Cultures Of Neural Crest Cells Research Articles

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Mechanical dissection and culture of mouse cranial neural crest cells.

Owing to the contribution of cranial neural crest cells (CNCCs) to the majority of craniofacial structures, they have been studied extensively for the pathogenesis of craniofacial diseases. To investigate and summarize how to isolate and culture the CNCCs from wild-type mice, a literature search was performed in online databases (PubMed and Web of Science) using optimized keywords "mouse," "cranial neural crest cell" and "culture." The literature was checked by two investigators according to the screening and exclusion criteria. Initially, 197 studies were retrieved from PubMed and 169 from Web of Science, and after excluding replicate studies, 293 articles were considered. Finally, 17 studies met all the criteria and were included in this review. The results showed that obtaining purified stem cells and balancing the need to promote cell growth and prevent unwanted early cell differentiation were the two key points in the isolation and culture of CNCCs. However, no standard criteria are available for answering these questions. Thus, it is important to emphasize the necessity for standardization of CNCC isolation, culture, and identification in research on craniofacial diseases.

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

Isolation and Culture of Cranial Neural Crest Cells from the First Branchial Arch of Mice.

Craniofacial anomalies (CFA) are a diverse group of deformities, which affect the growth of the head and face. Dysregulation of cranial neural crest cell (NCC) migration, proliferation, differentiation, and/or cell fate specification have been reported to contribute to CFA. Understanding of the mechanisms through which cranial NCCs contribute for craniofacial development may lead to identifying meaningful clinical targets for the prevention and treatment of CFA. Isolation and culture of cranial NCCs in vitro facilitates screening and analyses of molecular cellular mechanisms of cranial NCCs implicated in craniofacial development. Here, we present a method for the isolation and culture of cranial NCCs harvested from the first branchial arch at early embryonic stages. Morphology of isolated cranial NCCs was similar to O9-1 cells, a cell line for neural crest stem cells. Moreover, cranial NCCs isolated from a transgenic mouse line with enhanced bone morphogenetic protein (BMP) signaling in NCCs showed an increase in their chondrogenic differentiation capacity, suggesting maintenance of their in vivo differentiation potentials observed in vitro. Taken together, our established method is useful to visualize cellular behaviors of cranial NCCs.

Isolation and primary culture of chick embryonic neural crest cells

هدف: هدف از این مطالعه ارائه یک روش ساده و کارآمد جهت جداسازی و تعیین خصوصیت سلول‏های ستیغ عصبی از بافت لوله عصبی می‏باشد.مواد و روش‏ها: تخم مرغ‏های نطفه‏دار حدود 35 ساعت در دمای 38 درجه سانتی‏گراد و رطوبت نسبی 55 تا 60 درصد در داخل انکوباتور قرار داده شدند تا جنین‏ها به‏مراحل 10-12 طبق جدول تکاملی هامبورگر-هامیلتون رسیدند. سپس جنین‏ها از روی سطح زرده تخم مرغ جداسازی شده و لوله عصبی از جنین جوجه جدا و به‏مدت 24 ساعت در ظروف کشت سلولی، کشت داده شد تا سلول‏های ستیغ عصبی از آن جدا شوند. آن‏گاه لوله عصبی از کف ظروف کشت جدا و خارج شد و به سلول‏ها به‏مدت 5 روز اجازه تکثیر و ازدیاد داده شد. در نهایت این سلول‏ها جمع‏آوری شدند و جهت ارزیابی پروفایل بیان ژن، PCR صورت گرفت.نتایج: سلول‏های ستیغ عصبی از لوله عصبی جدا شده و در شرایط کشت گسترش یافتند. این سلول‏ها هم‏چنین نشانگرهای Slug، Sox9 و Sox10 را به‏روش RT-PCR بیان کردند.نتیجه‏گیری: سلول‏های ستیغ عصبی می‏توانند در محیط آزمایشگاهی از لوله عصبی رها شده و در شرایط کشت مناسب و ساده تکثیر و گسترش یابند.

Establishing Primary Cultures of Trunk Neural Crest Cells.

Neural crest cells constitute a unique population of progenitor cells with extensive stem cell capacities able to navigate throughout various environments in the embryo and are a source of multiple cell types, including neurons, glia, melanocytes, smooth muscles, endocrine cells, cardiac cells, and also skeletal and supportive tissues in the head. Neural crest cells are not restricted to the embryo but persist as well in adult tissues where they provide a reservoir of stem cells with great therapeutic promise. Many fundamental questions in cell, developmental, and stem cell biology can be addressed using this system. During the last decades there has been an increased availability of elaborated techniques, animal models, and molecular tools to tackle neural crest cell development. However, these approaches are often very challenging and difficult to establish and they are not adapted for rapid functional investigations of mechanisms driving cell migration and differentiation. In addition, they are not adequate for collecting pure populations of neural crest cells usable in large scale analyses and for stem cell studies. Transferring and adapting the neural crest system in tissue culture may then represent an attractive alternative, opening up numerous prospects. Here we describe a simple method for establishing primary cultures of neural crest cells derived from trunk neural tubes using the avian embryo as a source of cells. This protocol is suited for producing pure populations of neural crest cells that can be processed for cytological, cellular, and functional approaches aimed at characterizing their phenotype, behavior, and potential. © 2020 Wiley Periodicals LLC. Basic Protocol: Primary cultures of avian trunk neural crest cells Support Protocol: Adaptations for immunofluorescence labeling and videomicroscopy.

Differential effects of fluoxetine and venlafaxine in the neural embryonic stem cell test (ESTn) revealed by a cell lineage map

There is a need for in vitro tests for the evaluation of chemicals and pharmaceuticals that may cause developmental neurotoxicity (DNT) in humans. The neural embryonic stem cell test (ESTn) is such an in vitro test that mimics early neural differentiation. The aim of this study was to define the biological domain of ESTn based on the expression of selective markers for certain cell types, and to investigate the effects of two antidepressants, fluoxetine (FLX) and venlafaxine (VNX), on neural differentiation. A cell lineage map was made to track neural differentiation and the effects of FLX and VNX in ESTn. Whole transcriptome analysis revealed differentiation from an embryonic stem cell population to a mixed culture of neural progenitors, neurons and neural crest cells 7 days into differentiation. Maturing neurons, astrocytes and oligodendrocytes were present after 13 days. Exposure to FLX or VNX led to different expression patterns between compounds at both time points. On day 7, both compounds upregulated most of the stem cell- and immature neuron markers, but had distinct effects on neural subtype markers. FLX downregulated glycinergic markers and upregulated cholinergic markers, while VNX had the opposite effect. On day 13, FLX and VNX affected their specific therapeutic targets, represented by mainly serotonergic markers by FLX- and dopaminergic and noradrenergic markers in VNX-exposed cultures, as well as oligodendrocyte and glycinergic neuron markers. This proof of concept study shows the added value of assessing DNT in ESTn through a cell lineage map and gives mechanistic insight in the potential neurodevelopmental effects of FLX and VNX. More compounds should be tested to further evaluate the use of the cell lineage map.

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Zika virus induces abnormal cranial osteogenesis by negatively affecting cranial neural crest development

Zika virus (ZIKV) infection during gestation is deemed to be coupled to birth defects through direct impairment of the nervous system during neurogenesis. However, in this study, our data showed that ZIKV infection dramatically suppressed cranial osteogenesis, shown by Safranin O/Fast Green and alizarin red staining, in chick embryos, which provides another possibility that craniofacial bone malformation caused by ZIKV may be a major cause of ZIKV-mediated birth defects. By immunofluorescent staining and electron microcopy, we confirmed ZIKV infection in chick embryo neural tubes and sites of neural crest. Next, in vivo (chick embryos) and in vitro [primary culture of neural crest cells (NCC)] ZIKV and HNK-1 double immunofluorescent staining demonstrated that ZIKV infection inhibited the production of migratory NCC. The reduction of both AP-2α- and Pax7-positive NCC in HH10 chick embryos infected by ZIKV confirmed that abnormal development of cranial NCC also occurred in the migratory process. Whole mount in situ hybridization demonstrated that cadherin 6B expression was elevated and Slug, FoxD3, and BMP4/Msx1 expressions decreased in ZIKV-infected HH10 chick embryos, implying that epithelial–mesenchymal transition (EMT) of neural crest production was blocked by ZIKV infection. Moreover, in vivo and in vitro pHIS3 and Pax7 double immunofluorescent staining showed that NCC proliferation was repressed by ZIKV infection. C-caspase-3 and AP-2α double immunofluorescent staining in HH10 chick embryos and western blotting showed that NCC apoptosis increased following ZIKV infection. Finally, electron microscopy showed multiple autophagosomes in ZIKV-infected embryos, and western blot and LC3B immunofluorescent staining demonstrated that autophagy-related genes were activated by ZIKV infection. Taken together, our data first showed that ZIKV infection during embryogenesis could interfere with cranial neural crest development, which in turn causes aberrant cranial osteogenesis. Our results provided new insights into brain malformations induced by ZIKV infection.

Ex Vivo Culture and Manipulation of Mouse Neural Crest Cells from Primary Embryonic Tissue Explants.

In vitro culture of neural crest cells allows for the manipulation and study of neural crest cell function in a cell-autonomous manner. While several stable neural crest cell lines exist, the transformed nature of these cells may not closely reflect the in vivo properties of neural crest cells, hence making molecular and functional analyses using these cell lines difficult to interpret. Here we describe a robust method to culture primary mouse neural crest cells ex vivo for several days to weeks in culture. We further describe a method for siRNA knockdown in these cells to study gene function. This culture method can also be adapted for other molecular analyses, including addition of small-molecule inhibitors and/or growth factors to the culture media, as well as culturing neural crest cells from knockout or genetically modified mice.

In Vitro Maintenance of Multipotent Neural Crest Stem Cells as Crestospheres.

Neural crest cells are a critical source of many cell types of the vertebrate body. However, as a stem cell population they are peculiar because of the transient nature of their stem cell niche; soon after the multipotent neural crest cells are specified in the neuroepithelium, they become mesenchymal cells that migrate into various destinations in early embryos. These rapid in vivo changes during neural crest development complicate the studies on their stem cell properties. Crestospheres are in vitro maintained primary cultures of premigratory neural crest cells that maintain a mixture of neural crest stem and progenitor cells for weeks without spontaneous differentiation, including the multipotent neural crest stem cells. Here, we describe how crestosphere cultures are initiated from either cranial or trunk levels of chick embryos. Alternatively, the same culture conditions can be used to maintain human embryonic stem cell-derived neural crest cells as crestospheres. Thus, crestospheres provide a useful tool for studies on neural crest stemness.

Embryonic cell culture in zebrafish.

Zebrafish embryonic cell cultures have many useful properties that make them complementary to intact embryos for a wide range of studies. Embryonic cell cultures allow for maintenance of transient cell populations, control of chemical and mechanical cues received by cells, and facile chemical screening. Zebrafish cells can be cultured in either heterogeneous or homogeneous cultures from a wide range of developmental time points. Here we describe two methods with particular applicability to chemical screening: a method for the culture of blastomeres for directed differentiation toward the myogenic lineageand a method for the culture of neural crest cells in heterogeneous cultures from early somitogenesis embryos.

Rutin increases neural crest stem cell survival against damage caused by aflatoxin B1

The neural crest (NC) corresponds to a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potential. The derivatives of the NC at the trunk level include neurons and glial cells of the peripheral nervous system, melanocytes, smooth muscle cells and some endocrine cells. The present work investigated, for the first time, the influence of aflatoxin B1 (AFB1) and the flavonoid rutin on the survival and proliferation of NC and NC-derived melanocytes. Quail NC cell cultures were treated with AFB1 (30 μM) and/or rutin (20 μM) for 6 days. Cell viability was assessed by MTT and trypan blue analyses and cell proliferation by BrdU staining. Melanocytes were identified by immunocytochemistry against the melanocyte-specific cellular marker MelEM. The AFB1 treatment decreased both NC cell viability and proliferation. The total number of MelEM-positive cells was also reduced after this treatment, an effect partially prevented by the addition of rutin. On the other hand, rutin added alone did not influence the NC cell population. Our results demonstrated that rutin increases the survival of the NC after damage caused by AFB1. However, additional studies are needed to better understand the mechanisms involved in AFB1 and rutin interactions.

Influence of intracellular zinc on cultures of rat cardiac neural crest cells.

Developmental zinc (Zn) deficiency increases the incidence of heart anomalies in rat fetuses, in regions and structures derived from the outflow tract. Given that the development of the outflow tract requires the presence of cardiac neural crest cells (cNCC), we speculated that Zn deficiency selectively kills cNCC and could lead to heart malformations. Cardiac NCC were isolated from E10.5 rat embryos and cultured in control media (CTRL), media containing 3 μM of the cell permeable metal chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), or in TPEN-treated media supplemented with 3 μM Zn (TPEN + Zn). Cardiac NCC were collected after 6, 8, and 24 h of treatment to assess cell viability, proliferation, and apoptosis. The addition of TPEN to the culture media reduced free intracellular Zn pools and cell viability as assessed by low ATP production, compared to cells grown in control or Zn-supplemented media. There was an accumulation of reactive oxygen species, a release of mitochondrial cytochrome c into the cytoplasm, and an increased cellular expression of active caspase-3 in TPEN-treated cNCC compared to cNCC cultured in CTRL or TPEN + Zn media. Zn deficiency can result in oxidative stress in cNCC, and subsequent decreases in their population and metabolic activity. These data support the concept that Zn deficiency associated developmental heart defects may arise in part as a consequence of altered cNCC metabolism.

Bentonite protects neural crest stem cells from death caused by aflatoxin B1

Bentonites, which have been shown to be good mycotoxin adsorbents, have a wide range of biochemical and industrial uses. Although many in vitro studies have shown the effects of bentonite on a variety of cell types, there is no comparable information regarding its effects on neural crest (NC) stem cells. The NC is a collection of multipotent and oligopotent progenitors endowed with both neural and mesenchymal potentials. NC cells originated at trunk level can become peripheral nervous system cells, melanocytes and smooth muscle cells. For this reason, the NC is considered an ideal model for studying the role of different substances on cell differentiation, survival and growth. We have recently shown that aflatoxin B1 (AFB1) decreases the viability and reduces the population of NC cells. In the present work, we have characterized bentonite extracted from the South of Brazil and investigated its effects on NC differentiation and survival, when challenged with AFB1. The results have highlighted the high content of SiO2, Fe2O3 and K2O present in bentonite, with content of MnO, MgO, CaO, Na2O, TiO2 and P2O5 lower than 1%. These results have also shown that bentonite at 0.6mg/mL is not toxic to NC cells in culture. Furthermore, bentonite increased the viability of NC cells previously treated with AFB1 without affecting cell differentiation. The evidence suggests that AFB1 interacted with bentonite by occupying the interlayer spaces, which might have contributed to a reduction of AFB1 availability and, consequently, a reduction in NC damage and an increase in the total number of cells. Our study contributes to a better understanding of the effects of clay minerals on the NC and may help to create new, more effective pharmacological applications to reduce or eliminate the toxic effects of AFB1.

Down-regulation of RE-1 silencing transcription factor (REST) in advanced prostate cancer by hypoxia-induced miR-106b~25

Clinically aggressive prostate cancer (PCa) is linked to androgen resistance, metastasis, and expression of neuroendocrine markers. To understand mechanism(s) of neuroendocrine differentiation (NED) of PCa epithelia, we compared neuronal differentiation occurring during embryogenesis, in primary cultures of neural crest (NC) cells, and NED in PCa cell lines (LNCaP and PC3). We demonstrate, hypoxia promotes neuronal and neuroendocrine differentiation of NC cells and PCa cells, respectively, by inducing the miR-106b~25 cluster. In turn, miR-106b~25 comprised of miR-106b, miR-93 and miR-25, down-regulates the transcriptional repressor REST, which represses neuron-specific protein-coding and miRNA genes. In prostate tumors of high Gleason score (≥8), an inverse trend was observed between REST and miR-106b~25 induction. Employing miRNA PCR arrays, we identified miRNAs up-regulated by hypoxia in LNCaP cells and REST-knockdown in NC cells. Significantly, a subset of miRNAs (miR-9, miR-25, miR-30d and miR302b) is up-regulated in high Gleason score (≥8) PCa, suggesting a mechanism by which NED contributes to PCa malignancy. We propose that loss of REST and induction of this set of microRNAs can serve as potential novel clinical markers of advanced PCa.

Matrigel supports neural, melanocytic and chondrogenic differentiation of trunk neural crest cells

The neural crest (NC) is composed of highly multipotent precursor cells able to differentiate into both neural and mesenchymal phenotypes. Until now, most studies focusing on NC cell differentiation have been performed with traditional two-dimensional (2D) cell culture systems. However, such culture systems do not reflect the complex three-dimensional (3D) microenvironments of in vivo NC cells. To address this limitation, we have developed a method of Matrigel™ coating to create 2D and 3D microenvironments in the same culture well. When we performed cultures of trunk neural crest cells (TNCCs) on three different lots of basement membrane matrix (Matrigel™), we observed that all analyzed Matrigel™ lots were equally efficient in allowing the appearance of glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes. We further observed that chondrocytes were found predominantly in the 3D microenvironment, whereas smooth muscle cells were almost exclusively located in the 2D microenvironment. Glial cells were present in both environments, but with broader quantities on the 2D surface. Melanocytes and neurons were equally distributed in both 2D and 3D microenvironments, but with distinct morphologies. It is worth noting the higher frequency of chondrocytes detected in this study using the 3D Matrigel™ microenvironment compared to previous reports of chondrogenesis obtained from TNCCs on traditional 2D cultures. In conclusion, Matrigel™ represents an attractive scaffold to study NC multipotentiality and differentiation, since it permits the appearance of the major NC phenotypes.