Export citation and abstract BibTeX RIS
We thank Dr Izeta for his commentary and certainly agree with much of his sentiment. At no point did we intend to dismiss the utility of skin-derived precursor Schwann cells (SKP-SCs) for future therapeutic application. Certainly, sufficient precautions need to be implemented to safeguard their successful translation. These are not necessarily new concepts as the risk assessment for the clinical use of stem/progenitor cells has been frequently discussed [1].
We also agree that careful experimental design is paramount to ensure validity of pre-clinical research and that the adoptions of clinical standards to animal models should be promoted to bolster reproducibility and hasten clinical translation. Such an approach might help to avoid outcomes reported by Anderson et al [2], where cells prepared for clinical trials were no longer found to elicit efficacy.
Additionally, we are strong believers in the philosophy that research outcomes should be transparent and publicly available. As such, we felt it necessary to document our negative results to highlight to others the potential for harm and to acknowledge the impact of cellular age, time in culture and excessive in vitro expansion/manipulation on Schwann cell behavior.
Although we agree with the comments by Izeta, our data also suggest that safe in vitro cell expansion parameters may need to go beyond adoption of 'GMP-compliant protocols' and must be tailored to account for donor cell age and cell type.
Although routine for our human cell experiments, mycoplasma testing and plasmocin treatment are rarely included in quality control testing for rodent cell studies. This is partly because many cell culture reagents (media supplements, crude enzymes) are not tested and, in fact, contain mycoplasma. Our study further emphasizes the importance of adding similar quality control measures in all pre-clinical rodent work. It is important to note, however, adult rodent Schwann cells (whether they come from nerve or from skin) are still prone to transformation even with the best of culture conditions [3]. Our own unpublished observations have found the same results when adult Schwann cells are expanded beyond eight passages in defined (serum-free, mycoplasma-free) conditions (Stratton and Biernaskie, unpublished). As well, our own follow-up transplant studies using skin-derived Schwann cells exposed to minimal time in vitro and that were verified to be mycoplasma-free, did not generate growths following transplant to the injured spinal cord [4]. Thus, we suspect that limiting ex vivo manipulation and time in vitro, are paramount in maintaining Schwann cell integrity and overall safety. Future studies will need to identify the safety limits of in vitro expansion prior to genomic alteration in both rodent and human Schwann cells.
It was suggested by Izeta that 'any purity less than 100% may pose a substantial risk of mesenchymal precursor cells being injected as part of the SKP-SC cultures, and responsible for the cell masses reported in the study'. Here, we FACS isolated only p75NTR+ GFP+ cells and so these were theoretically 100% pure prior to transplant. That said, suggesting that safety can be ensured by cell selection with one or two markers is relying on a precision in the FACS technology that does not exist. Even the best of sorting/enrichment protocols cannot safeguard against a small number of contaminating cells that may remain p75NTR+ (or cd56+) but may have coincidentally acquired oncogenic mutations during cell expansion. Before transplantation of expanded cells can be safely and reliably implemented, intrinsic safeguards to identify pre- and post-transplant cells that may pose a risk of aberrant growth must be created. Schwann cells, because of their inherent plasticity and capacity to readily re-enter the cell cycle, may require greater scrutiny because of their potential for oncogenesis. Donor cells that are giving rise to a more terminal phenotype (e.g., skeletal muscle cells, neurons) may be less of a risk. Indeed, in neonatal skin-derived Schwann cells, we have successfully isolated cells that exhibit robust myelination and support improved functional outcome with no indication of aberrant growth (>60 recipient rats with cells derived from >10 independent litters) [5–7]. Yet, using the same p75NTR selection strategy here but in adult Schwann cells subject to extended in vitro expansion, we see excessive aberrant growth in vivo suggesting that marker expression is not a reliable indicator of cell integrity. Large-scale expansion of adult Schwann cells in artificial conditions may also pose additional concerns because unlike embryonic/early neonatal cells (or even adult stem cells) they may not harbor the same intrinsic mechanisms to protect against DNA damage that is a frequent consequence of extensive replication. This may endow aged cells with greater susceptibility to transformation.
In summary, we certainly agree with the commentary by Izeta that there is enormous therapeutic promise with skin progenitors for various clinical and commercial applications, including possibly neurologic repair. However, we caution that adult Schwann cells have the potential to exhibit unwanted growth particularly when grown for periods beyond 5 weeks in vitro, as indicated by our study. Because of their incredible plasticity, there are cautionary limitations that must be fully appreciated before their efficacy can be safely realized.