SUPPLEMENTATION OF ACELLULAR NERVE GRAFTS WITH SKIN DERIVED PRECURSOR CELLS PROMOTES PERIPHERAL NERVE REGENERATION S. WALSH, a * J. BIERNASKIE, b S. W. P. KEMP a AND R. MIDHA a a Department of Clinical Neuroscience and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Heritage Medical Research Building 109-3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1 b Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada M5G 1L7 Abstract—Introduction of autologous stem cells into the site of a nerve injury presents a promising therapy to promote axonal regeneration and remyelination following peripheral nerve damage. Given their documented ability to differentiate into Schwann cells (SCs) in vitro, we hypothesized that skin- derived precursor cells (SKPs) could represent a clinically- relevant source of transplantable cells that would enhance nerve regeneration following peripheral nerve injury. In this study, we examined the potential for SKP-derived Schwann cells (SKP–SCs) or nerve-derived SCs to improve nerve re- generation across a 12 mm gap created in the sciatic nerve of Lewis rats bridged by a freeze-thawed nerve graft. Immuno- histology after 4 weeks showed survival of both cell types and early regeneration in SKP seeded grafts was comparable to those seeded with SCs. Histomorphometrical and electro- physiological measurements of cell-treated nerve segments after 8 weeks survival all showed significant improvement as compared to diluent controls. A possible mechanistic expla- nation for the observed results of improved regenerative outcomes lies in SKP–SCs’ ability to secrete bioactive neu- rotrophins. We therefore conclude that SKPs represent an easily accessible, autologous source of stem cells for trans- plantation therapies which act as functional Schwann cells and show great promise in improving regeneration following nerve injury. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words : Peripheral nerve regeneration, acellular graft, transplantation, Schwann cells, stem cells. Satisfactory treatment of peripheral nerve injuries presents a challenge and outcomes are often less than ideal (Kelsey et al., 1997). Primary, tension free repair is performed when possible; however defects of peripheral nerves that arise as gaps in continuity, are repaired by autologous grafting with “expendable” nerve tissue which requires sac- rificing healthy nerve and causes further impairment (Lun- dborg, 2004). As such, several experimental studies have investigated the use of allografts, synthetic or biological guidance channels, and acellular graft materials (Bel- lamkonda, 2006; Fansa et al., 1999; Lundborg, 2004; Stang et al., 2005). Yet, since none exactly mimic the structural and functional characteristics of native periph- eral nerve, none of these grafts have appeared to be as effective as autologous nerve grafting. Moreover, many of these alternative graft materials lack viable Schwann cells (SCs), which are responsible for providing both trophic and structural support for regenerating axons (Bunge, 1994) and therefore tend to fail with increasing gap length (Lun- dborg et al., 1982). As a strategy to improve regeneration through alterna- tive conduits, many groups have supplemented various acellular graft materials with cultured SCs (Arino et al., 2008; Fansa and Keilhoff, 2004; Fox et al., 2005; Frerichs et al., 2002; Nishiura et al., 2004). SCs have proven suc- cessful to a certain extent, however as many authors have noted, human therapeutic Schwann cell culture is inher- ently flawed. First, Schwann cell culture is a lengthy pro- cess, requiring several weeks to obtain sufficient numbers (Nishiura et al., 2004). Second, to avoid the need for immunosuppression, autologous sources must be used, thus requiring the sacrifice of healthy nerve (Guest et al., 1997). As a result, several groups have turned their atten- tion to identifying more accessible sources of autologous SCs for therapeutic transplant. Emphasis has been placed specifically on exploring stem or progenitor cells that are easily accessible, rapidly expandable in culture, capable of survival and integration within the host tissue, and amena- ble to stable transfection and expression of exogenous genes (Azizi et al., 1998). This has lead to isolation of cells from bone marrow, adipose tissue, amniotic fluid, and hair follicle among others (for a review of studies to date, please see (Kemp et al., 2008; Walsh and Midha, 2009). The skin dermis contains neural crest-related precur- sor cells (termed skin-derived precursors, or skin-derived precursor cells (SKPs)) that differentiate into neural crest cell types in vitro when supplied with the appropriate cues, including those with characteristics of peripheral neurons and SCs (Fernandes et al., 2004; McKenzie et al., 2006; Toma et al., 2001, 2005). SKPs have been generated in neonatal and adult skin of both rodents (Fernandes et al., 2004; Toma et al., 2001) and humans (McKenzie et al., 2006; Toma et al., 2005), responding to environmental cues in a similar fashion. When cultured with neuregulin- 1 , an agent known to promote proliferation and differen- tiation of SCs from embryonic neural crest precursors, rodent and human SKPs generate bipolar, S100-positive cells that coexpress myelin basic protein, glial fibrillary *Corresponding author. Tel: +1-403-210-9367; fax: +1-403-270-7878. E-mail address: skwagg@ucalgary.ca (S. Walsh). Abbreviations: ELISA, enzyme-linked immunosorbent assay; GFAP, glial fibrillary acidic protein; NGF, nerve growth factor; PBS, phos- phate-buffered saline; SCs, Schwann cells; SKPs, skin-derived pre- cursor cells. Neuroscience 164 (2009) 1097–1107 0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.08.072 1097