The Generation of Olfactory Epithelial Neurospheres in Vitro Predicts Engraftment Capacity Following Transplantation in Vivo

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.