Lineage pathway differentiation of CNS progenitor cells

CNS祖细胞的谱系途径分化

• 批准号: 8940092
• 负责人: Avindra Nath
• 金额: $ 26.15万
• 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/8940092
• 关键词: Adult; Astrocytes; Automobile Driving; Binding Sites; Brain; C-terminal; Cell Culture Techniques; Cell Differentiation process; Cell Lineage; Cell Separation; Cell Therapy; Cell model; Cell surface; Cells; Characteristics; Clinical; Clone Cells; DNA Binding; DNA-Binding Proteins; Development; Differentiation Antigens; Flow Cytometry; Gene Chips; Gene Cluster; Gene Expression; Genes; Genomics; Glial Fibrillary Acidic Protein; Growth Factor; Human; Infection; Investigation; Journals; Laboratories; Link; Methodology; Microarray Analysis; Modeling; Myelin Basic Proteins; N-terminal; Nature; Neurons; Oligodendroglia; Pathway interactions; Pattern; Phenotype; Population; Predisposition; Process; Proteins; Protocols documentation; Publications; Reporter Genes; Sorting - Cell Movement; Staging; Staining method; Stains; Stem cells; System; Time; Transactivation; Tropism; Up-Regulation; Video Microscopy; Viral; Virus Diseases; Virus Inhibitors; Visual; base; brain cell; brain tissue; cell type; expression vector; insight; instrument; nestin protein; oligodendrocyte lineage; red fluorescent protein; research study; tool; transcription factor; viral DNA

We have identified and cultured human CNS derived progenitor cells from the human brain. These cells are nestin positive and do not express any other phenotypic marker of differentiation. Upon treatment of such cells with specific growth factors, these cells can be directed toward lineage pathways that make these cells neurons, astrocytes or oligodendrocytes. We are in the process of studying the genomics of this differentiation and the proteins that are uniquely made during the course of such differentiation. We use viral infection to mark the phenotypes of these cells and are able to increase infection or diminish infection depending upon the pathway that these cells are directed toward. We have recently made gene expression vectors that are only expressed in specific cell lineage pathways using reporter genes for either green or red fluorescent proteins. These gene products are visually recognized using video microscopy so they mark the pathway of cell differentiation. Such experiments will ultimately provide valuable insights into the nature of development of the brain and allow a key experimental tool for a large array of studies for cell therapy. We have also developed the tools to differentiate progenitor cells to oligodendrocytes, myelin basic protein producing cells with a phenotype similar to mature oligodendrocytes. However, upon infection with JCV that targets these cells in the adult brain, the differentiated oligodendrocytes are not susceptible to infection. These cells are missing the NF-1X protein transcription factor so that infection does not proceed. In addition to observations for NF-1X investigation, we have identified an inhibitor of viral expression, NF-1A, expressed at levels that block the up-regulation of JCV by NF-1X. These two DNA binding proteins have similar DNA binding sites at their N terminal end for the viral DNA but differ in their C terminal ends that are transactivation regions that recognize host cell factors. The laboratory has recently had an instructional video made as part of its publication in the Journal of Visual Experiments that details the methodology developed to differentiate the multipotential brain derived progenitor cells through lineages for oligodendrocytes. In addition to this approach, we have more recently applied gene expression microarrays at various time points during differentiation of progenitor cells to astrocytes. Preliminary evidence shows that clusters of genes get up-regulated quickly when the progenitor cells are exposed to selective medium driving differentiation to GFAP positive cells. Some of these genes are those involved in acquiring the characteristics of susceptibility to JCV infection. These observations make the link between phenotypes of cell seen in clinical brain tissue and cell models of infection in culture systems. We have further defined a protocol using flow cytometry to select population of cells and sort them based on stages of differentiation. For example, some cells will display phenotypes based on staining patterns of cell surface markers that allow sorting in the FACS Aria II instrument following infection by viruses. The identification of cells in the human developing brain then allows determination of not only stages of differentiation but also analysis of cell gene expression using microarrays analysis.

我们已经鉴定并培养了来自人脑的人类中枢神经系统衍生祖细胞。这些细胞是巢蛋白阳性，不表达任何其他分化表型标记。在用特定生长因子处理这些细胞后，这些细胞可以定向到使这些细胞成为神经元、星形胶质细胞或少突胶质细胞的谱系途径。我们正在研究这种分化的基因组学以及在这种分化过程中独特产生的蛋白质。我们使用病毒感染来标记这些细胞的表型，并能够根据这些细胞所指向的途径来增加或减少感染。我们最近利用绿色或红色荧光蛋白的报告基因制作了仅在特定细胞谱系途径中表达的基因表达载体。这些基因产物是用视频显微镜视觉识别的，因此它们标志着细胞分化的途径。这样的实验最终将为大脑发育的本质提供有价值的见解，并为大量细胞治疗研究提供关键的实验工具。