Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Exp Neurol. 2011 Jun;229(2):308-23. doi: 10.1016/j.expneurol.2011.02.014. Epub 2011 Mar 1.
The stem and progenitor cells of the olfactory epithelium maintain the tissue throughout life and effectuate epithelial reconstitution after injury. We have utilized free-floating olfactory neurosphere cultures to study factors influencing proliferation, differentiation, and transplantation potency of sphere-grown cells as a first step toward using them for therapeutic purposes. Olfactory neurospheres form best and expand most when grown from neonatal epithelium, although methyl bromide-injured or normal adult material is weakly spherogenic. The spheres contain the full range of epithelial cell types as marked by cytokeratins, neuron-specific antigens, E-cadherin, Sox2, and Sox9. Globose basal cells are also prominent constituents. Medium conditioned by growth of phorbol ester-stimulated, immortalized lamina propria-derived cells (LP(Imm)) significantly increases the percentage of Neurog1eGFP(+) progenitors and immature neurons in spheres. Sphere-forming capacity resides within selected populations; FACS-purified, Neurog1eGFP(+) cells were poorly spherogenic, while preparations from ΔSox2eGFP transgenic mice that are enriched for Sox2(+) basal cells formed spheres very efficiently. Finally, we compared the potency following transplantation of cells grown in spheres vs. cells derived from adherent cultures. The sphere-derived cells engrafted and produced colonies with multiple cell types that incorporated into and resembled host epithelium; cells from adherent cultures did not. Furthermore, cells from spheres grown in conditioned media from the phorbol ester-activated LP(Imm) line gave rise to significantly more neurons after transplantation as compared with control. The current findings demonstrate that sphere formation serves as a biomarker for engraftment capacity and multipotency of olfactory progenitors, which are requirements for their eventual translational use.
嗅上皮的干细胞和祖细胞在整个生命过程中维持组织，并在损伤后实现上皮重建。我们利用游离嗅神经球培养物来研究影响球体生长细胞增殖、分化和移植能力的因素，作为将其用于治疗目的的第一步。尽管溴甲烷损伤或正常成年材料的成球能力较弱，但从新生上皮中生长的嗅神经球形成最佳且扩增最多。这些球体包含细胞角蛋白、神经元特异性抗原、E-钙黏蛋白、Sox2 和 Sox9 标记的完整上皮细胞类型。球状基底细胞也是突出的组成部分。经佛波醇酯刺激的永生化固有层衍生细胞（LP（Imm））培养的条件培养基可显著增加球体中 Neurog1eGFP（+）祖细胞和未成熟神经元的比例。球形成能力存在于选定的群体中；FACS 纯化的 Neurog1eGFP（+）细胞成球能力差，而富含 Sox2（+）基底细胞的ΔSox2eGFP 转基因小鼠制备物则非常有效地形成球体。最后，我们比较了在球体中培养的细胞与从贴壁培养物中获得的细胞移植后的效力。球源性细胞植入并产生具有多种细胞类型的集落，这些细胞整合并类似于宿主上皮；来自贴壁培养物的细胞则没有。此外，与对照相比，来自在佛波醇酯激活的 LP（Imm）系的条件培养基中生长的球体的细胞在移植后产生了显著更多的神经元。目前的研究结果表明，球体形成是嗅前体细胞移植能力和多能性的生物标志物，这是它们最终转化应用的要求。
