Comparison of bulbar and mucosal olfactory ensheathing cells using FACS and simultaneous antigenic bivariate cell cycle analysis

Abstract

Transplantation of olfactory ensheathing cells (OECs) is a promising route for CNS repair. There have, however, been major discrepancies between the results from different groups. Part of this can be attributed to variations in cell sources and culture protocols. Accurate estimation of the proportions of OECs and their associated fibroblasts (ONFs) and their evolution with time in culture is an essential baseline for establishing the reparative properties of transplants. In this study, we compare the evolution of cultures from the superficial layers of the olfactory bulb with tissue from the olfactory mucosa, both whole and split into lamina propria and epithelial layer. We used FACS based on p75 and Thy1 to provide a robust and objective numerical estimate of the numbers of OECs and ONFs, respectively in the cultures. A novel four color simultaneous antigenic bivariate cell cycle analysis shows that proliferation of OECs is time-limited, and is unable to prevent an overall loss of OECs with time. Overall, the numbers of OECs in the cultures were inversely correlated with the deposition of fibronectin (FN). Further, culture of the cells purified by flow cytometry shows that, whereas the Thy1 population is terminally differentiated, the p75 population from the mucosal samples generates subpopulations with different antigenic phenotypes, including the reappearance of a subpopulation of p75 cells expressing FN. Culturing epithelial samples at high density reveals an unexpected transient stem cell-like population of rapidly proliferating p75 positive cells.

Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb

“…1C). Cytometry analyses were conducted for p75 expression, because this is the only referred marker known to be specifically and differentially expressed in vivo by OECs (Gueye et al, 2011;Kueh et al, 2011;Windus et al, 2010). Intracellular GFAP staining showed that all the p75 cells were GFAP positive, confirming the high purity of the OECs cultures (Supp.…”

“…In fact, because of their specific role during neurogenesis of primary olfactory system, OECs could be found in peripheral nervous system (PNS) and in CNS. Also, it has been previously demonstrated that specific populations of OECs exist in each compartment and that these cells exhibit specific behaviors in vitro and in vivo (Guerout et al, 2010;Kueh et al, 2011;Paviot et al, 2011;Richter et al, 2005;Windus et al, 2010). Specific microenvironment may explain the presence of distinct OECs subpopulations.…”

Olfactory mucosa: a rich source of cell therapy for central nervous system repair

“…GBCs are positive for early neuronal differentiation markers such as Mash1, Neurogenin1, N-CAM, Sox2, and nestin (Gordon et al, 1995;Beites et al, 2005;Thakur et al, 2014), hence confirming that they are neural progenitor cells, while HBCs express keratin 5 and keratin 14 but are negative for neuronal markers (Carter et al, 2004) (see Table 1). However, only specific antibodies GBC-2 and GBC III were used to isolate GBCs from the OE by FACS, according to recent data (Chen et al, 2004;Krolewski et al, 2013;Thakur et al, 2013Thakur et al, , 2014, while ICAM-1 was Brought to you by | New York University Bobst Library Technical Services Authenticated Download Date | 5/25/15 10:27 PM Human Gorrie et al, 2010;Kachramanoglou et al, 2013;Tabakow et al, 2013;Xie et al, 2013 Rat Rizek andKawaja, 2006;Kalincik et al, 2010;Centenaro et al, 2011;Kueh et al, 2011;Paviot et al, 2011;Stamegna et al, 2011;Tharion et al, 2011;Mayeur et al, 2013;Ishihara et al, 2014Dog Granger et al, 2012Ziege et Rat Kalincik et al, 2010;Centenaro et al, 2011;Paviot et al, 2011;Stamegna et al, 2011;Ishihara et al, 2014 S100 OECs Human Gorrie et al, 2010;Kachramanoglou et al, 2013;Tabakow et al, 2013Rat Centenaro et al, 2011 ( used to purify HBCs (Iwai et al, 2008;Fletcher et al, 2011;…”

“…OM-OECs and OB-OECs present different proliferative capacities (Kueh et al, 2011) and exhibit considerable overlap of antigenic markers in vitro (García-Escudero et al, 2012). It appears that OM-OECs and OB-OECs display distinct gene expression patterns.…”

Ultrastructural analysis of olfactory ensheathing cells derived from olfactory bulb and nerve of neonatal and juvenile rats

“…Microscopic studies of cultured OECs have shown morphologic differences among cultures obtained from lamina propria of nasal mucosa and from olfactory bulb (Kueh et al, 2011;Watanabe et al, 2006). However, there is little evidence regarding morphological ultrastructure particularities in different developmental stages and in different anatomical origins of OECs, including olfactory nerve.…”