Directed Differentiation of Stem Cell Transplants into Myelinating Glia

描述（由申请人提供）：少突胶质细胞替代和髓鞘再生是脊髓损伤（SCI）后功能恢复的必要条件。干细胞或祖细胞的治疗递送是一种很有前途的工具，因为这些细胞产生减少炎症的因子，提高现有少突胶质细胞的存活率，并具有分化成新的少突胶质细胞的能力。然而，脊髓损伤后形成的局部环境并不能促进分化为成熟细胞，使其能够与宿主组织和髓鞘再生轴突进行功能整合。本研究将开发一种策略来改变这种局部环境，以指导人类胎儿来源的神经干细胞（hCNS-SCns）向髓鞘少突胶质细胞的分化。促进少突胶质细胞分化所需的关键因素在很大程度上是未知的，目前的方案是低效的。在Aim 1中，我们将使用转录因子（TF）活性的高通量阵列来识别受控微环境中的这些关键因子。作为复杂的细胞内信号网络的输出，TF活性代表了细胞的功能状态（如分化阶段）。利用这种新颖的系统生物学方法，我们有能力量化功能性细胞对各种细胞外信号的反应，并确定最有效地激活诱导少突胶质细胞分化的信号通路的信号。在3D微环境中培养的活hCNS-SCns中，TF激活的动态变化将被监测，以同时呈现定义的细胞外线索的组合。这些线索的影响将被系统地评估，以确定最好的条件，直接少突胶质细胞分化。在Aim 2中，hCNS-SCns将被移植到SCI的体内模型中，并在微环境中显示可最大化少突胶质细胞分化的线索。微环境将被纳入谢伊实验室先前开发的生物材料平台。这些桥式支架表现出一种微结构，促进轴突引导穿过损伤并返回宿主组织。支架介导的神经营养因子编码基因传递进一步促进轴突生长。这个建议建立在这个成功的基础上，通过添加一个成分来替代脊髓损伤后的髓鞘少突胶质细胞。此外，Aim 2将研究植入时的分化阶段如何影响hCNS- SCns的髓鞘形成能力。结果将显著提高细胞移植修复脊髓损伤的临床应用潜力。此外，拟议的培训计划将为研究员在学术界的成功职业生涯做好充分准备。该研究员将在人类干细胞培养、脊髓损伤的体内模型和基因修饰活细胞的方法方面获得专业知识，作为了解生物机制的工具。此外，她将运用这些知识开发再生医学的新策略。该研究员还通过拟议的培训计划获得了大量的专业发展机会，包括教学和写作讲习班、参加科学会议和指导学生。

DESCRIPTION (provided by applicant): Oligodendrocyte replacement and remyelination are essential to functional recovery after spinal cord injury (SCI). Therapeutic delivery of stem or progenitor cells is a promising tool as these cells produce factors that reduce inflammation, enhance survival of existing oligodendrocytes and have the capacity to differentiate into new oligodendrocytes. However, the local environment that develops after SCI does not promote differentiation into mature cells capable of functionally integrating with the host tisue and myelinating regenerated axons. The proposed research will develop a strategy to alter this local environment to direct differentiation of human fetal-derived neural stem cells (hCNS-SCns) into myelinating oligodendrocytes. The key factors required to promote oligodendrocyte differentiation are largely unknown and current protocols are inefficient. In Aim 1, we wil use a high-throughput array of transcription factor (TF) activity to identify these key factors within controlled microenvironments. As the output of complex intracellular signaling networks, TF activity represents the functional state of a cell (e.g., stage of differentiation). Using this novl systems biology approach, we have the ability to quantify the functional cell response to various extracellular cues and identify cues which most efficiently activate the signaling pathways that induce oligodendrocyte differentiation. Dynamic changes in TF activation will be monitored in live hCNS-SCns cultured in 3D microenvironments tuned to present combinations of defined extracellular cues simultaneously. Effects of these cues will be systematically evaluated to identify conditions that best direct oligodendrocyte differentiation. In Aim 2, hCNS-SCns will be transplanted to an in vivo model of SCI within microenvironments displaying cues found to maximize oligodendroctye differentiation. Microenvironments will be incorporated into a biomaterial platform previously developed by the Shea laboratory. These bridge scaffolds exhibit a microarchitecture that promotes axon guidance across the injury and back into host tissue. Scaffold-mediated delivery of genes encoding for neurotrophic factors further enhances axon growth. This proposal builds upon this success by adding a component to replace myelinating oligodendrocytes after SCI. In addition, Aim 2 will investigate how the stage of differentiation at the time of implantation affects the myelination capacity of hCNS- SCns. Results will significantly advance the clinical potential of cell transplantation for SCI repair. I addition, the proposed training plan will more than adequately prepare the felow for a successful career in academia. The fellow will gain expertise in human stem cell cultures, in vivo models of SCI and methods to genetically modify living cells as a tool to understand biological mechanisms. Furthermore, she will apply this knowledge to develop novel strategies for regenerative medicine. The fellow also has significant opportunities for professional development through the proposed training plan, including teaching and writing workshops, attending scientific conferences and mentoring students.