Instructive role of Wnt/beta-catenin in sensory fate specification in neural crest stem cells

Wnt signaling has recently emerged as a key factor in controlling stem cell expansion. In contrast, we show here that Wnt/beta-catenin signal activation in emigrating neural crest stem cells (NCSCs) has little effect on the population size and instead regulates fate decisions. Sustained beta-catenin activity in neural crest cells promotes the formation of sensory neural cells in vivo at the expense of virtually all other neural crest derivatives. Moreover, Wnt1 is able to instruct early NCSCs (eNCSCs) to adopt a sensory neuronal fate in a beta-catenin-dependent manner. Thus, the role of Wnt/beta-catenin in stem cells is cell-type dependent.     

Cell line segregation during peripheral nervous system ontogeny

The peripheral nervous system of vertebrates arises from the neural crest and the ectodermal placodes. Construction of quail-chick chimaeras has provided significant information on the migration and fate of the neural crest and placodal cells. Transplantation of neural crest tissue to various sites in these chimaeras has demonstrated that the differentiation of neural crest cells is controlled by environmental influences during their migration and, particularly, during gangliogenesis. Experiments with in vitro and monoclonal antibody techniques have shown that these environmental cues act on a heterogeneous population of neural crest cells whose developmental potencies are partly restricted to definite differentiation pathways.     

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.     

大鼠胎儿神经干细胞的分离、培养及诱导分化

为了探讨神经干细胞(Neural stem cells,NSCs)在体外培养中的生物学特性,研究不同细胞因子对NSCs分化的影响,试验对大鼠胎儿神经干细胞进行了分离培养,并研究了10,50和100 ng/mL的神经生长因子(nerve growth factor,NGF)、脑源性神经营养因子(brain derived neruotrophic factor,BDNF)和胶质源性神经生长因子(glial cell line-derived neurotrophic factor,GDNF)对NSCs的定向诱导分化作用。结果表明,大鼠胎儿NSCs可在体外增殖并形成典型的神经球,指数增长期在传代后的第4~5天;10和50 ng/mL的NGF、BDNF和GDNF对NSCs的诱导分化作用不明显,100 ng/mL的NGF、BDNF和GDNF可诱导NSCs分化为神经元或胶质细胞。说明NGF和BDNF可诱导NSCs向神经元方向分化,GDNF诱导其向胶质细胞分化。     

Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning

Cranial neural crest cells generate the distinctive bone and connective tissues in the vertebrate head. Classical models of craniofacial development argue that the neural crest is prepatterned or preprogrammed to make specific head structures before its migration from the neural tube. In contrast, recent studies in several vertebrates have provided evidence for plasticity in patterning neural crest populations. Using tissue transposition and molecular analyses in avian embryos, we reconcile these findings by demonstrating that classical manipulation experiments, which form the basis of the prepatterning model, involved transplantation of a local signaling center, the isthmic organizer. FGF8 signaling from the isthmus alters Hoxa2 expression and consequently branchial arch patterning, demonstrating that neural crest cells are patterned by environmental signals.     

Neural crest cells contribute to normal aorticopulmonary septation

By analyzing the hearts of quail-chick chimeras, it was found that neural crest cells at the level of occipital somites 1 to 3 migrate to the region of the aorticopulmonary septum. Bilateral removal of this neural crest population prior to migration causes malformation of the aorticopulmonary septum resulting in common arterial outflow channels or transposition of the great vessels.     

The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification

The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.     

The cellular and molecular origins of beak morphology

Cellular and molecular mechanisms underlying differences in beak morphology likely involve interactions among multiple embryonic populations. We exchanged neural crest cells destined to participate in beak morphogenesis between two anatomically distinct species. Quail neural crest cells produced quail beaks in duck hosts and duck neural crest produced duck bills in quail hosts. These transformations involved morphological changes to non-neural crest host beak tissues. To achieve these changes, donor neural crest cells executed autonomous molecular programs and regulated gene expression in adjacent host tissues. Thus, neural crest cells are a source of molecular information that generates interspecific variation in beak morphology.     

Mouse B-type lamins are required for proper organogenesis but not by embryonic stem cells

B-type lamins, the major components of the nuclear lamina, are believed to be essential for cell proliferation and survival. We found that mouse embryonic stem cells (ESCs) do not need any lamins for self-renewal and pluripotency. Although genome-wide lamin-B binding profiles correlate with reduced gene expression, such binding is not directly required for gene silencing in ESCs or trophectoderm cells. However, B-type lamins are required for proper organogenesis. Defects in spindle orientation in neural progenitor cells and migration of neurons probably cause brain disorganizations found in lamin-B null mice. Thus, our studies not only disprove several prevailing views of lamin-Bs but also establish a foundation for redefining the function of the nuclear lamina in the context of tissue building and homeostasis.     

Local embryonic matrices determine region-specific phenotypes in neural crest cells

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.     

Ectodermal Wnt function as a neural crest inducer

Neural crest cells, which generate peripheral nervous system and facial skeleton, arise at the neural plate/ectodermal border via an inductive interaction between these tissues. Wnts and bone morphogenetic proteins (BMPs) play roles in neural crest induction in amphibians and zebrafish. Here, we show that, in avians, Wnt6 is localized in ectoderm and in vivo inhibition of Wnt signaling perturbs neural crest formation. Furthermore, Wnts induce neural crest from naive neural plates in vitro in a defined medium without added factors, whereas BMPs require additives. Our data suggest that Wnt molecules are necessary and sufficient to induce neural crest cells in avian embryos.     

GDNF和LIF对小鼠精原干细胞体外增殖的影响

【目的】探讨胶质细胞源性神经营养因子（GDNF）和白血病抑制因子（LIF）对小鼠精原干细胞（SSCs）体外增殖的影响,为后续SSCs的诱导分化、转基因动物生产、基因治疗等研究奠定基础。【方法】收集6～8日龄小鼠睾丸,采用机械法和2步酶消化法获得细胞悬液。通过多次差异贴壁法分离纯化SSCs和支持细胞,采用碱性磷酸酶（AP）染色和RTPCR检测Ngn3和Oct4基因2种方法对SSCs进行鉴定。采用单独添加GDNF（添加量为0,10,20,40 ng/mL）或LIF（添加量为0,500,1 000,1 500 U/mL）的无血清DMEM/F12培养基培养SSCs,于培养第3,5,7天取样,同时用添加GDNF和LIF（各因子单独添加量两两组合）的无血清DMEM/F12培养基培养SSCs,于培养第3,4,5天取样,采用甲基噻唑基四唑（MTT）法检测GDNF、LIF对SSCs体外增殖的单因子效应和配伍效应。【结果】与对照组相比,不论培养时间如何,单独添加20和40 ng/mL的GDNF可以显著促进SSCs增殖(P＜0.05),而单独添加不同量LIF对SSCs增殖的影响不显著(P＞0.05);同时添加20 ng/mL GDNF和1 000 U/mL LIF可以显著促进SSCs的增殖,在该条件下当精原干细胞接种密度为(6×10⁴)～(10×10⁴) mL^-1,共培养5 d时,其OD₄₉₀值为0.696。【结论】DMEM/F12培养基中单独添加20 ng/mL GDNF或同时添加20 ng/mL GDNF 和 1 000 U/mL LIF可以显著促进小鼠SSCs的体外增殖。     

Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells

Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes 1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.     

Generalized potential of adult neural stem cells

The differentiation potential of stem cells in tissues of the adult has been thought to be limited to cell lineages present in the organ from which they were derived, but there is evidence that some stem cells may have a broader differentiation repertoire. We show here that neural stem cells from the adult mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers. This demonstrates that an adult neural stem cell has a very broad developmental capacity and may potentially be used to generate a variety of cell types for transplantation in different diseases.     

精原干细胞体外培养体系的建立

精原干细胞(spermatogonial stem cells,SSCs)是雄性哺乳动物精子发生及具有生育能力的保障。精原干细胞的体外培养不仅为精子发生的研究提供材料,还有助于开发新的家畜保种方法和动物遗传修饰。为了探索猪精原干细胞体外培养体系的建立方法,本研究采用胶原酶Ⅳ-胰酶两步酶法对3~7日龄大白仔猪睾丸进行消化得到单细胞悬液,利用不同时间程序的差速贴壁对精原干细胞进行纯化,选择大白仔猪睾丸支持细胞作为饲养层,添加不同细胞因子研究精原干细胞的增殖情况以期得到最佳的培养体系。结果显示,通过差速贴壁得到的UCHL-1阳性生殖细胞比例最高为18.59%±0.94%;不同细胞因子组合添加试验发现精原干细胞添加20 ng·mL^-1 GDNF、10 ng·mL^-1 IGF和20 ng·mL^-1 bFGF的增殖效果最佳;以支持细胞作为饲养层、在DMEM/F12中添加1%FBS以及上述细胞因子组合对精原干细胞进行培养15 d后可见大量的精原干细胞集落,通过免疫荧光、AKP染色、荧光定量PCR等试验证明精原干细胞进行了大量增殖。本研究初步建立了猪精原干细胞的体外培养体系,可通过体外培养大量增殖精原干细胞,为后续精原干细胞的研究奠定基础。     

湖羊精原干细胞体外培养

研究了湖羊精原干细胞(SSCs)体外培养方法,湖羊睾丸曲精细管两步酶消化法制备细胞悬液,Percoll分离后比较SSCs纯化方法、FCS以及GDNF对SSCs体外培养与集落碱性磷酸酶(AKP)染色的影响。结果显示,虽然盘化法纯化的SSCs在集落形成时间与集落数上显著低于差速贴壁法(P<0.05),但是盘化法所得集落的AKP阳性率显著高于差速贴壁法(P<0.05);添加1% FCS的培养基可显著降低集落形成的时间,增加集落的数目与AKP阳性率(P<0.05),但集落形成时间与AKP阳性率在5%与1% FCS之间无显著变化(P>0.05);20 ng/mL GDNF处理组内集落数与AKP阳性率显著高于10 ng/mL GDNF(P<0.05),但集落形成时间与AKP阳性率与30～40 ng/mL GDNF的处理组无显著差别(P>0.05)。     

APP蛋白家族胞内段释放与神经干细胞向神经元细胞定向分化的关系研究

[目的]研究APP家族胞内段对神经细胞定向分化的影响。[方法]通过分离体外培养神经干细胞,转染导入APP蛋白家族胞内段以过表达后,研究其对神经干细胞向神经元定向分化的影响。[结果]APP家族蛋白胞内段的过表达可以抑制神经干细胞向神经元细胞的分化,其中APP和APLP2胞内段的影响尤为显著。突变试验表明,通过Capase水解释放末端31氨基酸片断是这个过程必需的。APP家族胞内段是通过释放APP-ICD31来抑制神经干细胞向神经元细胞的定向分化。[结论]该研究进一步证实了APP蛋白和老年痴呆病发生的联系,也为老年痴呆症的治疗提供新的靶点。     

小鼠胚胎中脑神经干细胞分离、培养及分化

目的:探讨小鼠胚胎中脑分离神经干细胞的体外培养方法,以获取高纯度的神经干细胞,为神经干细胞的深入研究提供试验材料。方法:无菌条件下分离孕12~13 d小鼠胚胎中脑曲,制成单细胞悬液,碱性成纤维生长因子(bFGF)和B27存在的培养基中培养扩增,通过免疫细胞化学染色鉴定神经干细胞及子代细胞的分化方向,流式细胞术检测TH阳性神经元比例。结果:培养的部分细胞体外分裂增殖,同时表达神经干细胞特异性抗原nestin,并向神经细胞和胶质细胞分化并经流式细胞仪检测自然分化为多巴胺能神经元的比例为3.25%。结论:小鼠胚脑中脑存在具有多分化潜能的神经干细胞,它们能在体外稳定培养、传代和分化。     

A stem cell molecular signature

Mechanisms regulating self-renewal and cell fate decisions in mammalian stem cells are poorly understood. We determined global gene expression profiles for mouse and human hematopoietic stem cells and other stages of the hematopoietic hierarchy. Murine and human hematopoietic stem cells share a number of expressed gene products, which define key conserved regulatory pathways in this developmental system. Moreover, in the mouse, a portion of the genetic program of hematopoietic stem cells is shared with embryonic and neural stem cells. This overlapping set of gene products represents a molecular signature of stem cells.