Cellular interactions during cartilage and bone development.

Both interactions between like cells, as between chondrogenic cells in a developing cartilaginous rudiment, and between unlike cells, as in epithelial-mesenchymal interactions, are dealt with in this review. Such interactions may involve direct apposition of cell membranes or may be mediated via interaction with peri- or extracellular matrices. An ontogenetic approach is taken in which cellular interactions involved in five processes of the development of cartilage and bone are discussed, the five being (1) origin of the cells, (2) migration of the cells within the embryo, (3) localization of the cells at their final embryonic site, (4) differentiation, and (5) morphogenesis. Some emphasis is placed on interactions affecting neural crest-derived cells both before and during their migration and on interactions, especially epithelial-mesenchymal interactions, that precede cytodifferentiation of chondroblasts or osteoblasts. Whether epithelial or mesenchymal specificity is required for such interactions to occur is discussed with reference to the otic vesicle-otic mesenchyme interaction that leads to differentiation and morphogenesis of the cartilaginous otic capsule.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Regenerative therapy for neuronal diseases with transplantation of somatic stem cells

Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described.     

Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos

Epithelial-mesenchymal interactions are crucial for the development of the endoderm of the pharyngeal pouches into the epithelia of thymus and parathyroid glands. Here we investigated the dynamics of epithelial-mesenchymal interactions that take place at the earliest stages of thymic and parathyroid organogenesis using the quail-chick model together with a co-culture system capable of reproducing these early events in vitro. The presumptive territories of thymus and parathyroid epithelia were identified in three-dimensionally preserved pharyngeal endoderm of embryonic day 4.5 chick embryos on the basis of the expression of Foxn1 and Gcm2, respectively: the thymic rudiment is located in the dorsal domain of the third and fourth pouches, while the parathyroid rudiment occupies a more medial/anterior pouch domain. Using in vitro quail-chick tissue associations combined with in ovo transplantations, we show that the somatopleural but not the limb bud mesenchyme, can mimic the role of neural crest-derived pharyngeal mesenchyme to sustain development of these glands up to terminal differentiation. Furthermore, mesenchymal-derived Bmp4 appears to be essential to promote early stages of endoderm development during a short window of time, irrespective of the mesenchymal source. In vivo studies using the quail-chick system and implantation of growth factor soaked-beads further showed that expression of Bmp4 by the mesenchyme is necessary during a 24 h-period of time. After this period however, Bmp4 is no longer required and another signalling factor produced by the mesenchyme, Fgf10, influences later differentiation of the pouch endoderm. These results show that morphological development and cell differentiation of thymus and parathyroid epithelia require a succession of signals emanating from the associated mesenchyme, among which Bmp4 plays a pivotal role for triggering thymic epithelium specification.     

Differentiation of human adipose-derived adult stem cells into neuronal tissue: Does it work?

Adipose tissue contains many cells and proteins that are of value not only for their potential therapeutic applications, but also for the low cost of their harvest and delivery. Mesenchymal stem cells (MSC) were originally isolated from the bone marrow, although similar populations have been isolated from adipose and other tissues. At one time, neural tissues were not regarded as regenerative populations of cells. Therefore, the identification of cell populations capable of neuronal differentiation has generated immense interest. Adipose tissue may represent an alternative source of cells that are capable of neuronal differentiation, potentially enhancing its use in the treatment of neurological disease. The aim of this review is to cover the current state of knowledge of the differentiation potential of human adipose-derived stem (ADAS) cells, specifically their ability to give rise to neuronal cells in vitro. This review presents and discusses different protocols used for inducing human ADAS cells to differentiate in vitro, and the neuronal markers utilized in each system.     

The potential of adipose-derived adult stem cells as a source of neuronal progenitor cells

Adipose-derived adult stem cells are a population of mesenchymal stem cells extracted from discarded adipose tissue. Many have reported the differentiation of adipose-derived stem cells into chondrocytes, myocytes, osteoblasts, and, most recently, neural progenitor cells. This article covers the current state of the potential of the differentiation of adipose-derived stem cells into neuronal cells and an overview of their potential as adult stem therapies for neurological disorders. It has been reported that adipose-derived stem cells are capable of undergoing neuronal differentiation using protocols similar to that of Woodbury et al., which reported the differentiation of bone marrow stromal cells specifically into neurons. However, the transdifferentiation of bone marrow stromal cells into neuronal cells has recently fallen under intense criticism, which will likely place the plasticity of adipose-derived stem cells under scrutiny as well. To date, no group has produced evidence that adipose-derived stem cells are capable of differentiating to mature, functional neuronal cells in vitro. However, recent in vivo studies with adipose-derived stem cells are promising.     

Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells

To determine if neural crest cells are pluripotent and establish whether differentiation occurs in the absence of noncrest cells, a cell culture method was devised in which differentiation could be examined in clones derived from single, isolated neural crest cells. Single neural crest cells, which were isolated before the onset of in vivo migration, gave rise to three types of clones: pigmented, unpigmented, and mixed. Pigmented clones consisted of melanocytes only, whereas some unpigmented cells in mixed and unpigmented clones contained catecholamines, identifying them as adrenergic cells. Extracellular matrix derived from quail somite or chick skin fibroblast cultures stimulated adrenergic differentiation and axon formation. These results demonstrate for the first time the existence of pluripotent quail neural crest cells that give rise to at least two progeny, melanocytes and neuronal cells. They also suggest that continuous direct interactions with noncrest cells are not required for the differentiation of these two cell types. However, components of the extracellular matrix derived from noncrest cells may play an important role in expression of the adrenergic phenotype.     

Human dental mesenchymal stem cells and neural regeneration

Nerve tissue presents inherent difficulties for its effective regeneration. Stem cell transplantation is considered an auspicious treatment for neuronal injuries. Recently, human dental mesenchymal stem cells (DMSCs) have received extensive attention in the field of regenerative medicine due to their accessibility and multipotency. Since their origin is within the neural crest, they can be differentiated into neural crest-derived cells including neuron and glia cells both in vitro and in vivo. DMSCs are also able to secrete a wide variety of neurotrophins and chemokines, which promote neuronal cells to survival and differentiation. Experimental evidence has shown that human DMSCs engraftment recovered neuronal tissue damage in animal models of central nervous system injuries. Human DMSCs can be a new hope for treatment of nervous system diseases and deficits such as spinal cord injury, stroke and Parkinson’s disease.     

Functional diversity of notch family genes in fetal lung development

In Drosophila, developmental signaling via the transmembrane Notch receptor modulates branching morphogenesis and neuronal differentiation. To determine whether the notch gene family can regulate mammalian organogenesis, including neuroendocrine cell differentiation, we evaluated developing murine lung. After demonstrating gene expression for notch-1, notch-2, notch-3, and the Notch ligands jagged-1 and jagged-2 in embryonic mouse lung, we tested whether altering expression of these genes can modulate branching morphogenesis. Branching of embryonic day (E) 11.5 lung buds increased when they were treated with notch-1 antisense oligodeoxynucleotides in culture compared with the corresponding sense controls, whereas notch-2, notch-3, jagged-1, or jagged-2 antisense oligos had no significant effect. To assess cell differentiation, we immunostained lung bud cultures for the neural/neuroendocrine marker PGP9.5. Antisense to notch-1 or jagged-1 markedly increased numbers of PGP9.5-positive neuroendocrine cells alone without affecting neural tissue, whereas only neural tissue was promoted by notch-3 antisense in culture. There was no significant effect on cell proliferation or apoptosis in these antisense experiments. Cumulatively, these observations suggest that interactions between distinct Notch family members can have diverse tissue-specific regulatory functions during development, arguing against simple functional redundancy.     

Canonical Wnt/β-Catenin Signalling Is Essential for Optic Cup Formation

A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.     

N-CAM expression and localization in PC12 cells modulated by extracellular peptides

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity.

We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells.

The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.     

Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells

Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin-integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP(+)). We here report that high levels of nestin promoter activity correlated with high expression levels of alpha(5)beta(1) integrin (alpha(5)beta(1)(high) cells). FACS analysis of nestin-GFP(+) cortical cells revealed an additional subpopulation with reduced expression of alpha(5)beta(1) integrin (alpha(5)beta(1)(low) cells). The size of the alpha(5)beta(1)(low) subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP(+) cortical progenitor cells were sorted into alpha(5)beta(1)(high) or alpha(5)beta(1)(low) populations, and each potential to differentiate was analyzed. We show that the nestin-GFP(+) alpha(5)beta(1)(high) population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP(+) alpha(5)beta(1)(low) cells appeared to be committed to a neuronal fate. These findings suggest that alpha(5)beta(1) expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.     

神经钙粘着蛋白在P19神经元分化中的作用

利用ＲＴ－ＰＣＲ技术,我们检测Ｐ１９细胞体外神经元分化过程中神经钙粘着蛋白（Ｎ－ｃａｄｈｅｒｉｎ）的表达模式。结果显示,该基因在上述过程中存在上调和下调过程,与体内中枢神经系统发育过程的表达模式十分相近。在此基础上,我们将神经钙粘着蛋白基因ｃＤＮＡ全长转入Ｐ１９细胞,通过药物筛选,得到稳定表达钙粘着蛋白的细胞株。     

The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction

The neural cell adhesion molecule NCAM and its glycosylation with polysialic acid (polySia) are crucially involved in proliferation, migration and differentiation of neural progenitors. Modification with polySia, homophilic and heterophilic interactions set the function of NCAM, but little is known on their interplay. We have shown recently that removal of polySia induces neuronal differentiation via heterophilic NCAM interactions at cell contacts between SH-SY5Y neuroblastoma cells. Here we analyze the additional impact of NCAM-positive fibroblasts as a ligand-presenting cellular environment, a model often used to demonstrate the neuritogenic effect of homophilic NCAM interactions. Native SH-SY5Y cells did not respond to interactions with fibroblast NCAM. However, after induction of neuronal differentiation by retinoic acid the previously ineffective NCAM signals activated extracellular signal-regulated kinase (ERK) and promoted neuritogenesis. Removal of polySia increased neuritogenesis in retinoic acid-treated cells additive to the NCAM substrate effect. The change in responsiveness to substrate NCAM was associated with a rearrangement of polysialylated NCAM away from its enrichment at homotypic cell-cell contacts and with the appearance of non-polysialylated NCAM, i.e. changes facilitating NCAM interactions with the substrate. Thus, heterophilic and homophilic NCAM interactions are integrated into the cell's response yet they have the capacity to independently trigger neuritogenesis. The actual occurrence of each of these interactions, however, depends on the cellular context, targeted cell surface presentation of NCAM and the dynamic regulation of its modification by polysialic acid. In summary, this study reveals how the complex interplay of NCAM interactions and polysialylation provides an elaborate system to regulate neuritogenesis.     

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain.     

FGF2 promotes skeletogenic differentiation of cranial neural crest cells

The cranial neural crest gives rise to most of the skeletal tissues of the skull. Matrix-mediated tissue interactions have been implicated in the skeletogenic differentiation of crest cells, but little is known of the role that growth factors might play in this process. The discovery that mutations in fibroblast growth factor receptors (FGFRs) cause the major craniosynostosis syndromes implicates FGF-mediated signalling in the skeletogenic differentiation of the cranial neural crest. We now show that, in vitro, mesencephalic neural crest cells respond to exogenous FGF2 in a dose-dependent manner, with 0.1 and 1 ng/ml causing enhanced proliferation, and 10 ng/ml inducing cartilage differentiation. In longer-term cultures, both endochondral and membrane bone are formed. FGFR1, FGFR2 and FGFR3 are all detectable by immunohistochemistry in the mesencephalic region, with particularly intense expression at the apices of the neural folds from which the neural crest arises. FGFRs are also expressed by subpopulations of neural crest cells in culture. Collectively, these findings suggest that FGFs are involved in the skeletogenic differentiation of the cranial neural crest.     

Embryonic stem cell-derived neurogenesis

Embryonic stem (ES) cells are able to differentiate in vitro into endodermal, mesodermal, and ectodermal cell types. However. the spontaneous development of neuronal cells from ES cells is rather limited. Therefore, specific protocols to increase the differentiation of neuronal cells have been established, such as retinoic acid (RA) induction and lineage selection of neuronal cells. High concentrations of RA resulted in efficient neuronal differentiation paralleled by the expression of tissue-specific genes, proteins, ion channels, and receptors in a developmentally controlled manner. Because the developmental pattern and survival capacity of RA-induced neuronal cells were limited, specific differentiation protocols by lineage selection of neuronal cells have been established using growth and extracellular matrix factors. After formation of cells of the three primary germ layers, mesodermal differentiation was inhibited by serum depletion, and neural precursor cells were generated by addition of basic fibroblast growth factor, followed by differentiation induction by neuronal differentiation factors. Further application of survival-promoting factors such as neurotrophic factors and cytokines at terminal stages resulted in a significant increase, survival, and maintenance of dopaminergic neurons. In the future, these cellular systems will be applicable: (1) for studying commitment and neuronal specification in vitro, (2) as pharmacological assays for drug screening, and (3) for the selective isolation of differentiated neuronal cells which may be used as a source for cell and tissue grafts.