An essential role for thymic mesenchyme in early T cell development

We show that the mesenchymal cells that surround the 12-d mouse embryo thymus are necessary for T cell differentiation. Thus, epithelial lobes with attached mesenchyme generate all T cell populations in vitro, whereas lobes from which mesenchyme has been removed show poor lymphopoiesis with few cells progressing beyond the CD4(-)CD8(-) stage of development. Interestingly, thymic mesenchyme is derived from neural crest cells, and extirpation of the region of the neural crest involved results in impaired thymic development and craniofacial abnormalities similar to the group of clinical defects found in the DiGeorge syndrome.Previous studies have suggested an inductive effect of mesenchyme on thymic epithelial morphogenesis. However, we have found that mesenchyme-derived fibroblasts are still required for early T cell development in the presence of mature epithelial cells, and hence mesenchyme might have a direct role in lymphopoiesis. We provide an anatomical basis for the role of mesenchyme by showing that mesenchymal cells migrate into the epithelial thymus to establish a network of fibroblasts and associated extracellular matrix. We propose that the latter might be important for T cell development through integrin and/or cytokine interactions with immature thymocytes.     

Role of Endothelin-1/Endothelin-A receptor-mediated signaling pathway in the aortic arch patterning in mice.

The intercellular signaling mediated by endothelins and their G protein-coupled receptors has recently been shown to be essential for the normal embryonic development of subsets of neural crest cell derivatives. Endothelin-1 (ET-1) is proteolytically generated from its inactive precursor by endothelin-converting enzyme-1 (ECE-1) and acts on the endothelin-A (ETA) receptor. Genetic disruption of this ET-1/ECE-1/ETA pathway results in defects in branchial arch- derived craniofacial tissues, as well as defects in cardiac outflow and great vessel structures, which are derived from cephalic (cardiac) neural crest. In this study, in situ hybridization of ETA-/- and ECE-1(-)/- embryos with a cardiac neural crest marker, cellular retinoic acid-binding protein-1, shows that the migration of neural crest cells from the neural tube to cardiac outflow tract is not affected in these embryos. Immunostaining of an endothelial marker, platelet endothelial cell adhesion molecule CD-31, shows that the initial formation of the branchial arch arteries is not disturbed in ETA-/- or ECE-1(-)/- embryos. To visualize the subsequent patterning of arch vessels in detail, we generated ETA-/- or ECE-1(-)/- embryos that expressed an SM22alpha-lacZ marker transgene in arterial smooth muscle cells. Wholemount X-gal staining of these mutant embryos reveals that the abnormal regression and persistence of specific arch arteries results in disturbance of asymmetrical remodeling of the arch arteries. These defects include abnormal regression of arch arteries 4 and 6, enlargement of arch artery 3, and abnormal persistence of the bilateral ductus caroticus and right dorsal aorta. These abnormalities eventually lead to various types of great vessel malformations highly similar to those seen in neural crest-ablated chick embryos and human congenital cardiac defects. This study demonstrates that ET-1/ETA-mediated signaling plays an essential role in a complex process of aortic arch patterning by affecting the postmigratory cardiac neural crest cell development.     

An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.     

Modulation of noncanonical TGF-β signaling prevents cleft palate in Tgfbr2 mutant mice

Patients with mutations in either TGF-β receptor type I (TGFBR1) or TGF-β receptor type II (TGFBR2), such as those with Loeys-Dietz syndrome, have craniofacial defects and signs of elevated TGF-β signaling. Similarly, mutations in TGF-β receptor gene family members cause craniofacial deformities, such as cleft palate, in mice. However, it is unknown whether TGF-β ligands are able to elicit signals in Tgfbr2 mutant mice. Here, we show that loss of Tgfbr2 in mouse cranial neural crest cells results in elevated expression of TGF-β2 and TGF-β receptor type III (TβRIII); activation of a TβRI/TβRIII-mediated, SMAD-independent, TRAF6/TAK1/p38 signaling pathway; and defective cell proliferation in the palatal mesenchyme. Strikingly, Tgfb2, Tgfbr1 (also known as Alk5), or Tak1 haploinsufficiency disrupted TβRI/TβRIII-mediated signaling and rescued craniofacial deformities in Tgfbr2 mutant mice, indicating that activation of this noncanonical TGF-β signaling pathway was responsible for craniofacial malformations in Tgfbr2 mutant mice. Thus, modulation of TGF-β signaling may be beneficial for the prevention of congenital craniofacial birth defects.     

哺乳动物颌突前体细胞的来源及其分化表型

背景:目前尚不能确定哺乳动物的外胚间充质干细胞是否也来源于迁移的神经嵴干细胞.目的:了解哺乳动物颌突前体细胞的表面标志及分化方向,探讨外胚间充质干细胞的来源及其分化表型.方法:采用免疫组织化学染色联合流式细胞仪分析,观察E9.5,E10.5,E11.5,E12.5d期间,SD大鼠颅面部外胚间充质细胞的分子表达谱及其变化.结果与结论:颌突的前体细胞表达多谱系标志,包括某些神经谱系的标志以及中胚层来源的标志,随着发育的进行,神经谱系标志下调,而中胚层谱系的标志出现或上调,提示该群细胞分化活跃,具有干细胞的特点;此外在该群细胞中检测到始终有3%～4%的细胞表达神经嵴干细胞特异性标志CD57和P75,提示哺乳动物外胚间充质干细胞来源于神经嵴,在定居后逐渐分化的同时,仍以某种机制维持自身数量的稳定.     

miR-21促进毛囊神经嵴干细胞分化为许旺细胞

背景：mi RNAs是一类长18-25个碱基的非编码单链小RNA分子,可以与m RNA分子的3’UTR上序列互补结合而调节目标基因的蛋白表达水平。大量证据表明,mi RNAs可能起到调节许旺细胞分化、髓鞘形成以及周围神经生长和发育的作用。目的：观察mi R-21在毛囊神经嵴干细胞分化为许旺细胞过程中的表达。方法：培养毛囊干细胞,通过流式分选法从人毛囊中分离神经嵴干细胞,并定向诱导为许旺细胞,在诱导过程中采用q RT-PCR检测mi R-21的表达水平。将毛囊神经嵴干细胞分为对照组、agomir-21组、agomir-NC组、antagomir-21组和antagomir-NC组。对照组无干预,agomir-21组加入mi R-21的激动剂,antagomir-21组加入mi R-21的抑制剂,agomir-NC组和antagomir-NC组分别为agomir-21和antagomir-21的阴性对照组,加入无活性的micro RNA类似物。最后,通过数据库寻找mi R-21可能的作用靶标。结果与结论：在毛囊神经嵴干细胞诱导分化为许旺细胞过程中,mi R-21表达水平逐渐升高。转染mi R-21激动剂agomir-21后,干细胞分化为许旺细胞的能力增强,而转染mi R-21抑制剂antagomir-21后可削弱干细胞的分化能力。通过数据库检索发现,SOX2可能是mi R-21重要靶基因并参与其调节干细胞分化的作用。结果提示,毛囊神经嵴干细胞可作为许旺细胞的一个重要来源,mi R-21可促进此分化过程。     

Regenerative effects of human embryonic stem cell‐derived neural crest cells for treatment of peripheral nerve injury

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up‐regulation of several regeneration‐related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair.     

Ontogeny of endothelins-1 and -3, their receptors, and endothelin converting enzyme-1 in the early human embryo.

The targeted gene inactivation of endothelins-1 and -3 (ET-1 and ET-3) and of one of their receptors, ETB, in the mouse causes severe defects in the embryonic development. These defects, cardiovascular and craniofacial malformations for ET-1, and colonic agangliogenesis associated with skin pigmentation anomalies for ET-3 and the ETB receptor, reproduce pathological phenotypes due to natural mutations of the same genes in the mouse and the human. The mutant phenotypes have been causatively linked to deficient migration/proliferation/differentiation of neural crest cells, i.e., neurocristopathies. To bring new insight about the exact roles of ETs in development and the involvement of neural crest cells in these processes, we have explored, by in situ hybridization, the ontogeny in the early human embryo of the ET system (ET-1 and ET-3, ETA and ETB receptors, ET converting enzyme-1). ET receptor mRNA expression in neural crest cells starts at 3 wk of gestation and continues during the entire period studied (up to 6 wk of gestation). During this period, ETA expression progressively spreads to undifferentiated mesodermal components of various structures and organs (head and axial skeleton, lateral and ventral subdermal mesoderm), whereas ETB expression remains more restricted to fewer differentiated cells (neural tube, sensory and sympathetic ganglia, endothelium). Some of these tissues and structures that express either one of the receptors do not appear to be of neural crest origin. In the digestive tract and the cardiovascular area, the present observations on the sources of ETs and their target cells in the young embryo provide the basis for a dynamic interpretation of the results of gene targeting of the mouse and the human phenotypes, and point to other possible roles of ETs in other ontogenetic processes. The results support the concept of local, rather than hormonal, interactions between the sources and targets of ETs during development.     

Environmental cues to guide stem cell fate decision for tissue engineering applications

The human body contains a variety of stem cells capable of both repeated self-renewal and production of specialised, differentiated progeny. Critical to the implementation of these cells in tissue engineering strategies is a thorough understanding of which external signals in the stem cell microenvironment provide cues to control their fate decision in terms of proliferation or differentiation into a desired, specific phenotype. These signals must then be incorporated into tissue regeneration approaches for regulated exposure to stem cells. The precise spatial and temporal presentation of factors directing stem cell behaviour is extremely important during embryogenesis, development and natural healing events, and it is possible that this level of control will be vital to the success of many regenerative therapies. This review covers existing tissue engineering approaches to guide the differentiation of three disparate stem cell populations: mesenchymal, neural and endothelial. These progenitor cells will be of central importance in many future connective, neural and vascular tissue regeneration technologies.     

Environmental cues to guide stem cell fate decision for tissue engineering applications

The human body contains a variety of stem cells capable of both repeated self-renewal and production of specialised, differentiated progeny. Critical to the implementation of these cells in tissue engineering strategies is a thorough understanding of which external signals in the stem cell microenvironment provide cues to control their fate decision in terms of proliferation or differentiation into a desired, specific phenotype. These signals must then be incorporated into tissue regeneration approaches for regulated exposure to stem cells. The precise spatial and temporal presentation of factors directing stem cell behaviour is extremely important during embryogenesis, development and natural healing events, and it is possible that this level of control will be vital to the success of many regenerative therapies. This review covers existing tissue engineering approaches to guide the differentiation of three disparate stem cell populations: mesenchymal, neural and endothelial. These progenitor cells will be of central importance in many future connective, neural and vascular tissue regeneration technologies.     

Dlx基因表达改变可影响颅神经嵴细胞迁移及第一鳃弓的发育？

背景：D反基因在颅神经嵴细胞中高表达,并调控颅神经嵴细胞的迁移及分化。目的：综述D反基因高表达对颅神经嵴细胞迁移及其分化的影响及机制。方法：由第一作者用计算机检索全国期刊全文数据库（CNKI）,Medline数据库,检索词分别为“神经嵴细胞,颅神经嵴细胞迁移,Dlx,Dlx高表达,Fgf,成软骨,成骨”和“cranialneual crest cells,cranial neural crest cells’migration,Dlx,Dlx overexpression,Fgf,chodrogenesis,osteogenesis”。从颅神经嵴细胞的迁移,Dlx表达改变对颅神经嵴细胞迁移的影响,Dlx基因与细胞环境的相互作用3个方面进行总结。共检索到63篇文章,按纳入和排除标准对文献进行筛选,共纳入43篇文章。结果与结论：Dlx基因表达改变导致细胞间黏附因子表达的改变,高表达会使得绝大多数颅神经嵴细胞聚集,错误迁移,致使畸形的发生。并且Dlx基因表达增高,也将导致骨或软骨的异常生成,其原因可能在于相关细胞因子间的相互作用。     

Adhesion molecules in neural crest development

Peripheral nerve cells, various endocrine and pigment cells and cranial connective tissue cells of vertebrates stem mainly from the embryonic neural crest. This originates with the central nervous system, but the crest cells detach from this tissue, via a decrease of cell-cell adhesion involving, particularly, a reduction of the adherens junction cell adhesive molecule A-CAM. This epithelio-mesenchymal transformation allows crest cells to migrate along pathways that are defined partly by the distribution of substrate adhesion molecules, the archetype being fibronectin, an extracellular matrix molecule recognized by integrin receptors on crest cells. Many other molecules, however, may act in the same way. In contrast, some molecules may define migration pathways by reducing adhesion; chpndroitin sulfate proteoglycan is a cadidate for this role. Pathways selection is most likely achieved by balanced combinations of molecules that promote and reduced adhesion. Cessation of migration, in the case of the nervous ganglia, correlated with re-expression of cell-cell adhesion molecules like A-CAM and others, consistent with an adhesive basis, although functional tests have not yet been peroformed. The development of the neural crest system provides a useful model that emphasizes the role of adhesion in morphogenesis.     

MicroRNA‐132 directs human periodontal ligament‐derived neural crest stem cell neural differentiation

Neurogenesis is the basis of stem cell tissue engineering and regenerative medicine for central nervous system (CNS) disorders. We have established differentiation protocols to direct human periodontal ligament‐derived stem cells (PDLSCs) into neuronal lineage, and we recently isolated the neural crest subpopulation from PDLSCs, which are pluripotent in nature. Here, we report the neural differentiation potential of these periodontal ligament‐derived neural crest stem cells (NCSCs) as well as its microRNA (miRNA) regulatory mechanism and function in NCSC neural differentiation. NCSCs, treated with basic fibroblast growth factor and epidermal growth factor‐based differentiation medium for 24 days, expressed neuronal and glial markers (βIII‐tubulin, neurofilament, NeuN, neuron‐specific enolase, GFAP, and S100) and exhibited glutamate‐induced calcium responses. The global miRNA expression profiling identified 60 upregulated and 19 downregulated human miRNAs after neural differentiation, and the gene ontology analysis of the miRNA target genes confirmed the neuronal differentiation‐related biological functions. In addition, overexpression of miR‐132 in NCSCs promoted the expression of neuronal markers and downregulated ZEB2 protein expression. Our results suggested that the pluripotent NCSCs from human periodontal ligament can be directed into neural lineage, which demonstrate its potential in tissue engineering and regenerative medicine for CNS disorders.     

The potential of cord blood stem cells for use in regenerative medicine

It is estimated that up to 128 million individuals might benefit from regenerative medicine therapy, or almost 1 in 3 individuals in the US. If accurate, the need to relieve suffering and reduce healthcare costs is an enormous motivator to rapidly bring stem cell therapies to the clinic. Unfortunately, embryonic stem (ES) cell therapies are limited at present by ethical and political constraints and, most importantly, by significant biologic hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend on the development of non-ES cell therapies. At present, non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). Each of these stem cells is being used to treat a variety of diseases. This review shows that CB contains multiple populations of pluripotent stem cells, and can be considered the best alternative to ES cells. CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopedic, neurologic and endocrine diseases.     

Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro

In some rodent models, there is evidence that hematopoietic stem cells (HSC) can differentiate into neural cells. However, it is not known whether humans share this potential, and, if so, what conditions are sufficient for this transdifferentiation to occur. We addressed this question by assessing the ability of fetal human liver CD34(+)/CD133(+)/CD3(-) hematopoietic stem cells to generate neural cells and astrocytes in culture. We cultured fetal liver-derived hematopoietic stem cells in human astrocyte culture-conditioned medium or using a method wherein growing human astrocytes were separated from cultured, nonadherent hematopoietic stem cells by a semipermeable membrane in a double-chamber co-culture system. Hematopoietic stem cell cultures were probed for neural progenitor cell marker expression (nestin and bone morphogenic protein-2 [BMP-2]) during growth in both culture conditions. RT-PCR, western blotting, and immunocytochemistry assays showed that cells cultured in either condition expressed nestin mRNA and protein and BMP-2 mRNA. HSC similarly cultured in nonconditioned medium or in the absence of astrocytes did not express either marker. Cells expressing these neural markers were transferred and cultured on poly-D-lysine-coated dishes with nonconditioned growth medium for further study. Immunocytochemistry demonstrated that these cells differentiated into astrocytes after 8 days in culture as indicated by their morphology and expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and S100, as well as by their rate of proliferation, which was identical to that of freshly isolated fetal brain astrocytes. These findings demonstrate that neural precursor gene expression can be induced when human hematopoietic stem cells are exposed to a suitable microenvironment. Furthermore, the neural stem cells generated in this environment can then differentiate into astrocytes. Therefore, human hematopoietic stem cells may be an alternative resource for generation of neural stem cells for therapy of central nervous system defects resulting from disease or trauma.     

The potential of cord blood stem cells for use in regenerative medicine

It is estimated that up to 128 million individuals might benefit from regenerative medicine therapy, or almost 1 in 3 individuals in the US. If accurate, the need to relieve suffering and reduce healthcare costs is an enormous motivator to rapidly bring stem cell therapies to the clinic. Unfortunately, embryonic stem (ES) cell therapies are limited at present by ethical and political constraints and, most importantly, by significant biologic hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend on the development of non-ES cell therapies. At present, non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). Each of these stem cells is being used to treat a variety of diseases. This review shows that CB contains multiple populations of pluripotent stem cells, and can be considered the best alternative to ES cells. CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopedic, neurologic and endocrine diseases.     

神经干细胞体外培养方法及影响因素研究进展

神经干细胞是指具有分化为神经元、星形胶质细胞和少突胶质细胞的能力,能自我更新并能提供大量脑组织细胞的细胞群。神经干细胞的发现对患有神经系统难治性疾病患者的治疗带来了福音,但体内内源性神经干细胞的数量极少,限制了其在治疗疾病中的应用,所以如何有效提高神经干细胞培养存活率、克隆形成率将是其在临床应用的重要基础和前提。本文就目前对神经干细胞体外培养方法及影响因素的研究进展做如下综述。