Strategies for Restoring Olfactory Neurogenesis

（修订摘要）说明（由申请人提供）：人类和其他动物的嗅觉功能的维持取决于嗅觉上皮神经发生在整个生命过程中的持续性。如果在嗅上皮损伤的过程中祖细胞群被破坏，则组织经历化生并重建为呼吸上皮。我们已经表明，球状基底细胞（GBC），其中既广泛有效的和神经元定向祖细胞，可以选择性地分离的流式细胞仪，并将在移植后移植到一个新的主机的上皮。因此，移植可能是恢复上皮细胞神经发生能力的可行策略。执行这一战略的进展取决于是否有足够多的祖细胞。为此，我们提出实验来测试祖细胞库是否可以在体外扩增，并且仍然在体内适当地移植和分化。 将有两个具体目标。首先，GBC将从正常小鼠中选择性分离， 普遍表达GFP和在收获前不久用溴甲烷气体损伤的其他细胞。在后一种情况下，病变的影响是激活嗅觉干细胞并使群体偏向多能性。GBC将在含有多种生长因子的成分确定的培养基中培养，这些生长因子已被证明可在异质外植体或分离培养物中驱动增殖和/或神经发生。其次，GBC将通过用编码来自SV 40的大T抗原癌基因的温度敏感型的逆转录病毒载体转染而条件性永生化。在转换至非允许温度后，将测定所得克隆细胞系的分化。扩增的原代分离物和在癌基因失活后分化良好的细胞系都将被移植，以确定它们在体内的分化能力（包括确定供体来源的神经元是否表达气味受体）。目标的成功完成将：1）阐明生长因子对嗅觉神经发生的控制的性质; 2）确定细胞替代是否是恢复神经发生的可行策略; 3）产生一组 用于进一步研究祖细胞能力的方案和细胞试剂， 神经元分化

(Revised Abstract) DESCRIPTION (provided by applicant): The maintenance of olfactory function in humans as in other animals depends on the persistence of neurogenesis in the olfactory epithelium throughout life. If the progenitor population is destroyed during the course of an injury to the olfactory epithelium, the tissue undergoes metaplasia and reconstitutes as respiratory epithelium instead. We have shown that the globose basal cells (GBCs), among which are both broadly potent and neuronal-committed progenitors, can be selectively isolated by FACS and will engraft in the epithelium of a new host after transplantation. Accordingly, transplantation may be a viable strategy for restoring the epithelium's capacity for neurogenesis. Progress in implementing that strategy depends on the availability of a sufficiently large population of progenitors. To that end we are proposing experiments to test whether the pool of progenitors can be expanded in vitro and still engraft and differentiate appropriately in vivo. Two Specific Aims will be pursued. First, GBCs will be selectively isolated from normal mice that ubiquitously express GFP and others that have been lesioned with methyl bromide gas shortly before harvest. In the later case, the effect of the lesion is activate olfactory stem cells and to bias the population toward multipotency. The GBCs will be cultured in defined media with a melange of growth factors that have been shown to drive proliferation and/or neurogenesis in heterogeneous explant or dissociated cultures. Second, GBCs will be conditionally immortalized by transfection with a retroviral vector that encodes a temperature sensitive version of the large T antigen oncogene from SV40. The differentiation of the resulting clonal cell lines will be assayed after switching to the non-permissive temperature. Both the expanded primary isolates and cell lines that differentiate well after inactivation of the oncogene will be transplanted in order to define their capacity for differentiation in vivo (including a determination of whether donor-derived neurons express odorant receptors). Successful completion of the Aims will; 1) clarify the nature of growth factor control on olfactory neurogenesis; 2) determine whether cellular replacement is a viable strategy for restoring neurogenesis; 3) generate a set of protocols and cellular reagents that are useful for further studies of progenitor cell capacity and neuronal differentiation.