Regulation of Growth and Differentiation in 3-D Cultures of Olfactory Epithelium

描述（由申请人提供）：嗅觉上皮（OE）中神经元和非神经元群体的再生能力是众所周知的，并贯穿整个生命周期。凭借其可及性，OE的神经活性干细胞和祖细胞是用于自体细胞疗法的有吸引力的候选者。控制这些过程的调节信号和机制还不清楚。为了充分利用干细胞和祖细胞，需要对嗅觉上皮细胞生成的调节进行深入分析，但进展缓慢。我们已经开发了一种组织培养测定法，其中来自新生儿或成人病变OE的细胞以条件依赖性方式在培养孔插入物的空气-培养基界面处形成的球体内生长。值得注意的是，在3维（3-D）中培养的细胞保留了参与动物体内OE再生的潜力和可塑性，这与2-D中生长的细胞无法做到这一点形成鲜明对比。提出了两个目的来探索利用球体测定的体内样性质来调节细胞生成和分化的线索。目标1将测试生长因子单独或组合如何影响OE细胞在3-D培养物中的生长。其中一些因素在OE中的周转和再生过程中具有先前确定的作用;它们将校准测定，提供体外和体内结果的登记。对于大多数人来说，他们在OE中的作用知之甚少，尽管他们在细胞出生或分化的其他地方发挥作用。相反，目标2将确定一个复杂的分子混合物，这是已知的，在调节OE中发挥重要作用的相关线索。在这种情况下，培养介质的固有层细胞系的生长条件（来自粘膜的一部分，是深入到OE）是需要从成人病变的OE插入球的形成。我们将采取候选分子和开放式方法来确定相关因素。 公共卫生相关性：这项工作的健康相关性在于实现嗅觉干细胞和祖细胞的可调控生长的最终目标。除了帮助患有嗅觉障碍或嗅觉缺失的患者外，神经活性组织干细胞可能有助于修复神经系统的其他部分，包括受伤后的脊髓。

DESCRIPTION (provided by applicant): The capacity for regeneration of neuronal and non-neuronal populations in the olfactory epithelium (OE) is well-known and extends throughout life. By virtue of their accessibility, the neurocompetent stem and progenitor cells of the OE are attractive candidates for use in autologous cellular therapies. The regulatory signals and mechanisms that govern these processes are not well understood. An in-depth analysis of the regulation of olfactory epitheliopoeisis is needed in order to exploit the stem and progenitor cells fully, but progress has been slow. We have developed a tissue culture assay in which cells from the neonatal or adult lesioned OE grow within spheres that form at the air-media interface of culture well inserts in a condition- dependent manner. Remarkably, cells that are cultivated in 3-dimensions (3-D) retain the potency and plasticity to participate in the regeneration of the OE in the animal, which stands in sharp contrast to the failure of cells grown in 2-D to do so. Two Aims are proposed to explore the cues regulating cell generation and differentiation taking advantage of the in vivo-like properties of the sphere assay. Aim 1 will test how growth factors, alone or in combination, will affect the growth of OE cells in the 3-D cultures. Some of the factors have a previously established role in the turnover and regeneration process in the OE; they will calibrate the assay, providing registration of the in vitro and in vivo results. For the majority, little is known of their role in the OE, although they function elsewhere in cell birth or differentiation. In contrast, Aim 2 will determine the relevant cues within a complex molecular mix that is known to play an important role in regulating the OE. In this case, culture media conditioned by the growth of a lamina propria cell line (derived from the part of the mucosa that is deep to the OE) is required for the formation of spheres from adult lesioned OE on inserts. We will take both a candidate molecule and an open-ended approach to identifying the relevant factors. PUBLIC HEALTH RELEVANCE: The health relevance of the work resides in the ultimate goal of achieving regulated controlled growth of olfactory stem cells and progenitor cells. Besides the goal of helping patients with dysosmias or anosmias, the neurocompetent tissue stem cells may prove useful for repairing other parts of the nervous system, including the spinal cord after it is injured.