Adult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells

Highlights

• Acute nerve injury re-activates early glial lineage genes in Schwann cells (SCs).

• Regenerative function is diminished in SCs after chronic denervation.

• Adult SKP-SCs resemble acutely injured SCs gene expression.

• SKP-SCs exhibit greater axon growth support and myelination compared to denervated SCs.

• SKP-SCs may be an accessible source of myelinating glia for nerve repair.

Abstract

Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

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.