Research PaperAdult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells
Graphical abstract
Introduction
Peripheral nerve injury is common and debilitating. Such injuries may occur in the context of trauma, either blunt or penetrating, or in specific disorders of peripheral nerves known as neuropathies. Although peripheral nerves regenerate better than neurons within the central nervous system, recovery is frequently incomplete, misdirected and/or associated with debilitating neuropathic pain (Sunderland, 1968). For instance, after median nerve suture repair, only 25% of patients recover full motor function and only 3% gain full sensory function (Kelsey et al., 1997). Nerve injury gaps and scarring within the injured nerve both prevent regenerating axons from effectively innervating the distal nerve stump (Addas and Midha, 2009). These are currently managed by resecting the scarred segment and replacing with an interposed autologous nerve graft (Millesi et al., 1972), thereby creating a distal environment that is rich with growth-promoting graft-derived Schwann cells (Aguayo et al., 1976, Lehmann et al., 2011).
Following nerve injury, Schwann cells (SCs) re-express genes associated with immature SCs and this allows them to play several key roles including: 1) macrophage recruitment and immune modulation via cytokine secretion (Tofaris et al., 2002), 2) phagocytosis of myelin debris during nerve injury (Stoll et al., 2002, Khuong et al., 2014) 3) secretion of extracellular matrix and growth factors that promote axonal protection and regeneration (Fu and Gordon, 1997), and 4) remyelination and node of Ranvier formation in regenerating axons (Black et al., 2006) all of which contribute to successful nerve regeneration. Notably however, restoration of functional recovery following nerve injury is highly dependent on timing of repair and the dynamic biology of Schwann cells (Hall, 1999). Although SCs persist within the denervated distal segment even following 6 months of denervation, their capacity to support axonal regeneration and remyelination is severely diminished (Fu and Gordon, 1995a, Bradley et al., 1998, Hall, 1999). Thus, maximal, albeit incomplete, regeneration and outcome are typically achieved following immediate nerve repair (Fu and Gordon, 1995b, Gulati, 1996, Evans, 2000, Sulaiman and Gordon, 2000, Sulaiman and Gordon, 2009).
These experimental findings parallel the poor clinical outcome after PNI when repair is delayed (Sunderland, 1978, Sunderland and Williams, 1992, Kline and Hudson, 1995, Samii et al., 1997, Samii et al., 2003, Jivan et al., 2009, Birch, 2011). Because most injured nerves remain in physical continuity and since some patients recover spontaneously, delayed repair (several weeks to months) of PNI occurs frequently in clinical practice. Indeed, even patients who have immediate nerve repair are subject to distal nerve denervation for considerable periods of time as the rate of regeneration is relatively low (approximately 1 mm/day in humans Sunderland, 1947), and the distances that regenerating nerve fibers need to grow are typically long. Hence, every severe nerve injury creates a situation in which the SCs of the distal nerve are denervated and less able to support axonal growth (Fu and Gordon, 1997). These studies strongly suggest that an inherent deficiency in denervated Schwann cells underlies the observed poor remyelination and repair, although a direct functional assessment of isolated Schwann cells (to eliminate confounding effects of the denervated environment) has not been done.
An attractive approach to enhance regeneration following temporal delay to repair, is to supplement the distal nerve segment with SCs that more closely resemble either the acutely injured SC phenotype or even an embryonic Schwann cell precursors, which reportedly exhibit enhanced myelination and provide a highly supportive environment for growing axons (Woodhoo et al., 2007). The hair follicle dermis, which can be obtained from patients with minimal morbidity, contains a reservoir of multipotent precursors (referred to as skin-derived precursors or ‘SKPs’) that exhibit remarkable similarity to embryonic neural crest cells, which are the developmental origin of SCs (Fernandes et al., 2004, Toma et al., 2005, Fernandes et al., 2008). SCs generated from neonatal SKPs are highly capable of myelinating both peripheral (McKenzie et al., 2006) and central axons (Biernaskie et al., 2007) and promoting partial sensorimotor recovery after a contusion spinal cord injury (Biernaskie et al., 2007). Most recently, we showed that neonatal SKP-SCs transplantations enhanced locomotor function following tibial nerve transection, even after extended and clinically relevant temporal delays to surgical repair (Khuong et al., 2014). For clinical use to become a practical reality, it is necessary to assess whether SKP-SCs derived from adult skin (aSKP-SCs) are able to restore nerve remyelination and support enhanced regeneration. Here, we performed detailed phenotypic and functional characterization of aSKP-SCs, comparing them to SCs derived from acute or chronically injured peripheral nerves and the gold standard embryonic Schwann cells. We identify several key transcription factors that are lost following chronic denervation, and we provide compelling evidence to suggest that due to their functional similarity to acutely denervated Schwann cells, aSKP-SCs may provide a unique and accessible source of cells to supplement the chronically injured nerve.
Section snippets
Animals
In this study we used 120 adult (8–12 week old) male Lewis rats (Charles River, Montreal, QC). We used pregnant females (n = 4) to obtain embryonic (E16) Schwann cells from developing sciatic nerves. NOD SCID mice (n = 35; Charles River, Montreal, QC) were used as recipients for cell transplantation in vivo. All procedures received prior approval from the University of Calgary Animal Care committee and were in accordance with guidelines provided by the Canadian Council for Animal Care.
Nerve transection injury
All rats were
Results
Previous studies have shown that chronic denervation following peripheral nerve injury results in poor regeneration and diminished functional outcome. This has been suggested to be due to deleterious changes in both axons and Schwann cell function (Fu and Gordon, 1995b, Fu and Gordon, 1997, Evans, 2000). To test this directly, we isolated Schwann cells from acutely (D5d) or chronically (D56d) denervated nerves and compared their capacity to: 1) proliferate in the presence of mitogen, 2) support
Discussion
Here we demonstrate that SKP-SCs, derived from adult skin, are potent myelinating glial cells, comparable to SCs obtained from embryonic day 16 (E16) nerves, and acutely injured nerve (5 days post-transection). By contrast, chronically denervated nerve (56 days post-transection) yielded SCs that were largely incapable of myelination and showed a diminished capacity to support axon growth in vitro. Previous studies have provided strong evidence in support of SKP-SC myelination of injured
Acknowledgments
The authors would like to acknowledge Waleed Rahmani for training in confocal microscopy, Dr. Michael Wegner for a generous gift of Sox-10 antibody and Dr. Akitsu Hotta for providing plasmids to construct GFP lentivirus. This research was supported through funding from the Canadian Institute for Health Research (CIHRMOP#106646 to JB), Alberta Innovates Health Solutions (AIHS CRIO to RM and JB) and endMS Research and Training Network grants to JB and RM and an AIHS studentship to RK.
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