Quantitative Multimodal Evaluation of Passaging Human Neural Crest Stem Cells for Peripheral Nerve Regeneration

Abstract

Peripheral nerve injury is a major burden to societies worldwide, however, current therapy options (e.g. autologous nerve grafts) are unable to produce satisfactory outcomes. Many studies have shown that stem cell transplantation holds great potential for peripheral nerve repair, and human neural crest stem cells (NCSCs), which give rise to a variety of tissues in the peripheral nervous system, are particularly promising. NCSCs are one of the best candidates for clinical translation, however, to ensure the viability and quality of NCSCs for research and clinical use, the effect of in vitro cell passaging on therapeutic effects need be evaluated given that passaging is required to expand NCSCs to meet the demands of transplantation in preclinical research and clinical trials. To date, no study has investigated the quality of NCSCs past the 5th passage in vivo. In this study, we employed a multimodal evaluation system to investigate changes in outcomes between transplantation with 5th (p5) and 6th passage (p6) NCSCs in a 15mm rat sciatic nerve injury and repair model. Using CatWalk gait analysis, gastrocnemius muscle index, electrophysiology, immunohistochemistry, and histomorphometric analysis, we showed that p6 NCSCs demonstrated decreased cell survival, Schwann-cell differentiation, axonal growth, and functional outcomes compared to p5 NCSCs (all p<0.05). In conclusion, p6 NCSCs showed significantly reduced therapeutic efficacy compared to p5 NCSCs for peripheral nerve regeneration.

Heparin-Poloxamer Thermosensitive Hydrogel Loaded with bFGF and NGF Enhances Peripheral Nerve Regeneration in Diabetic Rats

“…Electrophysiology assessment is an effective method to evaluate functional recovery of the regenerated sciatic nerve. These methods were widely adopted in many research papers [78–80], including our previous studies [81–83]. However, it also carries several limitations, including the need for larger sample sizes (therefore more animals) to obtain sufficient statistical power due to the high variance of measurements.…”

“…Bilateral gastrocnemius muscles were harvested and wet weights were measured as described before [38,39]. The muscle wet weight ratio (%) was calculated as: the muscle wet weight of the injured limb/the weight of the contralateral one×100%.…”

Optimal electrical stimulation boosts stem cell therapy in nerve regeneration

“…The TCCF was then compared against the mean TCCF of neighboring interphase cells in the same field of view, with results presented as fold increase over interphase levels [32]. The number of HuNu positive cells and the proportion of HuNu/S-100 double-labeled cells to HuNu positive cells per slide were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD) [33]. 6 animals per group (30 mm thickness, 6e8 sections per animal) were included in this analysis.…”

“…Peak-to-peak amplitude and latency of the largest peak in CMAP and M wave in MEP were analyzed offline in MATLAB. Peaks were detected with a threshold, which was set to two or three times the standard deviation above the mean of the signal, based on the signal-to-noise ratio [33,35]. Peaks below this threshold were considered noise.…”

Biomimetic neural scaffolds: a crucial step towards optimal peripheral nerve regeneration

“…40,137,138 However, while stem cell transplantation seems to show great potential, current technologies are yet to identify optimized conditions for clinical use in terms of both efficacy and safety. To achieve clinical efficacy, graft survival must be adequate.…”