Mammalian neural stem cells

Neural stem cells exist not only in the developing mammalian nervous system but also in the adult nervous system of all mammalian organisms, including humans. Neural stem cells can also be derived from more primitive embryonic stem cells. The location of the adult stem cells and the brain regions to which their progeny migrate in order to differentiate remain unresolved, although the number of viable locations is limited in the adult. The mechanisms that regulate endogenous stem cells are poorly understood. Potential uses of stem cells in repair include transplantation to repair missing cells and the activation of endogenous cells to provide "self-repair. " Before the full potential of neural stem cells can be realized, we need to learn what controls their proliferation, as well as the various pathways of differentiation available to their daughter cells.     

3种培养体系下人胚胎干细胞神经诱导分化的比较

【目的】比较人胚胎干细胞在人胚胎成纤维细胞、鼠胚胎成纤维细胞饲养层和无饲养层3个诱导体系下形成神经上皮祖细胞的能力。【方法】将人胚胎干细胞在人、鼠胚胎成纤维细胞饲养层或无饲养层体系中诱导10 d后,检测其胚胎干细胞和神经上皮祖细胞相关基因(Oct4、Nestin、Pax6、Sox1、Sox2、Sox3、β-actin)的表达,用免疫荧光染色法检测神经上皮祖细胞抗原标记Pax6、Musashi、Nestin和胚胎干细胞抗原标记SSEA4的表达。【结果】3种诱导体系下均有神经上皮祖细胞形成,均表达神经上皮祖细胞抗原标记Pax6、Musashi、Nestin及Pax6、Nes-tin、Sox家族基因。无饲养层体系诱导后的细胞,Oct4基因的表达高于有饲养层体系;其SSEA4染色阳性细胞比例为(26.3±3.5)%,高于有饲养层体系;其Pax6和Musashi染色阳性细胞比例分别为(81.0±3.0)%,(79.0±4.4)%,均低于有饲养层细胞体系。【结论】人胚胎干细胞在有饲养层和无饲养层体系中均可诱导分化形成神经上皮祖细胞,但无饲养层体系的效率相对较低,且有未分化胚胎干细胞残余。     

Mosaic organization of neural stem cells in the adult brain

The in vivo potential of neural stem cells in the postnatal mouse brain is not known, but because they produce many different types of neurons, they must be either very versatile or very diverse. By specifically targeting stem cells and following their progeny in vivo, we showed that postnatal stem cells in different regions produce different types of neurons, even when heterotopically grafted or grown in culture. This suggests that rather than being plastic and homogeneous, neural stem cells are a restricted and diverse population of progenitors.     

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.     

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.     

牛iPSCs向神经细胞的诱导分化

【目的】通过类胚体介导体外自由分化与诱导分化,验证所建立的牛诱导性多能干细胞(Bovine induced pluripotent stem cells,biPSCs)能否分化为外胚层的神经细胞。【方法】将biPSCs悬浮培养以制备类胚体,通过类胚体介导使biPSCs在添加全反式维甲酸(RA)和β巯基乙醇(β-Me)诱导体系中分化为神经细胞,比较其诱导效率;提取不同诱导体系的分化细胞与类胚体的总RNA,利用RT-PCR方法鉴定分化细胞中神经细胞特异性基因的表达。【结果】培养的biPSCs集落呈球形,中央隆起,周围界限清晰,与胚胎干细胞集落形态类似。biPSCs碱性磷酸酶(AP)染色为阳性,并表达SSEA-4干细胞特异性表面蛋白。biPSCs通过类胚体介导分化,在未添加化学诱导物的条件下,可自由分化为Nestin、GFAP与NSE阳性神经细胞,其阳性细胞率分别为(15.14±1.13)%,(6.25±0.35)%和(5.45±0.62)%;biPSCs在RA诱导组中分化的神经细胞的数量最多,诱导后表达Nestin、GFAP和NSE细胞的阳性率分别为(49.56±2.33)%,(16.58±1.28)%和(13.66±2.21)%;在β-Me诱导组中,表达Nestin的阳性细胞率为(42.23±1.25)%。2个诱导组的诱导效率与无化学诱导物组有显著差异(P0.05)。RT-PCR检测结果显示,不同诱导体系获得的分化细胞均能扩增到神经细胞特异性基因Nestin和βⅢ-Tubulin片段,产物长度与预期结果完全一致。【结论】biPSCs在体外具有向神经细胞发育的能力;RA是诱导biPSCs向神经细胞分化的良好诱导物。     

仔猪神经干细胞体外培养及鉴定

由仔猪脑部海马体齿状回分离得到神经干细胞,利用无血清培养基添加特定生长因子方式培养;采用RT-PCR鉴定神经干细胞和诱导分化后细胞生物特性;利用免疫荧光化学技术检测克隆的细胞抗原和分化后特异性成熟神经细胞抗原表达。由海马体齿状回分离的细胞群具有连续克隆能力;根据免疫荧光检测,脑神经干细胞表达巢蛋白、配对盒因子6和解整合素样金属蛋白酶10;体外培养下NSCs可被诱导分化为神经源性细胞,且表达微管相关蛋白2、胶质纤维酸性蛋白和髓鞘碱性蛋白。分离和培育NSCs细胞结果显示,其具有更新能力和分化为神经元和胶质细胞的潜能,可为干细胞移植提供基础数据。     

Primary bioassay of human tumor stem cells

A simple method has been developed to support human tumor stem cell colony growth in soft agar. The technique appears suitable for culture of a variety of neoplasms of differing histopathology. Tumor stem cell colonies arising from different types of cancer have differing growth characteristics and colony morphology. This bioassay should be suitable for clinical studies of effects of anticancer drugs or irradiation on human tumor stem cells.     

Segregation of sister chromatids in mammalian cells

Segregation of sister chromatids in embryonic mouse cells in primary tissue culture is not random. In mitosis those chromatids replicated on a DNA template synthesized during the preceding division cycle are separated from those constructed on a template synthesized two division cycles previously. Segregation in cells of the Chinese hamster follows a similar, but less pronounced, pattern.     

Multilineage potential of adult human mesenchymal stem cells

Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.     

Magnetic resonance spectroscopy identifies neural progenitor cells in the live human brain

The identification of neural stem and progenitor cells (NPCs) by in vivo brain imaging could have important implications for diagnostic, prognostic, and therapeutic purposes. We describe a metabolic biomarker for the detection and quantification of NPCs in the human brain in vivo. We used proton nuclear magnetic resonance spectroscopy to identify and characterize a biomarker in which NPCs are enriched and demonstrated its use as a reference for monitoring neurogenesis. To detect low concentrations of NPCs in vivo, we developed a signal processing method that enabled the use of magnetic resonance spectroscopy for the analysis of the NPC biomarker in both the rodent brain and the hippocampus of live humans. Our findings thus open the possibility of investigating the role of NPCs and neurogenesis in a wide variety of human brain disorders.     

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.     

Brain stem neurons in modified pathways for motor learning in the primate vestibulo-ocular reflex

The vestibulo-ocular reflex (VOR) stabilizes retinal images by generating smooth eye movements that are equal in amplitude and opposite in direction to head turns. Whenever image motion occurs persistently during head turns, the VOR undergoes motor learning; as a result image stability is gradually restored. A group of brain stem neurons that are in the modified pathways has now been described. The neurons express changes in firing in association with motor learning in the VOR and receive monosynaptic inhibition from the flocculus of the cerebellum. The changes in firing have an appropriate magnitude and are expressed at the correct latency to account for the altered VOR. The response properties of the neurons point to their brain stem vestibular inputs for further investigation of the site of motor learning.     

人胚胎干细胞不同冷冻保存方法的比较

【目的】比较人胚胎干细胞不同冷冻保存方法的复苏率,建立高效的冷冻保存方法。【方法】利用慢速冷冻、开放式和密闭式玻璃化冷冻3种方法保存人胚胎干细胞克隆,比较这3种方法的复苏效率,并通过免疫组化染色检测复苏后细胞的OCT-4表达。【结果】开放式快速冷冻方法的克隆复苏率为(97.5±5.0)%,密闭式快速冷冻方法的克隆复苏率为(96.7±5.8)%,均极显著高于慢速冻存方法(25.7±4.1)%。解冻后的胚胎干细胞克隆仍然保持胚胎干细胞的特性。【结论】开放式和密闭式玻璃化冻存方法能够高效保存人胚胎干细胞。     

The nasotemporal division in primate retina: the neural bases of macular sparing and splitting

In primates, each hemisphere contains a representation of the contralateral visual hemifield; unilateral damage to the visual pathways results in loss of vision in half of the visual field. Apparently similar severe, unilateral lesions to the central visual pathways can result in two qualitatively different central visual field defects termed macular sparing and macular splitting. In macular sparing a 2 degrees to 3 degrees region around the fovea is spared from the effects of unilateral damage to the visual pathways. In macular splitting there is no such spared region and the scotoma produced by unilateral brain damage bisects the fovea. The patterns of decussation of the different classes of retinal ganglion cells in both New World (Saimiri sciureus) and Old World (Macaca fascicularis) monkeys have been determined by horseradish peroxidase injection. In both species the distributions of ipsilaterally and contralaterally projecting ganglion cells in the central retina are different from those in other mammals and suggest neural bases for macular sparing and splitting, respectively.     

Patient-specific embryonic stem cells derived from human SCNT blastocysts

Patient-specific, immune-matched human embryonic stem cells (hESCs) are anticipated to be of great biomedical importance for studies of disease and development and to advance clinical deliberations regarding stem cell transplantation. Eleven hESC lines were established by somatic cell nuclear transfer (SCNT) of skin cells from patients with disease or injury into donated oocytes. These lines, nuclear transfer (NT)-hESCs, grown on human feeders from the same NT donor or from genetically unrelated individuals, were established at high rates, regardless of NT donor sex or age. NT-hESCs were pluripotent, chromosomally normal, and matched the NT patient's DNA. The major histocompatibility complex identity of each NT-hESC when compared to the patient's own showed immunological compatibility, which is important for eventual transplantation. With the generation of these NT-hESCs, evaluations of genetic and epigenetic stability can be made. Additional work remains to be done regarding the development of reliable directed differentiation and the elimination of remaining animal components. Before clinical use of these cells can occur, preclinical evidence is required to prove that transplantation of differentiated NT-hESCs can be safe, effective, and tolerated.