Growth Factor Regulation of Olfactory Neurospheres

描述(申请人提供)：人类和啮齿动物的嗅黏膜(OM)是一种可接触的组织，在损伤后表现出持续的神经发生和上皮重建，并显示出基于细胞的治疗的潜力。要实现OM的治疗潜力，需要一种培养系统，该系统可以用来扩大、了解祖细胞群体的调节，并作为祖细胞群体植入潜力的生物标志物。这一假设将得到检验，即嗅觉神经球的形成通过改变干细胞和祖细胞的“状态”以及作用于它们的生长因子影响，作为植入潜力的生物标记物。一个辅助目标是建立使用神经球作为一种手段来评估作用于体内干细胞和多潜能祖细胞的相互作用网络。OM来源的神经球将被测试为多能性和干性的生物标志物，使用从涉及不同内源性干细胞和祖细胞激活的体内条件分离的细胞的球体形成活性：正常成体、选择性神经元再生、甲基溴(MeBR)诱导的毒性损伤后上皮的完全重建以及出生后早期扩张。得到的球体将被量化，并确定它们的免疫表型。然后，嗅觉神经球将从构成表达GFP的小鼠中培养出来，并移植到MeBR损伤的宿主动物中，以确定植入的细胞是否产生OM细胞类型。为了测试作用于球状基底细胞(GBCs)或水平基底细胞(HBC)的因子是否能特异性地促进球的形成和植入潜能，嗅神经球将被各种生长因子处理，这些生长因子在OM中既有已知的取向，也有未知的功能。能够实时跟踪GBCs及其后代的报告小鼠将被用来获得嗅神经球上生长因子活性的读数。对球体大小、分布和荧光强度产生显着变化的生长因子将通过上述移植试验进行测试。OE的干细胞和祖细胞在成年人类中是可以获得的，并在整个成年期经历神经发生。与其他已将干细胞生物学转化为临床实践的系统一样，如造血系统，很明显，临床应用将需要扩大祖细胞群体的能力。这项工作将通过研究哪些因素可以用来促进这些干细胞群体的增殖和繁殖，来扩展初步研究，展示嗅觉粘膜来源细胞在治疗脊髓损伤和神经退行性疾病方面的临床潜力。

DESCRIPTION (provided by applicant): The olfactory mucosa (OM) of both humans and rodents is an accessible tissue that exhibits ongoing neurogenesis and epithelial reconstitution following injury with demonstrated potential for cell-based therapies. To realize the OM's therapeutic potential requires a culture system that can be used to expand, understand the regulation of, and serve as a biomarker for engraftment potential of progenitor populations. The hypothesis will be tested that olfactory neurosphere formation serves as a biomarker for engraftment potential by varying the "state" of the stem and progenitor cells and also the growth factor influences that act on them. A subsidiary goal is to establish the use of neurospheres as a means of evaluating the network of interactions acting on stem and multipotent progenitor cells in vivo. OM-derived neurospheres will be tested as biomarkers for multipotency and stemness using the sphere-forming activity of cells isolated from in vivo conditions that involve different endogenous stem and progenitor cell activation: normal adult, selective neuronal regeneration, full reconstitution of the epithelium after methyl bromide (MeBr)-induced toxic injury, and early postnatal expansion. Resulting spheres will be quantified and their immunophenotype determined. Olfactory neurospheres will then cultured from constitutively expressing GFP mice and transplanted into MeBr lesioned host animals to determine whether the engrafted cells produce OM cell types. To test whether factors that act on globose basal cells (GBCs) or horizontal basal cells (HBCs) specifically enhance sphere formation and engraftment potential, olfactory neurospheres will be treated with a variety of growth factors with both known tropism as well as unknown function in the OM. Reporter mice that allow for live tracking of GBCs and their progeny will be used to obtain readouts of growth factor activity on olfactory neurospheres. Growth factors that produce a significant change in the size distribution and fluorescence intensity of spheres will be tested via the aforementioned transplantation assay. the stem and progenitor cells ofthe OE are accessible in adult humans and undergo neurogenesis throughout adulthood. As in other systems that have translated stem cell biology to clinical practice, such as the hematopoietic system, it is apparent that the ability to expand the progenitor populations will be required for clinical applications. This work will expand on initial studies showing the clinical potential of olfactory mucosal-derived cells for treatment of spinal cord injury and neurodegenerative diseases by studying what factors can be used to enhance the proliferation and propagation of these stem cell populations.