Search

It has long been held that the main difference between cranial and trunk neural crest (CNC and TNC, respectively) was the potential of CNC to originate mesenchymal cell types, especially skeletogenic. This is an age-old question that continues to challenge researchers, even today. Unfortunately, to date, no consensus has concluded the extent of TNC mesenchymal potential, nor has a systematic review been conducted to organize current knowledge about this fascinating question. However, the number of studies related to this question have expanded and deepened considerably in the last few years thanks to several new different species of vertebrates employed, the generation of transgenic animal strains, the combination of cell markers, and also the improvement of cell culture conditions through the use of different substrates and signaling molecules. Therefore, this review summarizes the literature showing that TNCCs can generate a broad range of mesenchymal cell types, including skeletogenic. This potential can be unveiled by certain favorable in vitro conditions, but it also seems to be expressed in some animal structures in vivo , to which TNCCs contribute. We also present several works that offer a contrary view and do not detect any mesenchymal/skeletogenic contribution of TNCCs in vivo . Perhaps, it is the controversy itself that makes this subject even more exciting.

The neural crest (NC) is a transitory embryonic structure of vertebrates that gives rise to an astonishing variety of derivatives, encompassing both neural and mesenchymal cell types. Neural crest cells (NCCs) are an excellent model to study how environmental factors modulate features such as cell multipotentiality and differentiation. Tests with multifunctional substrates that allow NCCs to express their full potential, while promoting cell subcloning, are needed to advance knowledge about NCC self-renewal and to foster future biotechnological approaches. Here we show that a self-assembled peptide named PuraMatrix TM is an excellent substrate that allows the differentiation of NCCs based on the identification of seven different cell types. Depending on the PuraMatrix TM concentration employed, different frequencies and quantities of a given cell type were obtained. It is noteworthy that an enormous quantity and diversity of mesenchymal phenotypes, such as chondrocytes, could be observed. The quantity of adipocytes and osteocytes also increased with the use of mesenchymal differentiation factors (MDF), but PuraMatrix TM alone can support the appearance of these mesenchymal cell types. PuraMatrix TM will promote advances in studies related to multipotentiality, self-renewal and control of NCC differentiation, since it is an extremely simple and versatile material which can be employed for both in vivo and in vitro experiments.

The developmental mechanisms that control the building of the complex head of vertebrates and particularly, facial skeletogenesis, remain poorly known. Progenitor cells derived from the embryonic neural crest (NC) are the major constituents and players of facial tissue development. Deciphering the cellular and molecular machinery that controls NC cell (NCC) differentiation into bone, cartilage, fat and other mesenchymal tissues, is thus a main issue for understanding vertebrate facial variations. In this work, we investigated the effects of fibroblast growth factor 8 (FGF8) and Sonic Hedgehog (Shh), two signaling molecules essential for craniofacial development, on the in vitro differentiation and multipotentiality of mesencephalic NCCs (MNCCs) isolated from the quail embryo. Comparison of distinct temporal treatments with FGF8 and/or Shh showed that both promoted chondrogenesis of MNCCs by increasing the amount and size of cartilage nodules. Higher rates of chondrogenesis were observed when MNCCs were treated with FGF8 during the migration phase, thus mimicking the in vivo exposure of migrating NCCs to FGF8 secreted by the isthmic brain signaling center. An in vitro cell cloning assay revealed that, after concomitant treatment with FGF8 and Shh, about 80% of NC progenitors displayed chondrogenic potential, while in untreated cultures, only 18% exhibited this potential. In addition, colony analysis showed for the first time the existence of a highly multipotent progenitor able to clonally give rise to adipocytes in addition to other cephalic NC phenotypes (i.e. glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes) (GNMFCA progenitor). This progenitor was observed only when clonal cultures were treated with both FGF8 and Shh. Several other types of multipotent cells, which generated four, five or six distinct phenotypes, accounted for 55% of the progenitors in FGF8 and Shh treated cultures, versus 13,5% in the untreated ones. Together, these data reveal an essential role for both FGF8 and Shh together in maintenance of MNCC multipotentiality by favoring the development of NC progenitors endowed with a broad array of mesectodermal potentials., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In the neural primordium of vertebrate embryos, the neural crest (NC) displays a unique character: the capacity of its component cells to leave the neural primordium, migrate along definite (and, for long, not identified) routes in the developing embryo and invade virtually all tissues and organs, while producing a large array of differentiated cell types. The most striking diversity of the NC derivatives is found in its cephalic domain that produces, not only melanocytes and peripheral nerves and ganglia, but also various mesenchymal derivatives (connective tissues, bones, cartilages…) which, in other parts of the body, are mesoderm-derived. The aim of this article was to review the large amount of work that has been devoted to solving the problem of the differentiation capacities of individual NC cells (NCC) arising from both the cephalic and trunk levels of the neural axis. A variety of experimental designs applied to NCC either in vivo or in vitro are evaluated, including the possibility to culture them in crestospheres, a technique previously designed for cells of the CNS, and which reinforces the notion, previously put forward, of the existence of NC stem cells. At the trunk level, the developmental potentialities of the NCC are more restricted than in their cephalic counterparts, but, in addition to the neural-melanocytic fate that they exclusively express in vivo, it was clearly shown that they harbor mesenchymal capacities that can be revealed in vitro. Finally, a large amount of evidence has been obtained that, during the migration process, most of the NCC are multipotent with a variable array of potentialities among the cells considered. Investigations carried out in adults have shown that multipotent NC stem cells persist in the various sites of the body occupied by NCC. Enlightening new developments concerning the invasive capacity of NCC, the growing peripheral nerves were revealed as migration routes for NCC travelling to distant ventrolateral regions of the body. Designated "Schwann cell precursors" in the mouse embryo, these NCC can leave the nerves and are able to convert to a novel fate. The convertibility of the NC-derived cells, particularly evident in the Schwann cell-melanocyte lineage transition, has also been demonstrated for neuroendocrine cells of the adult carotid body and for the differentiation of parasympathetic neurons of ganglia distant from their origin, the NC. All these new developments attest the vitality of the research on the NC, a field that characterizes vertebrate development and for which the interest has constantly increased during the last decades., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Carrageenan is a thermoreversible polymer of natural origin widely used in food and pharmaceutical industry that presents a glycosaminoglycan-like structure. Herein, we show that kappa-type carrageenan extracted by a semi-refined process from the red seaweed Kappaphycus alvarezii displayed both chemical and structural properties similar to a commercial carrageenan. Moreover, both extracted carrageenan hydrogel and commercial carrageenan hydrogel can serve as a scaffold for in vitro culture of human skin-derived multipotent stromal cells, demonstrating considerable potential as cell-carrier materials for cell delivery in tissue engineering. Skin-derived multipotent stromal cells cultured inside the carrageenan hydrogels showed a round shape morphology and maintained their growth and viability for at least one week in culture. Next, the effect of the extracted carrageenan hydrogel loaded with human skin-derived multipotent stromal cells was evaluated in a mouse model of full-thickness skin wound. Macroscopic and histological analyses revealed some pointed ameliorated features, such as reduced inflammatory process, faster initial recovery of wounded area, and improved extracellular matrix deposition. These results indicate that extracted carrageenan hydrogel can serve as a scaffold for in vitro growth and maintenance of human SD-MSCs, being also able to act as a delivery system of cells to wounded skin. Thus, evaluation of the properties discussed in this study contribute to a further understanding and specificities of the potential use of carrageenan hydrogel as a delivery system for several applications, further to skin wound healing.

The neural crest (NC) is an attractive system for investigating the mechanisms underlying cell lineage diversification in higher vertebrates. The NC contains a mixed population of already defined precursors and multipotent cells that can give rise to a great variety of cell types, including glial cells and neurons of the peripheral nervous system, melanocytes, and smooth muscle cells (SMCs). Microenvironmental factors, such as the fibroblast growth factor 2 (FGF2), found along migratory paths and in target tissues, strongly influence the fate of multipotent NC precursors. We have previously demonstrated that the FGF2 promotes the differentiation of NC cells to glial phenotypes, while the epidermal growth factor induces NC differentiation to neurons and melanocytes. In the present study, we used mass cultures and single-cell culture assays to demonstrate that FGF2 influences NC cell differentiation and increases the proportion of multipotent progenitors. Furthermore, we demonstrate for the first time that avian tripotent glial, melanocyte and smooth muscle NC progenitors, as well as bipotent melanocyte and smooth muscle NC progenitors, are capable of self-renewal. FGF2 significantly stimulated the self-renewal of bipotent progenitor cells with glial cells and SMC potentials. These cells propagated for many generations and behaved as stem cells. These results suggest an important role of FGF2 in maintaining the stemness of avian NC 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.

In the amniote embryo, the neural crest (NC) has the unique capacity to give rise to neuronal and glial cells in the peripheral nervous system (PNS), melanocytes and mesenchymal cells including those forming the head skeleton and connective tissues. In the trunk, mesenchymal cells are derived from the mesoderm. The question was raised whether the NC-derived head mesenchyme arises from a lineage separate from the neural-melanocytic one, or if both skeletogenic and neural-melanocytic derivatives originate from a common putative stem cell in the early cephalic NC. We discuss here these issues and present experimental data that provide evidence for the multipotency of NC cells (NCC), focusing on those at the origin of the craniofacial skeleton. Recent work of in vitro clonal culture revealed that the vast majority (92% of clonogenic cells) of the cephalic quail NCC are capable to yield osteoblasts together with neurones, glial cells and melanocytes. A common pluripotent progenitor for chondrocytes, osteocytes, neurones, glial cells, melanocytes and myofibroblasts has been identified and is present in the early cephalic NC at the frequency of 7 to 13% of clonogenic cells depending on the environmental conditions. Together with recent reports that multipotent NC-related progenitors persist in adult tissues in rodents and humans, these results reinforce a stem cell model for the generation and maintenance of NC-derived lineages during embryogenesis and in adult tissue homeostasis.

The neural crest (NC) is a vertebrate innovation that distinguishes vertebrates from other chordates and was critical for the development and evolution of a "New Head and Brain." In early vertebrates, the NC was the source of dermal armor of fossil jawless fish. In extant vertebrates, including mammals, the NC forms the peripheral nervous system, melanocytes, and the cartilage and bone of the face. Here, we show that in avian embryos, a large majority of cephalic NC cells (CNCCs) have the ability to differentiate into cell types as diverse as neurons, melanocytes, osteocytes, and chondrocytes. Moreover, we find that the morphogen Sonic hedgehog (Shh) acts on CNCCs to increase endochondral osteogenesis while having no effect on osteoblasts prone to membranous ossification. We have developed culture conditions that demonstrate that "neural-mesenchymal" differentiation abilities are present in more than 90% of CNCCs. A highly multipotent progenitor (able to yield neurons, glia, melanocytes, myofibroblasts, chondrocytes, and osteocytes) comprises 7-13% of the clonogenic cells in the absence and presence of Shh, respectively. This progenitor is a good candidate for a cephalic NC stem cell.

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.

In the vertebrate embryo, the neurectodermal neural crest cells (NCC) have remarkably broad potencies, giving rise, after a migratory phase, to neurons and glial cells in the peripheral nervous system, and to skin melanocytes, being all designated here as "neural" derivatives. NC-derived cells also include non-neural, "mesenchymal" cell types like chondrocytes and bone cells, myofibroblasts and adipocytes, which largely contribute to the head structures in amniotes. Similar to the blood cell system, the NC is therefore a valuable model to investigate the mechanisms of cell lineage diversification in vertebrates. Whether NCC are endowed with multiple differentiation potentials or if, conversely, they are a mosaic of different committed cells is an important ongoing issue to understand the ontogeny of NC derivatives in normal development and pathological conditions. Here we focus on recent findings that established the presence in the early migratory NC of the avian embryo, of a multipotent progenitor endowed with both mesenchymal and neural differentiation capacities. This "mesenchymal-neural" clonogenic cell lies upstream of all the other NC progenitors known so far and shows increased frequency when single cell cultures are treated with the Sonic Hedgehog signaling molecule. These findings are discussed in the context of the broad potentials of NC stem cells recently evidenced in certain adult mammalian tissues.

In the vertebrate embryo, the cephalic neural crest cells (CNCCs) produce cells belonging to two main lineages: the neural [including neurons, glial cells of the peripheral nervous system (PNS), and melanocytes] and the mesenchymal (chondrocytes, osteoblasts, smooth muscle cells, and connective tissue cells), whereas the trunk NCCs (TNCCs) in amniotes yield only neural derivatives. Although multipotent cells have previously been evidenced by in vitro clonal analysis, the issue as to whether all of the mesenchymal and neural phenotypes can be derived from a unique NC stem cell has remained elusive. In the present work, we devised culture conditions that led us to identify a highly multipotent NCC endowed with both neural and mesenchymal potentials, which lies upstream of all the other NC progenitors known so far. We found that addition of recombinant Sonic Hedgehog (Shh) increased the number of CNCC progenitors yielding both mesenchymal and neural lineages and promoted the development of such precursors from the TNCC. Shh decreased the neural-restricted precursors without affecting the overall CNCC survival and proliferation. By showing a differential positive effect of Shh on the expression of mesenchymal phenotypes (i.e., chondrocytes and smooth muscle cells) by multipotent CNCCs, these results shed insights on the in vivo requirement of Shh for craniofacial morphogenesis. Together with evolutionary considerations, these data also suggest that the mesenchymal-neural precursor represents the ancestral form of the NC stem cell, which in extinct forms of vertebrates (the ostracoderms) was able to yield both the PNS and superficial skeleton.

Thyroid hormone deficiency during the critical period of neural differentiation produces permanent and severe alterations in the morphology and function of the nervous system leading to cretinism. Perinatal hypothyroidism results in permanent alterations of hippocampal synaptic functions in adult rats consequently causing learning and memory impairment. Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that regulate essential cellular activities ranging from gene expression, mitosis, programmed cell death to plasticity and memory formation, but their involvement in perinatal hypothyroidism is not determined. The present work was designed to investigate MAPKs phosphorylation in hippocampus of congenital neonatal hypothyroid rats. Congenital hypothyroidism promotes an increase in extracellular signal-regulated kinases 1/2 (ERK 1/2) phosphorylation (+50%) and a decrease in p38(MAPK) phosphorylation (-50%) without changing in Jun N-terminal kinases (JNK) phosphorylation. Therefore, the congenital hypothyroidism model disturbs ERK 1/2 and p38(MAPK) phosphorylation pathways causing an important molecular alteration in the hippocampus. This event might be related, at least partially, to the deficits in hippocampal development and cognitive functions due neonatal congenital hypothyroidism.

The effects of thyroid hormone (T3) deficiency on extracellular matrix protein expression were analyzed in newborn rat brain. In hypothyroid animals, a marked increase in the expression of 62 kDa protein was observed in cerebral hemispheres and midbrain, while the 51.6 kDa protein was increased in cerebral hemispheres and decreased in midbrain and the 44.5 kDa protein was down regulated in both structures. On the basis of molecular weights, these proteins may be the proteoglycans cerebroglycan, glypican and N-Syndecan, respectively. In addition, hypothyroidism reduced fibronectin expression in midbrain (-59,7%), but not in cerebral hemispheres. T3 deficiency affects differently the expression of proteins in different brain regions. This may be involved in brain impairment caused by hypothyroidism.