Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications

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-MycER^TAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycER^TAM-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 (p75^NTR, 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-MycER^TAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells.

Figure 1

c-MycER^TAM transduction characterisation and karyotyping of human olfactory mucosa cells. (A) The incorporation of the c-MycER^TAM construct in PA5 cells was analyzed by genomic DNA amplification (gDNA) using specific primers (lanes 4–6). CTX0E03 cells were used as a positive control and primary cells from the olfactory mucosa (phOMCs) as a negative control. Amplification of endogenous c-Myc was performed to verify amplification of gDNA samples (lanes 1–3). Finally, gDNA and primers were fractionated to verify integrity (lanes 7–12). Expression of the c-MycER^TAM protein is shown by western blotting using (B) anti-c-Myc and (C) anti-ERα primary antibodies. CTX0E03 cells were used as a positive control and primary cells (phOMCs) as a negative control. (D) PA5 cells had a normal 46, XY karyotype, whereas (E) PA7 cells had a mosaic karyotype with hypodiploidy (45, X) in 3 of 20 cells.

Figure 2

(A) Growth of PA5 and PA7 c-MycER^TAM-derived human olfactory mucosa cells was followed for 60 days. (B) Morphology of PA5 and PA7 cells at 100 × total magnification. Scale bar represents 400 µm.

Figure 3

Biomarker expression was assessed by immunocytochemistry for PA5 c-MycER^TAM-derived hOMCs at passage 8, and primary cells (phOMCs) at passage 3. Both cell populations stained positive for OEC markers (p75^NTR, S100ß and oligodendrocyte marker O4), neural markers (nestin and ß-III-tubulin), and fibroblast-associated markers (fibronectin, CD90/Thy1) for PA5 and primary hOMCs. Primary hOMCs were negative for GFAP, whereas PA5 hOMCs were positive for this glial marker. Scale bars represent 200 µm.

Figure 4

Co-culture of PA5 hOMCs at passage 8 (PDL 10) and passage 15 (PDL 18) with NG108–15 cells. (A) Timeline of the co-cultures with NG108-15 cells. (B) Mean neurite length, (C) mean longest neurite, and (D) mean number of neurites per neuron measurements were performed manually. (E) Representative images of co-cultures stained with ß-III-tubulin at 100 × total magnification and zoomed regions with NG108-15 cells. Scale bar represents 400 µm at 100 × total magnification, and 200 µm at the zoomed regions. Data are mean SEM, n = 3.

Figure 5

Co-culture of PA5 hOMCs with DRG neurons. (A) Timeline of the co-cultures with DRG neurons. (B) Mean neurite length, (C) mean longest neurite, and (D) mean number of neurites per neuron measurements were performed manually. Representative images of co-culture stained with ß-III-tubulin (red) and S100ß (green) at 100 × total magnification and zoomed regions with DRG neurons. Scale bar represents 400 µm at 100 × total magnification, and 200 µm at the zoomed regions. Data are mean SEM, n = 3.

Figure 6

Structure of the c-MycER^TAM provirus.

Figure 7

Generation of c-MycER^TAM - derived populations of human olfactory mucosa cells (hOMCs).