A Neuroregenerative Human Ensheathing Glia Cell Line with Conditional Rapid Growth

Abstract

Ensheathing glia have been demonstrated to have neuroregenerative properties but this cell type from human sources has not been extensively studied because tissue samples are not easily obtained, primary cultures are slow growing, and human cell lines are not available. We previously isolated immortalized ensheathing glia by gene transfer of BMI1 and telomerase catalytic subunit into primary cultures derived from olfactory bulbs of an elderly human cadaver donor. These cells escape the replicative senescence characteristic of primary human cells while conserving antigenic and neuroregenerative properties of ensheathing glia, but their low proliferative rate in culture complicates their utility as cell models and their application for preclinical cell therapy experiments. In this study we describe the use of a conditional SV40 T antigen (TAg) transgene to generate human ensheathing glia cell lines, which are easy to maintain due to their robust growth in culture. Although these fast growing clones exhibited polyploid karyotypes frequently observed in cells immortalized by TAg, they did not acquire a transformed phenotype, all of them maintaining neuroregenerative capacity and antigenic markers typical of ensheathing glia. These markers were also retained even after elimination of the TAg transgene using Cre/LoxP technology, although the cells died shortly after, confirming that their survival depended on the presence of the immortalizing genes. We have also demonstrated here the feasibility of using these human cell lines in animal models by genetically marking the cells with GFP and implanting them into the injured spinal cord of immunosuppressed rats. Our conditionally immortalized human ensheathing glia cell lines will thus serve as useful tools for advancing cell therapy approaches and understanding neuroregenerative mechanisms of this unique cell type.

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).…”

“…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.…”

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

“…Examples include: (a) the source of the tissue: olfactory bulb (OB), (Li et al, 1997;Ram on-Cueto et al, 1998), olfactory mucosa or lamina propria (LP); (Au and Roskams, 2002;F eron et al, 1999), (b) the age of the donor [adult, neonatal, or fetal; (Barnett and Roskams, 2008;Huang et al, 2008)], (c) the time in tissue culture (Garcia-Escudero et al, 2010a;Llamusi et al, 2010;Novikova et al, 2010), (d) whether the OECs are purified [in most studies, e.g., (Barnett and Chang, 2004;Chuah and Au, 1993)], or used as mixed primary cultures (Deumens et al, 2006;Raisman and Li, 2007;Yui et al, 2011), or cell lines (Garcia-Escudero et al, 2010b), (e) the use of growth factors (Bianco et al, 2004;Pollock et al, 1999), (f) the species: apart from the rat, other OECs have been prepared from mouse , pig (Radtke et al, 2004), dog (Ito et al, 2006), monkey (Guest et al, 2008), and human (Barnett et al, 2000;F eron et al, 1998).…”

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.…”

“…Methods for the routine culture and infection of human cells were performed essentially as previously described (38), with the following modifications. Human OECs were maintained in Dulbecco's modified Eagle's medium (DMEM)-F12 (1:1; Gibco, Grand Island, NY) supplemented with 10% (vol/vol) fetal bovine serum (Moregate BioTec, Bulimba, QLD, Australia), 2 mM GlutaMAX (Gibco), 20 g/ml bovine pituitary extract (Life Technologies, Carlsbad, CA), 2 M forskolin (Cell Signaling Technology, Danvers, MA), and 100 U/ml penicillin-streptomycin (Gibco) (36). OECs were used to seed 24-well plates and incubated at 37°C in 5% CO 2 until approximately 80% confluence.…”