Directed In Vivo Differentiation of Neural Stem Cells for Repair of Brain Lesion

描述：神经干细胞或祖细胞移植作为促进组织再生和重建创伤性脑损伤损伤腔的一种有前景的策略已被越来越多地提出。然而，移植后对人类神经干细胞/祖细胞（hNSCs）分化的控制不佳，移植细胞在病变部位的低存活率和低整合严重限制了细胞治疗的成功。本研究的目的是开发一种纳米颗粒介导的转录激活方法，以指导hNSCs在体内向功能性神经元和少突胶质细胞的分化。该方法将更好地控制和提高功能细胞在体内的存活、分化、成熟和整合的效率。我们将通过在特制透明质酸水凝胶中移植转录激活的hNSCs到创伤性脑损伤（TBI）大鼠模型中，并通过检查细胞存活、移植细胞的分化和整合以及病变部位的组织再生结果来证明这种方法的优势。基于最近的研究发现，通过病毒转染过度表达关键转录因子可诱导快速有效地产生功能性神经元，以及可生物降解的纳米颗粒转染方法能够介导干细胞和祖细胞中高效的转基因表达，我们计划验证人类诱导多能干细胞（hiPSC）衍生的NSCs在移植前短暂表达阶段特异性转录因子神经原素2 （Ngn2）和少突胶质细胞转录因子Olig2将分别促进其在体内向功能神经元和少突胶质细胞分化的假设。我们将在这一探索性资助中追求以下具体目标：(1)建立高效纳米颗粒，分别通过编码转录因子Ngn2和Olig2的质粒转染hipsc衍生的NSCs，并分别研究Ngn2和Olig2表达对神经元和少突胶质细胞分化的影响；(2)证明ngn2转染和olig2转染的hNSCs在体内的定向分化，以及水凝胶递送在促进转染细胞的存活、分化和成熟以及促进大鼠TBI模型中组织再生方面的优势。本研究不仅将促进我们对转录因子激活在体内调节移植的hipsc来源的NSCs的存活、分化和成熟的理解，而且还将为脑组织再生治疗各种创伤性损伤和神经退行性疾病提供新的策略。这种新的纳米颗粒方法与具有确定组合物的原位形成水凝胶一起用于临床翻译是非常理想的。

DESCRIPTION: Transplantation of neural stem or progenitor cells has been increasing proposed as a promising strategy to promote tissue regeneration and reconstruct the lesion cavity of TBI. However, poor control over the differentiation of human neural stem/progenitor cells (hNSCs) following transplantation, low survival and integration of the transplanted cells at the lesion site has severely limited the success of cell-based therapies. The objective of this study is to develop a nanoparticle-mediated transcription activation approach to direct the differentiation of hNSCs in vivo into functional neurons and oligodendrocytes. This new method will afford better control and higher efficiency of the survival, differentiation, maturation, and integration of the functional cells in vivo. We will demonstrate the advantages of this approach by transplanting transcriptionally activated hNSCs in a tailored hyaluronic acid hydrogel into a rat model of traumatic brain injury (TBI), and by examining cell survival, differentiation and integration of the transplanted cells, and tissue regeneration outcomes at the lesion site. Based on recent findings that over-expression of key transcriptional factors by viral transfection induces rapid and efficient production of functional neurons, and biodegradable nanoparticle transfection method capable of mediating efficient transgene expression in stem and progenitor cells, we plan to test the hypothesis that transient expression of stage-specific transcriptional factor neurogenin 2 (Ngn2) and oligodendrocyte transcription factor Olig2 in human induced pluripotent stem cell (hiPSC)-derived NSCs prior to transplantation will promote their in vivo differentiation towards functional neurons and oligodendrocytes, respectively. We will pursue the following specific aims in this exploratory grant: (1) to establish a highly effective nanoparticles for transfection of hiPSC-derived NSCs by plasmids encoding transcription factor Ngn2 and Olig2, respectively, and to investigate the effect of Ngn2 and Olig2 expression on neuronal and oligodendrocyte differentiation, respectively; (2) to demonstrate the directed differentiation of Ngn2-transfected and Olig2-transfected hNSCs derived from hiPSCs in vivo, and the advantage of hydrogel delivery in promoting the survival, differentiation, and maturation of the transfected cells and in enhancing tissue regeneration in a rat TBI model. This study will not only advance our understanding of transcription factor activation in regulating the survival, differentiation, and maturation of transplanted hiPSC-derived NSCs in vivo, but also offer new strategies for brain tissue regeneration in treating a wide range of traumatic injuries and neurodegenerative diseases. This new nanoparticle method together with the in situ forming hydrogels with defined compositions is highly desirable for clinical translation.