Seminal Biologies of Neural Stem Cells

神经干细胞的精液生物学

• 批准号: 7969506
• 负责人: JEFFERY BARKER
• 金额: $ 84.42万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969506
• 关键词: Accounting; Apoptosis; Apoptotic; Astrocytes; Binding Sites; Biology; CSPG4 gene; Cell Cycle Stage; Cell Lineage; Cells; Cellular biology; Cholera Toxin; Collaborations; Complex; Consensus; DNA; Development; Dyes; Exclusion; Exhibits; Ganglioside GD3; Gangliosides; Gene Expression; Generic Drugs; Genes; In Vitro; Integrin alpha3; Integrin alpha6beta1; Integrin beta Chains; Integrins; Investigation; Label; Life; Link; Literature; Methods; Mitotic; Molecular; Molecular Profiling; NGFR gene; National Institute of Neurological Disorders and Stroke; Neuroepithelial Cells; Neuroglia; Neurons; Nuclear; Pan Genus; Pattern; Phase; Phenotype; Ploidies; Population; Proliferating; Property; Protocols documentation; Radial; Reagent; Relative (related person); Research Personnel; Resolution; Role; Sampling; Seminal; Sorting - Cell Movement; Source; Staging; Staining method; Stains; Stem cells; Sum; Surface; Surveys; Tetanus Toxin; Time; Tubulin; Ventricular; base; cDNA Arrays; hippocampal pyramidal neuron; interest; nerve stem cell; neurogenesis; novel; polysialyl neural cell adhesion molecule; progenitor; prospective; relating to nervous system; self-renewal; stem; stem cell biology; tool; transcription factor; transcriptional coactivator p75

During CNS development NSCs are considered to be the cellular compartment that both expands through self-renewal and generates lineage-restricted progenitors. Progenitors ultimately differentiate into the neural phenotypes composing different stages of CNS development. Although the seminal biology of NSCs has come under intensive investigation, there is still no consensus regarding exactly which cells are actually NSCs, since specific markers do not exist. So, neuroepithelial cells are widely used as the primary source of NSCs, and often as if they all were NSCs. And, NSCs are widely identified functionally as cells that can both self-renew and differentiate into multiple neural phenotypes. Thus, there is a general consensus that further elucidation of NSC biology first requires their phenotypic identification since this could provide direct experimental access to them for prospective rather than retrospective investigation. During FY2009 we focused entirely on relating our phenotyping strategy to all of the available markers currently used in the field. Different investigators use different markers and methods to try to identify putative NSCs from retrospective analyses of heterogeneous cell populations exhibiting the generic functional endpoints of putative NSC phenotypes (self-renewal and multipotent differentiation). The results have led to a continuing lack of consensus regarding the seminal properties of true NSCs and their lineal relationships. So, many putative NSC phenotypes are simply lumped together, since investigators have not resolved progenitors from NSCs. Our specific aim has been to resolve NSC and progenitor populations in a clear and convincing manner using comprehensive immunostaining protocols with commercially available markers used in the field. Previously, we had identified NSCs at the onset of cortical development based on their complete lack of surface expression of several known neural markers (tetanus toxin (TxTx) fragment C and cholera toxin B complex ganglioside binding sites, A2B5 (GD3 ganglioside) and CD15/LeX/SSEA-1). We have added surface (CD133, NG2, PSA-NCAM, CD24, CD57, NGFR p75, integrin alpha 3, integrin alpha 6, integrin beta 1), cytoskeletal/cytoplasmic (beta 4 tublin, alpha 1 tubulin, Aldefluor) and nuclear markers (HuD, Ngn1, Ngn3, Mcm2, FoxG1, D1x2, Olig2, pax6, prox1, Sox1, Sox2, Hoechst), which are all of used in the field. We combined pairs of alpha and beta integrin subnits with CD57, a pan-specific marker of CD15+ CD24+ CDw60+ PSA-NCAM+ A2B5+ neural progenitors, and TnTx, a pan-specific neuronal lineage marker. This novel combination provided an unprecedented level of resolution and revealed the lineal relationships among all the major populations throughout corticogenesis. The co-expression of six surface markers effectively links six disparate literatures on neural progenitors together for the first time into the same lineage. Since these cells also co-expressed the transcription factor Tbr2, we identified them as intermediate progenitor cells (IPCs), thereby establishing multiple IPC-related surface markers. NSCs were identified as CD57- TnTx- cells co-expressing specific integrin heterodimers. These cells rapidly decreased in relative abundance during the early period of corticogenesis as they either self-renewed, died via apoptosis or differentiated into different CD57+/- and TnTx+ neural progenitors. We also included a live-cell DNA stain to quantify total DNA content. The most proliferative subsets were found in IPCs expressing heterodimers, while the most abundant post-mitotic cells were found in the neuronal progenitors devoid of either integrin subunit. This comprehensive surface phenotyping strategy provides, for the first time, a complete ex vivo account of the proliferating, quiescent/differentiating and apoptotic phases that accompany the intermediate stages of lineage progressions derived from NSCs. Analysis of the four bivariate FACS plots (CD57+/- TnTx+/-) associated with the four integrin subunit-labeled populations (alpha+/- beta+/-) revealed clear linkages among each of the neural populations either devoid of markers (CD57- TnTx- NSCs) or expressing either one or both markers. We conclude that NSCs initially give rise to both neuronal progenitors (CD57- TnTx+) and IPCs (CD57+ TnTx-), which , in turn, produce other neuronal progenitors (CD57+ TnTx+). The surface markers allowed us to sort select populations for retrospective analysis of their seminal properties using clonal cultures. The results obtained in vitro closely recapitulate the lineage progressions inferred from FACS analyses of populations phenotyped ex vivo, indicating that seminal properties are effectively preserved and hence accessible for cellular and molecular studies. NSCs self-renew and first produce IPC-lineage-negative neuronal progenitors followed by IPCs and their neuronal progenitor progeny. Subsequently, astrocytes, both lineage-negative and lineage-positive, are produced along with lineage-negative radial glial cells, which themselves give rise to lineage-negative neurons and astrocytes. It is clear that the seminal properties, including self-renewal, apoptosis, and multipotent differentiation, are all expressed by NSCs, IPCs and radial glia. Thus, each of these distinct, lineally-related phenotypes exhibits similar, but not identical seminal properties typically ascribed to NSCs. We also quantified the distributions of three other surface markers (CD133, NG2, NGFR p75) in the context of the beta integrin subunit, CD57 and TnTx. Each of these markers is expressed by rare populations throughout development (0-7%). CD133+ cells primarily reside at the ventricular interface in proliferating cells and may only be expressed at specific stage(s) of the cell cycle. The other two markers are mostly expressed by cells devoid of neural markers. Survey of several cytoplasmic markers revealed that alpha 1 tubulin is primarily expressed by IPCs and thus equates them with "short neural precursors" in the literature, while beta 4 tubulin is restricted to oligodendroglial progeny. Neither Aldefluor labeling nor Hoechst dye exclusion were specific, since subpopulations of NSCs and IPCs were found to be Aldefluor+ and excluded Hoechst dye. Survey of NSC-associated transcription factors (TFs) showed that Pax6, Sox1 and Sox2 label the majority of NSCs and IPCs. These TFs are also expressed by radial glia, but are down-regulated among the differentiating neuronal phenotypes generated by these three proliferating populations. Other TFs (HuD, Ngn1, Ngn3, Prox1) emerge primarily among differentiating neuronal progenitors, especially those derived from IPCs, while Mcm2 and FoxG1 are co-expressed by these cells. In sum, lineage-negative NSCs self-renew and predominate at the earliest stages of corticogenesis and generate neuronal progenitors and IPCs, which differentiate into transiently-expressed pioneer neurons and transiently-expressed Cajal-Retzius neurons, respectively. Later IPCs generate the permanent pyramidal neurons composing the cortical layers. Self-renewing NSCs also differentiate into radial glia, which give rise to pyramidal neurons and astroglia. All of these cells co-express integrin subunits at varying levels. Our analysis has revealed 20 distinct populations with some transiently appearing, then disappearing and others persisting during cortical development. Using multi-marker phenotyping thus provides the first complete characterization of all emerging populations and reveals the dynamic lineal relationships that exist among them. This novel, all-inclusive framework is based on results using a tool kit of commercially available reagents, thus making the strategy accessible to all investigators interested in neural stem and progenitor cell biology.

在中枢神经系统发育期间，NSC被认为是通过自我更新并产生谱系限制祖细胞扩展的细胞室。祖细胞最终分化为构成CNS发育不同阶段的神经表型。尽管NSC的开创性生物学已经进行了深入的研究，但由于具体标记不存在，因此仍然尚无共识。因此，神经上皮细胞被广泛用作NSC的主要来源，并且通常都是NSC。并且，NSC在功能上被广泛鉴定为可以自我更新和分化为多种神经表型的细胞。 因此，有一个普遍的共识，即进一步阐明NSC生物学首先需要它们的表型识别，因为这可以直接与它们进行实验访问，以进行前瞻性研究，而不是回顾性研究。 在2009财年期间，我们完全专注于将表型策略与当前在现场使用的所有可用标记联系起来。 不同的研究人员使用不同的标记和方法来试图通过对推定NSC表型（自我更新和多功能分化）的一般功能终点的回顾性分析来鉴定推定的NSC。 结果导致关于真实NSC的开创性及其线性关系的开创性持续缺乏共识。 因此，许多假定的NSC表型被简单地结合在一起，因为研究人员没有解决NSC的祖细胞。 我们的具体目的是使用该领域中使用的市售标记以全面的免疫染色方案来清晰而令人信服的方式解决NSC和祖先种群。 以前，我们基于皮质发育开始时已经确定了NSC，因为它们完全缺乏几种已知神经标记（Tetanus毒素（TXTX）片段C和霍乱毒素B复杂的神经节苷脂结合位点，A2B5（GD3 Ganglioside）（GD3 Ganglioside）和CD15/Lex/SSSEA）的表面表达。 We have added surface (CD133, NG2, PSA-NCAM, CD24, CD57, NGFR p75, integrin alpha 3, integrin alpha 6, integrin beta 1), cytoskeletal/cytoplasmic (beta 4 tublin, alpha 1 tubulin, Aldefluor) and nuclear markers (HuD, Ngn1, Ngn3, Mcm2, FoxG1, d1x2，olig2，pax6，prox1，sox1，sox2，hoechst），这些都在现场中使用。 我们将一对α和β整合蛋白子Nits与CD57（CD15+ CD24+ CDW60+ PSA-NCAM+ A2B5+ A2B5+神经祖细胞和TNTX）和PAN特异性神经元谱系标记物结合在一起。 这种新颖的组合提供了前所未有的分辨率水平，并揭示了整个皮质生成的所有主要种群之间的线性关系。 六个表面标记的共表达有效地将神经祖细胞上的六个不同文献联系在一起，这是第一次与同一谱系联系在一起。 由于这些细胞也共同表达了转录因子TBR2，因此我们将它们鉴定为中间祖细胞（IPC），从而建立了多个与IPC相关的表面标记。 NSC被确定为CD57-TNTX-细胞共表达特定的整合素异二聚体。 在皮质生成的早期，这些细胞的相对丰度迅速降低，因为它们是自我更新，通过凋亡死亡或分化为不同CD57 +/-和TNTX+神经祖细胞的。 我们还包括一个活细胞DNA染色以量化总DNA含量。 在表达异二聚体的IPC中发现了最大的子集，而在没有整合素亚基的神经元祖细胞中发现了最丰富的有丝质细胞。 这种全面的表面表型策略首次提供了伴随NSC谱系中间阶段的增殖，静止/区分和凋亡阶段的完整实体描述。 分析与四个与四个整合素亚基标记的种群（Alpha +/- beta +/-）相关的四个双变量FACS图（CD57 +/- TNTX +/-）揭示了每个神经种群之间没有标记物（CD57- TNTX- NSC）或两个或两个标记的神经群体之间的明显联系。 我们得出的结论是，NSC最初会引起神经元的祖细胞（CD57-TNTX+）和IPC（CD57+ TNTX-），从而产生其他神经元祖细胞（CD57+ TNTX+）。 表面标记使我们能够使用克隆培养物对其精确特性进行回顾性分析。 在体外获得的结果紧密地概括了从FACS分析表型的表型外体的谱系进程，这表明有效地保留了精液特性，因此可以用于细胞和分子研究。 NSC自我更新和首先产生IPC-LINEGE阴性神经元祖细胞，其次是IPC及其神经祖细胞后代。 随后，谱系阴性和谱系阳性的星形胶质细胞与谱系阴性径向神经胶质细胞一起产生，这些细胞本身会引起谱系阴性神经元和星形胶质细胞。 显然，包括自我更新，细胞凋亡和多能分化在内的精确特性均由NSC，IPC和径向胶质胶质素表达。 因此，这些独特的直线相关表型中的每一个表现出相似但不相同的精液特性，通常归因于NSC。 我们还量化了其他三个表面标记（CD133，NG2，NGFR P75）的分布，在β整合素亚基CD57和TNTX的背景下。 这些标记中的每一个都由稀有种群在整个发展中表达（0-7％）。 CD133+细胞主要驻留在增殖细胞中的心室界面上，只能在细胞周期的特定阶段表达。 其他两个标记主要由没有神经标记的细胞表达。 对几个细胞质标记的调查表明，α1微管蛋白主要由IPC表达，因此将它们等同于文献中的“短神经前体”，而β4微管蛋白仅限于寡头后代。 由于发现NSC和IPC的亚群为Aldefluor+和排除的Hoechst染料，因此Aldefluor标记和Hoechst染料排除均未具体。 NSC相关转录因子（TFS）的调查表明，PAX6，SOX1和SOX2标记了大多数NSC和IPC。 这些TF也用径向神经胶质表达，但在这三个增殖种群产生的分化神经元表型中被下调。 其他TF（HUD，NGN1，NGN3，Prox1）主要出现在区分神经元祖细胞中，尤其是从IPC衍生的祖细胞，而MCM2和FOXG1则由这些细胞共表达。 总而言之，谱系阴性的NSC自我更新和占主导地位，并在皮质生成的最早阶段占主导地位，并产生神经元的祖细胞和IPC，分别分别为暂时表达的先锋神经元，并分别瞬时表达的Cajal-Retzius神经元。 后来，IPC会生成构成皮质层的永久性金字塔神经元。 自我更新的NSC也分化为径向神经胶质，这会引起金字塔神经元和星形胶质细胞。 所有这些细胞在不同水平上共表达整联蛋白亚基。 我们的分析揭示了20个不同的人群，有些瞬时出现，然后消失，而另一些人则在皮质发育过程中持续存在。 因此，使用多标志物表型提供了所有新兴人群的第一个完整表征，并揭示了它们之间存在的动态直系关系。 这个新颖的全包框架基于使用市售试剂的工具包的结果，从而使所有对神经茎和祖细胞生物学感兴趣的研究者都可以访问该策略。