Reversibly immortalized human olfactory ensheathing glia from an elderly donor maintain neuroregenerative capacity

Abstract

A continuous normal function of olfactory ensheathing glia (OEG) is to promote axonal regeneration from the olfactory neuroepithelium to the brain, and their neuroregenerative potential in other CNS sites such as the injured spinal cord has been studied for over a decade. However, human OEG are difficult to obtain in large amounts directly from tissues, and the derived primary cultures have a limited duplication capacity. Thus, although auto-transplantation may be an obvious option for initial proof-of-concept trials, alternatives must be explored to obtain large quantities of homogeneous, pre-characterized OEG for wide-scale therapeutic use. We have cultured primary human OEG derived from olfactory bulbs (OB) obtained by necropsy and successfully extended the replicative lifespan of these cells using lentivectors encoding Bmi-1 and TERT transgenes flanked by loxP sites. In contrast to the primary cells which could only be expanded for a limited number of passages (approximately 12), adult human OEG immortalized Bmi-1/TERT divided indefinitely in culture. Clonal lines were isolated and the floxed transgenes could be excised by lentivector-mediated Cre recombinase delivery. Primary, immortalized, and deimmortalized human OEG all expressed typical markers of this cell type and importantly, were all able to promote axonal regeneration of adult rat retinal ganglion neurons (RGN) in co-culture assays.

Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration

“…1b) to levels similar to that observed using primary cultured OEGs derived from elderly and young human donors (Fig. 1c) and other human OEG lines that also exhibit this proregenerative capacity (Garcia-Escudero et al, 2011;Lim et al, 2010).…”

“…Primary cultures of OEG were prepared from human olfactory bulbs derived from young and elderly cadaver donors as described Lim et al, 2010) and employing a method described previously with modifications (Moreno-Flores et al, 2003a,b). The cultures established in this way contained >99% cells that expressed the S100b protein and diffuse glial fibrillary acidic protein (GFAP) , detected by antibodies against S100b (Sigma) and GFAP (Chemicon International, Temecula, CA).…”

“…Previously, we established immortalized OEG clonal cell lines conserving the proregenerative capacity of primary OEGs with promise for their therapeutic use , 2011Lim et al, 2010;Moreno-Flores et al, 2003a,b, 2006. The mechanisms by which OEGs facilitate axonal outgrowth are still not fully understood.…”

The Impact of Oxidative Stress Factors on the Viability, Senescence, and Methylation Status of Olfactory Bulb-Derived Glial Cells Isolated from Human Cadaver Donors

“…Another approach for enhancing the expansion of OECs is their reversible immortalization [Lim et al, 2010], and/or the application of induced pluripotent stem cells [Lee-Kubli and Lu, 2015], or even mesenchymal stem cells [Dasari et al, 2014]. We also noticed the smallest percentage of senescence cells and the highest percentage of living cells in the culture of the first group of donors when compared to the second group.…”

Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells

“…To analyze the interactions of B. pseudomallei WT MSHR520 and the CPS I-deficient mutant with human cells, we used human OECs (36,37). These assays were used to define the adhesion and invasion of WT MSHR520 and the ⌬cap mutant and to determine the immune responses of these cells to B. pseudomallei.…”