Survival & differentiation of ESNLCs after transplant

描述（改编自摘要）：项目3提出探索通过离体药理学和/或遗传操作诱导神经胶质细胞谱系的小鼠ES细胞是否可以在移植到脊髓空洞或挫伤的成年大鼠脊髓中存活;分化为神经元，少突胶质细胞。成年大鼠脊髓损伤;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ES细胞移植到空洞后（挫伤后9天）死亡的机制，并通过保护机制减少死亡，无论是基于遗传还是基于遗传。初步研究表明，在移植到挫伤诱导的空洞后，细胞分化为神经胶质（主要）和神经元（少量），但也表明移植后供体ES细胞大量死亡.研究人员假设，这种细胞死亡可能限制了植入程序的功能益处。因此，他们提出了如何研究最小化原位死亡，特别是由凋亡（主要在创伤后第一个48小时内）和兴奋性毒性（主要在损伤后第一个2天后）机制介导的死亡，通过特定的药理学和遗传干预推测这种结果将优化功能恢复。基因操作（例如，bcl-2过表达或Bax或caspase 9基因缺失）将在ES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中进行的研究（例如，泛半胱天冬酶抑制剂ZVAD或谷氨酸受体拮抗剂MK-801）。移植后，将以规定的时间间隔定量评估供体ES细胞的存活和分化命运（通过多种标记技术鉴定，包括细胞或细胞类型特异性结构固有的lacZ转基因、供体细胞的BrdU预孵育、非扩散性荧光细胞质染料、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物）。ES移植的解剖学、生理学和功能影响将结合项目IV（“移植的ESNLC在脊髓回路中的整合”）和“损伤和功能评估核心”来确定。

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".