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，并且贯穿整个生命。由于其可及性，OE的神经活性干细胞和祖细胞是自体细胞治疗中有吸引力的候选者。控制这些过程的调控信号和机制尚不清楚。为了充分利用干细胞和祖细胞，需要对嗅上皮形成的调控进行深入的分析，但进展缓慢。我们已经开发了一种组织培养试验，其中来自新生儿或成人损伤的OE的细胞在培养孔插入的空气介质界面形成的球体中以条件依赖的方式生长。值得注意的是，在3维（3-D）中培养的细胞保留了参与动物OE再生的效力和可塑性，这与在2-D中培养的细胞无法做到这一点形成鲜明对比。提出了两个目的，以探索调节细胞产生和分化的线索，利用球试验的体内样特性。目的1将测试生长因子（单独或组合）如何影响3d培养中OE细胞的生长。有些因素在OE的周转和再生过程中具有先前确定的作用；他们将校准分析，提供体外和体内结果的注册。对于大多数人来说，尽管它们在细胞出生或分化的其他地方起作用，但对它们在OE中的作用知之甚少。相反，Aim 2将确定已知在调节OE中起重要作用的复杂分子混合物中的相关线索。在这种情况下，需要由固有层细胞系（来自深至OE的粘膜部分）生长的培养基，才能从成人病变的OE在插入物上形成球体。我们将采用候选分子和开放式方法来确定相关因素。

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.