cultured on siPAX9-transfected HPDLC-3U displayed downregulation of iPDLSC-associated marker expression and adipocytic differentiation capacity relative to controls. Our findings suggest that PAX9 is one of the transcription factors regulating ECM production in human PDL cells, which is responsible for the differentiation of iNCs into iPDLSCs.

The effect of BMP4, FGF8 and WNT3a on mouse iPS cells differentiating to odontoblast-like cells.

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells.

IPSC-derived cranial neural crest-like cells can replicate dental pulp tissue with the aid of angiogenic hydrogel

The dental pulp has irreplaceable roles in maintaining healthy teeth and its regeneration is a primary aim of regenerative endodontics. This study aimed to replicate the characteristics of dental pulp tissue by using cranial neural crest (CNC)-like cells (CNCLCs); these cells were generated by modifying several steps of a previously established method for deriving NC-like cells from induced pluripotent stem cells (iPSCs). CNC is the anterior region of the neural crest in vertebrate embryos, which contains the primordium of dental pulp cells or odontoblasts. The produced CNCLCs showed approximately 2.5–12,000-fold upregulations of major CNC marker genes. Furthermore, the CNCLCs exhibited remarkable odontoblastic differentiation ability, especially when treated with a combination of the fibroblast growth factors (FGFs) FGF4 and FGF9. The FGFs induced odontoblast marker genes by 1.7–5.0-fold, as compared to bone morphogenetic protein 4 (BMP4) treatment. In a mouse subcutaneous implant model, the CNCLCs briefly fated with FGF4 + FGF9 replicated dental pulp tissue characteristics, such as harboring odontoblast-like cells, a dentin-like layer, and vast neovascularization, induced by the angiogenic self-assembling peptide hydrogel (SAPH), SLan. SLan acts as a versatile biocompatible scaffold in the canal space. This study demonstrated a successful collaboration between regenerative medicine and SAPH technology.

Abstract IA01: Strategies for targeted and immune therapy in melanoma

Abstract Melanoma cells activate pathways that are also activated in proliferating fibroblasts, endothelial cells, or even inflammatory and immune cells. The tumor-secreted proteins and expressed cell surface receptors are associated with growth, migration, invasion, and antiapoptosis. Then there is a group of molecules that is not associated with the phenotype of differentiated cells in skin or tumor stroma but that represents re-expressed developmental genes. Several developmental pathway genes, such as those associated with noncanonical Wnt or Notch signaling, are commonly found on stem cells from the dermis that can give rise to melanocytes and on melanoma cells, but they are absent from or not activated in epidermal melanocytes. We have recently identified a G protein-coupled receptor, LPAR1, that is highly activated in neural crest-like stem cells and melanoma cells and absent from melanocytes. Expression is associated with resistance to BRAFV600E inhibition. When other subpopulations of melanoma cells were characterized that were associated with resistance to BRAF, MEK, or ERK small-molecule inhibitors, their major common property was very slow proliferation, which was in contrast to the bulk of the tumor cells. These “label-retaining cells,” characterized by expression of the H3K4 demethylase JARID1B and the protease SERPIN E2, and the EGF and NGF receptors, are not only drug resistant but also highly invasive in vitro and in experimental animal model. Nonproliferating, drug-resistant malignant cells may pre-exist in tumors. As the majority of melanoma cells are killed by kinase inhibitors, multikinase receptor-expressing cells begin proliferating to repopulate the tumor to its original heterogeneity. Thus, melanomas contain small subpopulations representing only 0.1-5% of all malignant cells that are critical for growth, invasion, and therapy resistance. To achieve total elimination of all malignant cells, we have to develop strategies to target not only the bulk of the tumor cells but also those with intrinsic resistance to common drugs. To closely mimic the conditions in patients’ tumors, we have developed >500 patient-derived xenografts. Together with the >300 permanent cell lines, they represent all clinical, genetic, and biologic groups of the disease. Since the human tumors only proliferate in immune-deficient mice, we have developed two humanized models that recapitulate the infiltration by human leukocytes into the human tumors. Inhibition of checkpoints with clinical-grade inhibitors stimulated the immune response, leading to focal or general tumor regression; thus, the models allow investigating the mechanisms of both stimulation and suppression of the immune system. Citation Format: Meenhard Herlyn. Strategies for targeted and immune therapy in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA01.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Lineage-specific differentiation of osteogenic progenitors from pluripotent stem cells reveals the FGF1-RUNX2 association in neural crest-derived osteoprogenitors.

Human pluripotent stem cells (hPSCs) can provide a platform to model bone organogenesis and disease. To reflect the developmental process of the human skeleton, hPSC differentiation methods should include osteogenic progenitors (OPs) arising from three distinct embryonic lineages: the paraxial mesoderm, lateral plate mesoderm, and neural crest. Although OP differentiation protocols have been developed, the lineage from which they are derived, as well as characterization of their genetic and molecular differences, has not been well reported. Therefore, to generate lineage‐specific OPs from human embryonic stem cells and human induced pluripotent stem cells, we employed stepwise differentiation of paraxial mesoderm‐like cells, lateral plate mesoderm‐like cells, and neural crest‐like cells toward their respective OP subpopulation. Successful differentiation, confirmed through gene expression and in vivo assays, permitted the identification of transcriptomic signatures of all three cell populations. We also report, for the first time, high FGF1 levels in neural crest‐derived OPs—a notable finding given the critical role of fibroblast growth factors (FGFs) in osteogenesis and mineral homeostasis. Our results indicate that FGF1 influences RUNX2 levels, with concomitant changes in ERK1/2 signaling. Overall, our study further validates hPSCs' power to model bone development and disease and reveals new, potentially important pathways influencing these processes.

Investigate the Odontogenic Differentiation and Dentin-Pulp Tissue Regeneration Potential of Neural Crest Cells.

Stem cell-based developmental engineering has been considered as a promising strategy for tissue/organ regeneration. Tooth is formed by sequential reciprocal interactions between epithelium derived from surface ectoderm and mesenchymal cells derived from cranial neural crest. The neural crest cell is an appealing cell source for tooth development and regeneration research. In this study, we investigated the odontogenic differentiation and dentin-pulp complex regeneration potential of neural crest cells. Our results showed that neural crest cells (O9-1 mouse cranial neural crest cell line) can sequentially differentiate into dentin matrix acidic phosphoprotein 1 (DMP-1)-positive odontoblasts within a developing tooth germ in vitro. Moreover, O9-1 cells and induced pluripotent stem cell (iPSC)-derived neural crest-like cells (iNCLCs) can form well-organized vascularized dentin-pulp complex when transplanted in vivo with tooth scaffold. Furthermore, both O9-1 cells and iNCLCs can be differentiated into odontoblast-like cells, positive staining with odontogenic-related markers DMP-1 and dentin sialophosphoprotein (DSPP), under odontogenic induction with the administration of bone morphogenetic protein 4 (BMP-4). These results demonstrated that neural crest cells, especially the unlimited iNCLCs, are a promising cell source for tooth development and dental tissue/tooth organ regeneration studies.

The genetic program to specify ectodermal cells in ascidian embryos.

The ascidian belongs to the sister group of vertebrates and shares many features with them. The gene regulatory network (GRN) controlling gene expression in ascidian embryonic development leading to the tadpole larva has revealed evolutionarily conserved gene circuits between ascidians and vertebrates. These conserved mechanisms are indeed useful to infer the original developmental programs of the ancestral chordates. Simultaneously, these studies have revealed which gene circuits are missing in the ascidian GRN; these gene circuits may have been acquired in the vertebrate lineage. In particular, the GRN responsible for gene expression in ectodermal cells of ascidian embryos has revealed the genetic programs that regulate the regionalization of the brain, formation of palps derived from placode-like cells, and differentiation of sensory neurons derived from neural crest-like cells. We here discuss how these studies have given insights into the evolution of these traits.

Abstract ES10-2: Understanding breast cancer using a developmental perspective

Abstract Parallels among embryonic development, stem cells, and cancer have long been recognized. We identified, isolated, and characterized stem cells that first become committed to a mammary fate during embryogenesis; we refer to these cells as fetal mammary stem cells (fMaSCs). Lineage tracing, in vitro sphere formation, and in vivo transplantation studies by our group and many others all confirm that cells in the embryo are the bipotent progenitors of the mammary gland. There is debate, however, on whether such bipotent cells persist into the adult, or whether the luminal and basal lineages are maintained by unipotent progenitors. To gain insight into the relationships between fMaSCs and breast cancer, and to investigate their potential persistence in the adult, we have applied bulk and single cell RNA-sequencing (sc-RNA-seq) and single nucleus ATAC-sequencing (snATAC-seq) throughout mammary development. The results to be discussed demonstrate that fMaSC transcriptomes are heterogeneous, but all share co-expression of genes associated with luminal and basal cell fates. This fits a model in which the bipotent state is created by a balance of lineage specifiers. We also find that the fMaSC transcriptome is highly enriched in basal-like human breast cancers and identify potential embryonic pathways that correlate with poor prognosis. We used a variety of computational tools to infer the gene expression programs that ensue when fMaSCs commit to luminal and basal states. The data from scRNA-seq and snATAC-seq demonstrate that the transitions are gradual, not precipitous, and that luminal and basal cells exhibit significant transcriptomic and epigenetic heterogeneity. This challenges the notion that the mammary gland consists of discrete cell types defined by rigid transcriptomic parameters, and reveals a potential for intrinsic phenotypic plasticity of normal mammary cells. Using the combined databases, we identified Sox10 as a significantly differentially expressed cell state regulator. We show that tumors are heterogeneous with regard to Sox10 expression, and that locally invasive cells tend to express high Sox10 levels. Elevated Sox10 correlates with acquisition of a neural-crest like, EMT-related state. Implications for interception of metastasis by targeting neural crest-like cells will be discussed. Finally, we have generated a web resource that is available to the scientific community to enable the transcription and epigenetic characteristics of any gene of interest to be tracked through mammary development (https://wahl-labsalk.shinyapps.io/Mammary_snATAC/). Citation Format: GM Wahl, Z Ma, C Chung, C Dravis, BT Spike, RR Giraddi, O Balcioglu, C Fan, B Hagos, R Heinz, Herrera-Valdez J, X Hou, J Hwang, R Lasken, G Luna, NE Lytle, EM Mehrabad, M Novotny, CM Perou, O Poirion, S Preissl, B Ren, T Reya, CL Trejo, KT Varley. Understanding breast cancer using a developmental perspective [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES10-2.

The origin and evolution of vertebrate neural crest cells.

The neural crest is a vertebrate-specific migratory stem cell population that generates a remarkably diverse set of cell types and structures. Because many of the morphological, physiological and behavioural novelties of vertebrates are derived from neural crest cells, it is thought that the origin of this cell population was an important milestone in early vertebrate history. An outstanding question in the field of vertebrate evolutionary-developmental biology (evo-devo) is how this cell type evolved in ancestral vertebrates. In this review, we briefly summarize neural crest developmental genetics in vertebrates, focusing in particular on the gene regulatory interactions instructing their early formation within and migration from the dorsal neural tube. We then discuss how studies searching for homologues of neural crest cells in invertebrate chordates led to the discovery of neural crest-like cells in tunicates and the potential implications this has for tracing the pre-vertebrate origins of the neural crest population. Finally, we synthesize this information to propose a model to explain the origin of neural crest cells. We suggest that at least some of the regulatory components of early stages of neural crest development long pre-date vertebrate origins, perhaps dating back to the last common bilaterian ancestor. These components, originally directing neuroectodermal patterning and cell migration, served as a gene regulatory ‘scaffold' upon which neural crest-like cells with limited migration and potency evolved in the last common ancestor of tunicates and vertebrates. Finally, the acquisition of regulatory programmes controlling multipotency and long-range, directed migration led to the transition from neural crest-like cells in invertebrate chordates to multipotent migratory neural crest in the first vertebrates.

Molecular Characterization of Embryonic Stem Cell-Derived Cardiac Neural Crest-Like Cells Revealed a Spatiotemporal Expression of an Mlc-3 Isoform.

Background and ObjectivesPluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the ‘fourth’ germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands’ development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations.MethodsMurine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level.ResultsComparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved.ConclusionsOur findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

CD49fhigh Defines a Distinct Skin Mesenchymal Stem Cell Population Capable of Hair Follicle Epithelial Cell Maintenance

The dermis harbors distinct mesenchymal stem cell (MSC) populations, which play equally important roles as epidermal stem cells in skin homeostasis and regeneration. However, to reliably identify and directly isolate the invivo counterpart of these cells is still challenging. Using the epidermal stem cell marker CD49f, we defined a CD49fhigh distinct mesenchymal subpopulation in the dermis. Invitro and invivo differentiation assays, and transcriptome analysis demonstrated that CD49fhigh cells possess neural crest-like cell characteristics. Our results showed that the formation of hair follicle-like budding structure and the expressions of key genes regulating hair follicle development were induced when hair follicle epithelial cells were co-cultured with CD49fhigh cells. We also found that CD49fhigh cells activated Notch signaling in co-cultured hair follicle epithelial cells, whereas the inhibition of Notch signaling resulted in epidermal cyst-like spheres and loss of maintenance of hair follicle epithelial cell characteristics. Furthermore, we identified Itga7 and CD49f as an efficient biomarker panel for direct selection of CD49fhigh skin MSCs. Our results lead to a deeper understanding of heterogeneity and the function of MSCs in the skin and may facilitate potential translational applications of these cells.

Stage-dependent differential gene expression profiles of cranial neural crest-like cells derived from mouse-induced pluripotent stem cells

Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS proto-oncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration.