Maintaining epitheliopoietic potency when culturing olfactory progenitors

Abstract

The olfactory epithelium is remarkable for the persistence of multipotent, neurocompetent progenitor and stem cells throughout life that can replace all of the various cell types of the epithelium following injury. The therapeutic exploitation of the neurocompetent stem cells of the adult olfactory epithelium would be facilitated by the development of a culture system that maintains the in vivo potency of the progenitors while they are expanded and/or manipulated. We have used an air-liquid interface culture protocol, in which a feeder cell layer of 3T3 cells is established on the underside of a culture insert and FACS-isolated or unsorted progenitor cells from the methyl bromide-lesioned adult rodent epithelium are seeded on upper side. Under these conditions, epithelial cells other than HBCs are capable of organizing themselves into complex 3-dimensional, epithelium-lined spheres, which can be passaged. The spheres contain cells with the molecular phenotype of globose basal cells, horizontal basal cells, sustentacular cells and neurons. Spheres derived from mice that express the green fluorescent protein constitutively can be dissociated after 6 days in vitro and directly transplanted into the epithelium of wild type, methyl bromide-lesioned mice via nasal infusion. The resulting clones contain the various cell types observed in aggregate when globose basal cells are transplanted acutely. In contrast, the same cells cultured as two dimensional, submerged cultures undergo fibroblastic transition after transplantation and do not integrate into the epithelium. In conclusion, the culture system described here maintains the potency of progenitors, which can then participate in epitheliopoiesis in vivo.

Contribution of Polycomb group proteins to olfactory basal stem cell self-renewal in a novel c-KIT+ culture model and in vivo

“…As mentioned above, we found that suspension or sphere cultures grew poorly with attempted passaging, consistent with prior reports (Jang et al, 2008;Krolewski et al, 2011). Therefore, we transitioned to adherent substrates, such as vitronectin, but found cells remained difficult to expand with standard neurosphere medium (Reynolds and Weiss, 1992).…”

“…Despite formation of primary spheres in standard neurosphere medium, attempts at expanding sphere-forming cultures were unsuccessful, consistent with prior reports (Jang et al, 2008). Thus, we abandoned floating cultures and allowed cultures derived Table 1 and Fig.…”

“…One major limitation in the study of olfactory basal stem cell regulatory mechanisms, or in manipulating OE stem cells for other potential uses, has been the inability to efficiently expand adult OE basal cells in culture (Jang et al, 2008;Krolewski et al, 2011;Nivet et al, 2011). Existing culture models have generally involved primary or short-term studies.…”

“…Existing culture models have generally involved primary or short-term studies. Short-term embryonic or postnatal OE explants have provided key insights into the regulation of migratory neural precursor basal cells (Calof and Chikaraishi, 1989;Gokoffski et al, 2011;Jang et al, 2008). Other models have utilized mesenchymal-like cells capable of ongoing renewal in culture (Murrell et al, 2005(Murrell et al, , 2008.…”

Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

“…In such cultures, Sus cells can be visualized as coherent epithelial sheets in which virtually all cells express cytokeratin 18 (CYT18; also known as keratin 18); Fig. 7D shows CYT18 staining of Sus cells in the OE in vivo (see Pixley, 1992;Jang et al, 2008). To determine the effects of ACTB and GDF11 on Sus cell development in vitro, cultures were grown for 48 hours in the presence or absence of these factors.…”