Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury
Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury
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DOI：
10.3791/50641
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发表时间：
2014-07-01
影响因子：
1.2
通讯作者：
Tuszynski, Mark
中科院分区：
文献类型：
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作者：
Lu, Paul;Graham, Lori;Tuszynski, Mark
神经干细胞（Neural stem cells，NSCs）具有自我更新、分化为神经元和胶质细胞的能力。移植的神经干细胞可以替代脊髓损伤（SCI）后丢失的神经元和神经胶质细胞，并可以形成功能中继，以重新连接病变上方和下方的脊髓节段。以前的研究移植神经干细胞受到脊髓损伤腔内移植物存活不完全的限制。此外，移植细胞存活、分化和工艺扩展的跟踪尚未优化。最后，在以前的研究中，培养的大鼠神经干细胞通常被报道在移植到受伤的脊髓时分化成胶质细胞，而不是神经元，除非命运被驱动到特定的细胞类型。为了解决这些问题，我们开发了新的方法来改善神经干细胞的存活，整合和分化，甚至严重的SCI的网站。从表达绿色荧光蛋白（GFP）的稳定转基因Fischer 344大鼠系的胚胎第14天脊髓（E14）新鲜分离NSC，并将其包埋到含有生长因子的纤维蛋白基质中;该制剂旨在将移植细胞保留在病变腔中并支持细胞存活。纤维蛋白/生长因子混合物中的NSC在胸3级（T3）完全脊髓横断后两周植入，从而避免炎症的高峰期。所得移植物完全填充病变腔并分化成神经元和胶质细胞，神经元将轴突延伸到宿主脊髓中非常长的距离。表达GFP的培养的人NSC的移植物导致类似的发现。因此，定义了用于改善神经干细胞移植、存活和体内结果分析的方法。
Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.