Survival & differentiation of ESNLCs after transplant

生存

• 批准号: 6565294
• 负责人: JOHN W MCDONALD
• 金额: $ 17.53万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565294
• 关键词: apoptosis; astrocytes; cell differentiation; cell line; cell proliferation; embryonic stem cell; fluorescent dye /probe; laboratory rat; nervous system regeneration; neurons; oligodendroglia; spinal cord injury; stem cell transplantation

DESCRIPTION (adapted from the abstract): Project 3 proposes to explore whether murine ES cells, induced down a neuroglial lineage by ex vivo pharmacological and/or genetic manipulation, can survive transplantation into the syrinx or a contused adult rat spinal cord; differentiate into neurons, oligodendrocytes. contused adult rat spinal cord; differentiate into neurons, oligodendrocytes, and astrocytes functional recovery. The particular focus of this grant is to understand the mechanisms of ES cell death following transplantation into the syrinx (9 days following contusion) and to reduce death by protective mechanisms, both pharmacologically and genetically based. Preliminary studies, while suggesting glial (predominantly) and neuronal (minor amounts) differentiation by the cells following engraftment into a contusion- induced syrinx, also demonstrated that substantial donor ES cell death occurred after transplantation. The investigators hypothesize that this cell death is likely limiting functional benefit from the engraftment procedure. Therefore, they propose how to investigate minimizing that death in situ, particularly death mediated by apoptotic (predominantly in the first post traumatic 48 hours) and excitotoxic (predominantly after the first 2 post-injury days) mechanisms, by specific pharmacological and genetic interventions speculating that such an outcome will optimize functional recovery. Genetic manipulations (e.g., over-expression of bcl-2 or deletion of Bax or caspase 9 genes) will be created in the ES Cell Genetic Engineering Core B; pharmacological approaches will be derived from the studies performed in Projects I & II (e.g., the pan-caspase inhibitor ZVAD or the glutamate receptor antagonist MK-801). The survival and differentiation fate of donor ES cells (identified by a number of marking techniques, including a lacZ transgene intrinsic to the cell or to cell type- specific structures, BrdU pre-incubation of donor cells, non-diffusible fluorescent cytoplasmic dyes, mouse-specific chromosomal sequences or cell surface markers that will not cross-react with rat host cells) will be assessed quantitatively at defined intervals after transplantation. The anatomical, physiological, and functional impact of ES transplantation will be determined in conjunction with Project IV ("Integration of transplanted ESNLCs in spinal circuits) and the "Injury and functional assessment core".

描述（改编自摘要）：项目3旨在探索通过离体药理学和/或基因操作诱导小鼠胚胎干细胞沿神经胶质谱系下行，移植到注射器或挫伤的成年大鼠脊髓中是否能够存活；分化为神经元、少突胶质细胞。成年大鼠脊髓挫伤；分化为神经元、少突胶质细胞和星形胶质细胞功能恢复。这项资助的特别重点是了解胚胎干细胞移植到鼻腔后（挫伤后9天）死亡的机制，并通过药理学和遗传学上的保护机制减少死亡。虽然初步研究表明，在植入挫伤诱导的鼻窦后，细胞会分化为胶质细胞（主要）和神经元细胞（少量），但也表明移植后供体胚胎干细胞会发生大量死亡。研究人员推测，这种细胞死亡可能限制了移植过程的功能益处。因此，他们提出了如何通过特定的药理学和遗传干预来研究最小化原位死亡，特别是由凋亡（主要发生在创伤后48小时内）和兴奋毒性（主要发生在损伤后的前2天）机制介导的死亡，推测这样的结果将优化功能恢复。基因操作（例如，bcl-2的过表达或Bax或caspase 9基因的缺失）将在胚胎干细胞基因工程核心B中创建；药理学方法将源自项目I和II的研究（例如，泛半胱天冬酶抑制剂ZVAD或谷氨酸受体拮抗剂MK-801）。供体胚胎干细胞的存活和分化命运（通过许多标记技术鉴定，包括细胞固有的lacZ转基因或细胞类型特异性结构，供体细胞的BrdU预孵育，非扩散荧光细胞质染料，小鼠特异性染色体序列或不会与大鼠宿主细胞交叉反应的细胞表面标记）将在移植后的规定间隔内进行定量评估。胚胎干细胞移植的解剖、生理和功能影响将与项目IV（“将移植的胚胎干细胞整合到脊髓回路中”）和“损伤和功能评估核心”一起确定。