Strategies for Restoring Olfactory Neurogenesis

(修订摘要)描述(由申请人提供)：人类和其他动物一样，嗅觉功能的维持取决于嗅觉上皮细胞在整个生命过程中神经发生的持久性。如果祖细胞群在嗅觉上皮损伤过程中被破坏，该组织将经历化生并重建为呼吸道上皮。我们已经证明，球状基底细胞(GBCs)可以被FACS选择性地分离出来，并在移植后移植到新宿主的上皮中，其中GBCs具有广泛的潜能和神经元定位的前体细胞。因此，移植可能是恢复上皮细胞神经发生能力的可行策略。在执行这一战略方面的进展取决于是否有足够多的前身。为此，我们提议进行实验，以测试祖细胞池是否可以在体外扩大，并在体内移植和适当分化。 我们将追求两个具体目标。首先，将有选择地从正常小鼠中分离出GBCS 无处不在地表达GFP和其他在收获前不久受到甲基溴气体损害的产品。在后一种情况下，损伤的影响是激活嗅觉干细胞，并使群体偏向多能性。GBCs将在含有多种生长因子的特定培养基中培养，这些生长因子已被证明能在异种外植体或分离培养中促进增殖和/或神经发生。其次，通过将编码来自SV40的大T抗原癌基因的温度敏感版本的逆转录病毒载体导入GBCS，将有条件地使GBCS永生化。在切换到不允许的温度后，将对所产生的克隆细胞系的分化进行检测。扩大的原代分离株和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)。这些目标的成功完成将：1)阐明生长因子控制嗅神经发生的本质；2)确定细胞替代是否是恢复神经发生的可行策略；3)产生一套 有助于进一步研究祖细胞能力和细胞活性的方案和细胞试剂 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.