Abstract
Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive “off-the-shelf” alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.
Highlights
Spinal cord injury (SCI) is a devastating condition affecting 250,000 to 500,000 people a year worldwide[1]
After selection of stably transduced late-adherent human olfactory mucosa cells (hOMCs) with geneticin (G418), successful incorporation of c-MycERTAM to PA5 hOMCs was confirmed at both DNA and protein levels
When amplification was carried out with primers across the junction between c-Myc and ERTAM, identical sized bands were obtained for PA5 hOMCs and CTX030E (Fig. 1A lanes 4 and 6), whereas no product was obtained for primary hOMCs (Fig. 1A lane 5)
Summary
Spinal cord injury (SCI) is a devastating condition affecting 250,000 to 500,000 people a year worldwide[1]. Under non-permissive conditions, cells lose their immortality and return to a “normal” phenotype[28] Such an approach has been successfully applied to generate neural stem cell lines from human fetal cortical neuroepithelial cells following retroviral transduction with c-MycERTAM gene[29]. To mimic a cell contact regeneration process, co-cultures of PA5 hOMC monolayers were performed with the NG108-15 cell line, a hybrid rat glioma mouse neuroblastoma cell line, and with primary dorsal root ganglion (DRG) neurons. Clones from polyclonal populations such as PA5 c-MycERTAM-derived hOMCs could be further derived, expanded, banked, and screened to generate an allogeneic cell therapy product for the treatment of spinal cord injury
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