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Neuroscience 164 (2009) 1097–1107

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UPPLEMENTATION OF ACELLULAR NERVE GRAFTS WITH SKINERIVED PRECURSOR CELLS PROMOTES PERIPHERAL NERVE

EGENERATION

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. WALSH,a* J. BIERNASKIE,b S. W. P. KEMPa AND

. MIDHAa

Department of Clinical Neuroscience and Hotchkiss Brain Institute,aculty of Medicine, University of Calgary, Heritage Medical Researchuilding 109-3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1

Developmental and Stem Cell Biology, Hospital for Sick Children,oronto, ON, Canada M5G 1L7

bstract—Introduction of autologous stem cells into the sitef a nerve injury presents a promising therapy to promotexonal regeneration and remyelination following peripheralerve damage. Given their documented ability to differentiate

nto Schwann cells (SCs) in vitro, we hypothesized that skin-erived precursor cells (SKPs) could represent a clinically-elevant source of transplantable cells that would enhanceerve regeneration following peripheral nerve injury. In thistudy, we examined the potential for SKP-derived Schwannells (SKP–SCs) or nerve-derived SCs to improve nerve re-eneration across a 12 mm gap created in the sciatic nerve ofewis rats bridged by a freeze-thawed nerve graft. Immuno-istology after 4 weeks showed survival of both cell typesnd early regeneration in SKP seeded grafts was comparableo those seeded with SCs. Histomorphometrical and electro-hysiological measurements of cell-treated nerve segmentsfter 8 weeks survival all showed significant improvement asompared to diluent controls. A possible mechanistic expla-ation for the observed results of improved regenerativeutcomes lies in SKP–SCs’ ability to secrete bioactive neu-otrophins. We therefore conclude that SKPs represent anasily accessible, autologous source of stem cells for trans-lantation therapies which act as functional Schwann cellsnd show great promise in improving regeneration followingerve injury. © 2009 IBRO. Published by Elsevier Ltd. Allights reserved.

ey words : Peripheral nerve regeneration, acellular graft,ransplantation, Schwann cells, stem cells.

atisfactory treatment of peripheral nerve injuries presentschallenge and outcomes are often less than ideal (Kelseyt al., 1997). Primary, tension free repair is performedhen possible; however defects of peripheral nerves thatrise as gaps in continuity, are repaired by autologousrafting with “expendable” nerve tissue which requires sac-ificing healthy nerve and causes further impairment (Lun-borg, 2004). As such, several experimental studies have

nvestigated the use of allografts, synthetic or biological

Corresponding author. Tel: �1-403-210-9367; fax: �1-403-270-7878.-mail address: skwagg@ucalgary.ca (S. Walsh).bbreviations: ELISA, enzyme-linked immunosorbent assay; GFAP,lial fibrillary acidic protein; NGF, nerve growth factor; PBS, phos-

chate-buffered saline; SCs, Schwann cells; SKPs, skin-derived pre-ursor cells.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.08.072

1097

uidance channels, and acellular graft materials (Bel-amkonda, 2006; Fansa et al., 1999; Lundborg, 2004;tang et al., 2005). Yet, since none exactly mimic thetructural and functional characteristics of native periph-ral nerve, none of these grafts have appeared to be asffective as autologous nerve grafting. Moreover, many ofhese alternative graft materials lack viable Schwann cellsSCs), which are responsible for providing both trophic andtructural support for regenerating axons (Bunge, 1994)nd therefore tend to fail with increasing gap length (Lun-borg et al., 1982).

As a strategy to improve regeneration through alterna-ive conduits, many groups have supplemented variouscellular graft materials with cultured SCs (Arino et al.,008; Fansa and Keilhoff, 2004; Fox et al., 2005; Frerichst al., 2002; Nishiura et al., 2004). SCs have proven suc-essful to a certain extent, however as many authors haveoted, human therapeutic Schwann cell culture is inher-ntly flawed. First, Schwann cell culture is a lengthy pro-ess, requiring several weeks to obtain sufficient numbersNishiura et al., 2004). Second, to avoid the need formmunosuppression, autologous sources must be used,hus requiring the sacrifice of healthy nerve (Guest et al.,997). As a result, several groups have turned their atten-ion to identifying more accessible sources of autologousCs for therapeutic transplant. Emphasis has been placedpecifically on exploring stem or progenitor cells that areasily accessible, rapidly expandable in culture, capable ofurvival and integration within the host tissue, and amena-le to stable transfection and expression of exogenousenes (Azizi et al., 1998). This has lead to isolation of cellsrom bone marrow, adipose tissue, amniotic fluid, and hairollicle among others (for a review of studies to date,lease see (Kemp et al., 2008; Walsh and Midha, 2009).

The skin dermis contains neural crest-related precur-or cells (termed skin-derived precursors, or skin-derivedrecursor cells (SKPs)) that differentiate into neural crestell types in vitro when supplied with the appropriate cues,

ncluding those with characteristics of peripheral neuronsnd SCs (Fernandes et al., 2004; McKenzie et al., 2006;oma et al., 2001, 2005). SKPs have been generated ineonatal and adult skin of both rodents (Fernandes et al.,004; Toma et al., 2001) and humans (McKenzie et al.,006; Toma et al., 2005), responding to environmentalues in a similar fashion. When cultured with neuregulin-�, an agent known to promote proliferation and differen-iation of SCs from embryonic neural crest precursors,odent and human SKPs generate bipolar, S100�-positive

ells that coexpress myelin basic protein, glial fibrillary

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cidic protein (GFAP) and p75 NTR (i.e., a SC-like phe-otype) (Biernaskie et al., 2006; Toma et al., 2001, 2005).KP-derived cells that are Schwann cell-like in their ap-arent differentiation (SKP–SC), survive and associateith axons within both normal mouse sciatic nerve andistal to crush, where they express a myelinating pheno-

ype (McKenzie et al., 2006). Yet, it is still unknown to whategree SKP–SCs can act like bona fide SCs in promotingxonal regeneration following transection injuries. Given

heir ability to generate potentially autologous SCs, SKPsre of great relevance to acellullar graft supplementationtrategies that may be able to overcome the previouslyescribed difficulties presented by immunosuppressionnd increasing gap length.

In the present study, we used a previously describedechnique of repetitive freezing and thawing nerve tissuerom donor animals (Ide, 1983) to generate acellularsografts which we then used to bridge a 12 mm gapreated in the sciatic nerve of Lewis rats. We then injectedKP-SCs, nerve-derived SCs or culture media into theraft to determine whether regenerative success throughhe initially acellular graft could be comparable to autograft,he current gold standard of repair. We performed histo-ogical assessment at 4 weeks and demonstrated an earlyaucity of regenerating axons in media group compared toll others. After 8 weeks, morphometrical and electrophys-

ological parameters established that SKP–SCs offer sim-lar regenerative success as autograft and age-matchederve-derived SCs in this model. Moreover, the SKP–SCsurvived over the study period and maintained their ex-ression of SC markers. Overall, the results suggest thatKP–SCs present a promising cell type for enriching acel-

ular grafts used for nerve repair.

EXPERIMENTAL PROCEDURES

ell culture

KPs were generated from dermis of postnatal day 2 Lewis ratsnd cultured according to published protocols (Toma et al., 2001).riefly, pups were quickly decapitated and skin on the dorsal torsoas sterilized with a 70% EtOH swab prior to removal with sterilecissors. Collected tissue was minced in Hank’s balanced saltolution (HBSS; GIBCO, Burlington, ON, Canada) on ice and thenncubated for approximately 45 min in 0.1% collagenase at 37 °C.kin pieces were mechanically dissociated, washed in coldMEM (GIBCO, Burlington, ON, Canada) and passed through a0 �m cell strainer. Filtrate was centrifuged at 1200 rpm and theellet was triturated and resuspended to a concentration of 50,000ells/ml in culture media (DMEM-F12(GIBCO, Burlington, ON,anada) 3:1, 1% penicillin/streptomycin (Sigma, Oakville, ON,anada)) containing B-27, 20 ng/ml EGF and 40 ng/ml bFGF (all

rom GIBCO). Cells were cultured and passaged three times asndifferentiated spheres in 25 cm2 tissue culture flasks (Corning,owell, MA, USA) in a 37 °C, 5% CO2 tissue-culture incubator. To

nduce differentiation towards Schwann cells, spheres were tritu-ated and replated on poly-D-lysine/laminin coated culture dishesCorning) in DMEM/F12 media with 4 �M forksolin, 10 ng/mleregulin 1�, and 1% N2 supplement (Gibco). After incubation forweek, cells appearing under phase contrast to have bipolar SCorphology (confirmed with GFAP staining of sister cultures) were

solated with cloning cylinders and expanded in the same medium

ntil �95% purity was achieved. c

Schwann cells were obtained either from sciatic nerve of P2eonates or adults following predegeneration period of 5 daysollowing proximal conditioning lesion according to modificationsf established protocols (Komiyama et al., 2003; Morrissey et al.,991). Briefly, sciatic nerves were excised, stripped of theirpineurium, and cut into 1 mm2 pieces. Segments were placed onoly-D lysine coated 35 mm culture dishes in DMEM/F12 mediaith 10% FBS and 1% penicillin/streptomycin for 3 days, allowingostly fibroblasts to migrate out from the nerve. Media was then

hanged to serum-free DMEM containing 1% N2, 10 ng/ml HRG-B, 4 �M forskolin for another 3 days to encourage Schwann cellutgrowth. At day 6, fragments were removed from the dish andxplanted onto another 35-mm dish in DMEM/F12 media with.5% FBS, a serum concentration that is ideal for suppression ofbroblast proliferation and maximum outgrowth and proliferationf Schwann cells (Komiyama et al., 2003). The explant procedureas repeated until little outgrowth of fibroblasts was observed,fter which point explants were discarded and all dishes contain-

ng SCs were purified in serum-free DMEM/F12 containing mito-ens as above.

All cells were labeled 20 min prior to injection with CellTrackerM-DiI (Invitrogen, Burlington, ON, Canada) according to theanufacturer’s guidelines. Briefly, cells were trypsinized from cul-

ure vessels and resuspended to 1�106 cells/ml in media contain-ng 1 �M DiI staining solution. Following incubation for 20 min inhe dark, cells were washed three times in DMEM and stored once until injection.

ssessment of neurotrophin production andioactivity

o obtain conditioned media, cell numbers (SKP spheres, SKP–Cs, and age-matched nerve SCs cultured as described above)ere standardized in each T75 flask, grown to approximately 70%onfluence after which media were exchanged for DMEM mediaor 48 h. This conditioned media were collected, filtered through a0 �m syringe filter apparatus (Millipore, Billerica, MA, USA) and

rozen immediately at �80 °C until use. Media were collected fromhree separate cultures for each condition for both following ex-eriments.

Enzyme-linked immunosorbent assays (ELISA) for nerverowth factor (NGF), neurotrophin-3 (NT-3) and brain-derived neu-otrophic factor (BDNF) were performed using Emax® ImmunoAssayystem kits from Promega (Madison, WI, USA). Total protein wasuantified from conditioned media samples using a standard BioRadicroplate assay, and samples were diluted to equal concentra-

ions using sample buffer. ELISA reactions were carried out on6-well plates according to manufacturer’s instructions with eachample analyzed in triplicate. Plates were read with a microplateeader at 450 nm and concentrations were calculated from absor-ance values extrapolated from a standard curve generated byerial dilutions of neurotrophin standards provided, following sub-raction of absorbance from negative control (DMEM). To accountor any variances in ultimate cell number between flasks, alloncentration values were expressed as a fraction of total proteinn the sample.

Rat pheochromocytoma (PC12) cells were purchased fromhe American Type Culture Collection (Cedarlane Laboratories,urlington, ON, Canada) and expanded in F12 media with 15%orse serum and 2.5% fetal bovine serum (all from Gibco–BRL).ells were then plated at low density into each well of four 4-wellhamber slides coated with poly-D-lysine and allowed to attachvernight. Media in each slide were then switched to one of thexperimental conditions: DMEM with 1% FBS (�50 ng/ml NGF) orMEM previously conditioned, as above, by either SKP–SCs orerve-derived SCs (with 1% FBS added). Fresh media were pro-ided every 2 days for a total incubation period after 10 days, afterhich chamber slides were imaged live at 400� under phase

ontrast microscopy. Ten high-powered fields per well were quan-

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ified by an observer blinded to the treatment and the total numberf neurite bearing cells/field was quantified (neurites were identi-ed as being projections greater than 1� the cell body diameter.

nimals

total of 37 male Lewis rats weighing 225–250 g (Charles River,C, Canada) were used in this study (as detailed below). Animalsere maintained in a temperature and humidity controlled envi-

onment with a 12-h light/dark cycle. Food (Purina, Mississauga,N, Canada) and water were available ad libitum. Surgical inter-

entions were carried out under inhalation anaesthetic (Isoflu-oane, 99.9% Halocarbon Laboratories, River Edge, NJ, USA) andain control was provided by means of i.p. or oral administration ofuprinorphene. Surgical procedures were carried out aseptically,nd standard microsurgical techniques were used with an oper-ting microscope (Wild M651; Wild Leitz, Willowdale, ON, Can-da). Animals were sacrificed at endpoint under deep anaesthesiasing an overdose of intracardiac Euthanol (Bimeda–MTC, Cam-ridge, ON, Canada). All efforts were made to minimize sufferingnd animal numbers by using appropriate protocols. The protocolas approved and monitored by the University of Calgary animalare committee and adhered strictly to guidelines set by theanadian Council on Animal Care.

reparation of acellular grafts

erve graft material was obtained from 14 deeply anaesthetizeddult rats by exposing the sciatic nerve bilaterally and excising a

ig. 1. Experimental paradigm: Skin-derived precursor cells were pr

ubsequently transplanted into isografts rendered acellular by repetitive freeze-nterpretation of the references to color in this figure legend, the reader is refe

0 mm segment from each side, following which animals wereuthanized. Harvested nerves were rinsed in sterile saline andivided into two 15 mm segments, yielding a total of four graftsbtained from each animal. Segments were placed in sterile cryo-ubes and subjected to five cycles of freezing in liquid nitrogen (2in) and thawing in 37 °C water bath (2 min) (Hall, 1986a; Ide,983). They were subsequently frozen at �80 °C until use.

xperimental groups and surgical methods

n the remaining 18 animals (nine sides per group, making a totalf 36 sides), the sciatic nerve was exposed bilaterally andransected in two locations proximal to the trifurcation to create a2-mm gap. Control animals received an immediate repair withhe transected segment (autograft), secured with 9–0 prolineicrosutures. Each of the three experimental groups was repaireds above with the freeze-thawed nerve graft (previously thawed at7 °C and trimmed to 13 mm). At this time, 3 �l of SKP–SCs,erve-derived SCs or carrier medium (DMEM) were injected intooth the distal and proximal 3 mm of the acellular grafts, giving aotal of 5�105 cells delivered in 6 �l of media. Indices of regen-ration were assessed at 4 weeks (immunohistochemistry) and 8eeks (electrophysiology and histomorphometry) (see Fig. 1).

mmunohistochemistry

t four (n�3/group) or 8 weeks (n�6/group, after electrophysiologi-al recording) following graft and cell implantation, the proximal andistal 4 mm of graft plus 3 mm of attached host nerve were removed

ntiated towards a Schwann cell phenotype (SKP–SCs) in vitro, and

e-differe thawing in order to determine their ability to support regeneration. Forrred to the Web version of this article.

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nd fixed overnight in 2% parafomaldehyde in phosphate-bufferedaline (PBS). Samples were washed three times in PBS, cryopro-ected in 20% sucrose and embedded in optimal cutting temperatureOCT) compound (Sakura Fine technical Co., Torrance, CA, USA).6 �m sections were cut with a cryostat (Leica Microsystems Inc.,ichmond Hill, ON, Canada) at �22 °C and mounted on Superfrostlides (Fisher Scientific). Sections were blocked with 1% BSA and

ncubated overnight in primary antibody. Antibodies used were anti-eurofilament-200 (1:800) anti-laminin (1:400) and anti S100-� (1:00) (all from Sigma). Following a wash with PBS, slides were

ncubated with secondary antibody (Alexa Fluor antimouse IgG 488r antirabbit IgG 488, 1:400; anti-rabbit IgG 350, 1:400, both Molec-lar Probes, Inc.) for 2 h. Slides were then washed and coverslippedsing Fluorosave reagent (Calbiochem, San Diego, CA, USA) andiewed under a fluorescence microscope (Olympus BX51, Centeralley, PA, USA) or with a Nikon C1si spectral confocal laser-scan-ing microscope. Omission of primary or secondary antibody wassed as negative control for the staining process. Four-week axonounts (by an observer blinded to the treatment group) were takenrom three points throughout the thickness of each distal graft seg-ent. In each of these sections, a line was drawn perpendicular to

he centre of the section and NF-200 positive profiles crossing thisine were counted and summed, similar to previously described

ethods (Kemp et al., 2009). Eight-week sections were used tossess phenotype of SKP–SCs by counting S100�/DiI positive cells

n a defined area throughout the thickness of the graft.

lectrophysiology

t the 8 week study termination timepoint, animals were anaes-hetized with isofluorane and nerve conduction recordings werebtained using a Sierra 6200 A electrodiagnostic system (Cadwellaboratories, Kennewick, WA, USA). Bipolar stimulating elec-

rodes were placed on the nerve 5 mm from the proximal sutureite of the graft while recordings were obtained 3 mm distal to theraft from nerve and from the belly of the gastrocnemius muscle.ecordings of compound muscle action potential amplitude and

atency as well as nerve conduction velocity were recorded fol-owing a supramaximal stimulus delivered by the proximal elec-rodes. Procedures were performed on a heating blanket to main-ain temperature of 37 °C.

istomorphometry

ollowing electrophysiological recording, 3-mm segments of mid-raft and nerve 5 mm distal to the graft segment (sampling asepicted in Fig. 1) was harvested and fixed overnight in 2.5% glutar-ldehyde buffered in cacodylate (0.025 M). Washed samples wereostfixed in osmium tetroxide (2%) for 2 h, and then embedded inpon. Semithin transverse sections (approximately 1 �m) were cutnd stained with toluidine blue. Seven fields of view per section werehotographed at high power light microscopy (400�; OlympusX51) for analysis. Images were digitized with a Wacom Intuos3igitizing tablet (Vancouver, WA, USA) and analyzed using Imagero Plus software (Media Cybernetics, Bethesda, MD, USA). Param-ters measured included number of myelinated axons (estimatedsing the formula: total estimated count�# of myelinated axonsounted in sampling area�cross sectional area/sampling area), fiberiameters, g-ratios (axon diameter/fiber diameter), and cross-sec-ional area of nerve tissue containing myelinated fibers. Data fromistal nerve were reported in this manuscript.

tatistical analysis

ifferences between groups were compared using a one-wayNOVA, with post hoc Tukey tests applied as appropriate ortudent’s t-tests. Statistical significance was accepted at P�0.05,

ith all results presented as the mean�SEM. u

RESULTS

n all animals, the surgical procedures were well-toleratednd wounds healed without complication or signs of pain oriscomfort. Graft repair remained intact in every case andhere was no evidence of excessive fibrosis in any of theroups.

kin derived precursor cells can be harvested fromeonatal dermis and differentiate into functional SCs

n vitro and in vivo

SKP phenotype in vitro. Within 1 week of culture inGF/bFGF -containing media, spheres formed in flasks con-

aining dissociated dermal cells. As described elsewhereToma et al., 2005) when these spheres were dissociated intoingle cell suspension, they generated secondary or tertiarypheres, thus identifying them as SKPs. After 1 week ofulture in DMEM/F12 media supplemented by forskolin anderegulin 1�, dissociated SKP spheres differentiated intomall colonies of bipolar cells with the morphological charac-eristics of SCs (Fig. 2A). By utilizing cloning cylinders, weere able to obtain cultures of SKP–SCs that were approxi-ately 95% pure for Schwann cell markers p75, GFAP and-100� (as assessed in sister cultures) within approximatelyto 3 weeks of culture initiation (data not shown).

SKP phenotype in vivo. Histological sections at both 4nd 8 weeks confirmed survival of diI-labeled SKP–SCs thatigrated several millimeters from injection sites to form aear continuous cord throughout the graft (Fig. 2B). Immu-ohistochemical analysis of frozen sections of graft tissueFig. 2C) demonstrated that many surviving SKP–SCs main-ained an elongated, bipolar morphology and aligned closelyith NF-200 stained regenerating axons. Approximately 65%f all diI labeled SKPs expressed SC marker S100� after 8eeks, demonstrating long-term maintenance of SC pheno-

ype. Moreover, the total number of SCs in SKP–SC treatedrafts (both SKP and host-derived) equaled the number ofCs in the control autografts (host-derived only). SKP–SCsppeared to align within the basal lamina tubes that remainedfter freeze-thawing and some were visible within these de-ellularized tubes in laminin-stained cross sections (Fig. 2D).

KP–SCs secrete bioactive neurotrophins

potential application of stem cells in nerve injuries is toncourage direct axonal outgrowth via release of neuro-rophic factors. ELISA analysis of cell culture supernatantemonstrated detectable levels of neurotrophin secretiony all three cell types assessed (Fig. 3A). Interestingly,KP–SCs produced consistently higher levels of neurotro-hins with respect to total protein concentration than eitherndifferentiated SKP spheres or nerve-derived SCs: As aunctional bioassay of secreted neurotrophins, PC12 cellsere cultured in the presence of media conditioned byither SKP–SCs or nerve-derived SCs (Fig. 3A). Both SCnd SKP–SC conditioned media elicited robust neuriteutgrowth to the level matching that of NGF positive con-rol, while were significantly fewer neurite bearing cells in

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ffect of SKP–SC treatment on early axonalegeneration through acellular isografts

amples of the graft and attached nerve stumps werebtained at the 4-week timepoint to assess early regener-tion in the form of axon counts. NF-200 immunostainingt 4 weeks following transplantation revealed successfulegeneration through the distal graft in all cases. Axonounts in the distal portion of graft and nerve were notignificantly different between autograft and either of theell-treated groups, however media-treated grafts demon-trated significantly diminished regeneration at this time-oint as compared to the other three groups (Fig. 4).

KP–SCs improve regeneration through acellularsografts versus media alone

Morphometrical analysis of graft and distal nerve.ight weeks following graft repair, we evaluated histomor-hometric parameters of regeneration on toluidine blue

ig. 2. Phenotype of SKP–SCs in vitro and in vivo. (A) SKP–SCsrominent nucleus, and bipolar morphology and align parallel to oagnification; scale bar�50 �m. (B) eight weeks following injection in

he graft, forming a near-continuous cord of cells. Confocal image, 100losely with NF-200 stained axons (green). Immunostaining of consegreen; co-labeling of S100� in diI labeled SKP–SCs appears yellow). Creezing and thawing of donor nerve rendered it acellular (D), with “cellular graft, with some aligned within the basal lamina tubes anluorescent microscopy of laminin-immunostained cross section of geferences to color in this figure legend, the reader is referred to the

tained semithin sections from the centre of the graft and g

rom the nerve distal to the graft. Fig. 5 demonstratesepresentative images of sections that were used for anal-sis. Qualitatively, the sections from SKP–SC treatedrafts exhibited numerous large, well myelinated axons,nd were indistinguishable from the autograft group. TheC group also demonstrated many mature regeneratedbers, however there was some variability in the distribu-ion of these fibers, with some regions throughout theerve cross section having very few and others havingany. The media-treated groups had noticeably fewer,

maller axon profiles in both graft and distal nerve seg-ents. In the distal nerve, the total number of axons was

ignificantly greater in the autograft and SKP–SC groupshan the media group (Fig. 6A). In the parameters of crossectional area enclosing myelinated fibers (Fig. 6B) and-ratio (Fig. 6C), the autograft and both cell-treated groupshowed significant improvement over media group. A plotf frequency of fiber diameters in cross sections of each

nerve derived SCs (A=) demonstrate similar oval-shaped cell body,er in confluent culture. Phase contrast microscopy, 200� originalft, diI-labeled SKP–SCs (red) persist and migrate along the length of

fication; scale bar�100 �m. (C) DiI labeled SKP–SCs (red) associatections (C=) demonstrated that these SKP–SCs also express S100�

images, 400� original magnification; scale bar�20 �m. (D) Repetitiveasal lamina scaffolds. Injected SKP–SCs are visible throughout the

exterior (D=) (Blue: laminin staining, Red: DiI-labeled SKP–SCs).original magnification; scale bar�20 �m. For interpretation of the

ion of this article.

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S. Walsh et al. / Neuroscience 164 (2009) 1097–11071102

n the SKP–SC and autograft control groups, while SC andedia groups were more frequently associated with smaller-iameter fibers.

ig. 3. SKP–SCs secrete bioactive neurotrophins into culture media.f NGF, BDNF, and NT-3, however SKP–SCs secrete significantly moonditioned media (either by SKP–SC or SCs) or NGF-containing medontrol media do not. Phase contrast microscopy, 400� original magnells grown in conditioned media demonstrates that the number of nef the other three groups. Data represent mean�SEM, * P�0.05, ** P

ig. 4. Quantitative analysis of early nerve regeneration. Four weeksollowing graft implantation and experimental treatment, the number ofF-200 positive axonal profiles was quantified from the distal portionf the nerve graft segment (see scheme in Fig. 1 for sampling). Whilehere was no difference in mean axon counts between the autograft

rnd cell treated groups, all were significantly greater than the mediaontrol group. Data represent mean�SEM, * P�0.05; Tukey test.

Electrophysiology of graft repaired nerves. After 8eeks, the SKP–SC grafted nerves demonstrated superior

ecovery of electrophysiological parameters of regenera-ion compared to media controls. Most significantly, theeak amplitude of compound muscle action potential in theKP–SC group approached that of the autograft grouphereas this was not observed in either SC or mediaroups (Fig. 7). The difference in amplitude betweenKP–SC and media groups achieved statistical signifi-ance. While measurements of nerve conduction velocityaralleled these findings, there was no significant differ-nce observed between groups.

DISCUSSION

he major findings in this study were (1) Skin-derivedrecursor cells pre-differentiated towards a SC phenotype

n vitro survived, migrated, and maintained the expressionf SC markers for at least 8 weeks when transplanted inton acellular (freeze-thawed) isografts (2) After 4 weeksKP–SCs supported axonal elongation to the same extents autograft and SC treatment; and (3) provided superiorxon regeneration, myelination, and electrophysiological

SCs, SKP spheres and nerve-derived SCs secrete detectable levelsnd NT-3 than either SCs or SKP spheres. (B) PC12 cells cultured inays extend neurites, while those cultured in the low-serum, NGF-freescale bar�50 �m. (C) Quantification of neurite outgrowth from PC12ring cells per field is significantly less in the NGF-free media than all*** P�0.01; Tukey test.

(A) SKP–re NGF aia for 10 dification;

ecovery as compared to media control at the 8-week

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ollowup timepoint. Finally, (4) SKP–SCs secrete neurotro-hic factors, which are known to stimulate peripheral nerveegeneration. Moreover, this is the first study to directlyompare the ability of SKP–SCs versus age-matchederve SCs to enhance peripheral nerve regeneration.

Nerve grafts provide a pathway for regenerating axonsrom the proximal nerve stump to innervate the distaltump (Midha and Mackay, 1999). The best option forepairing extensive lesions in peripheral nerves thus faries in the use of autografts obtained from additional nerveissue. However, success in the case of grafting is oftenompromised by incomplete or nonspecific regenerationBellamkonda, 2006; Frerichs et al., 2002; Nichols et al.,004). Also, the process involves sacrifice of one or moreealthy nerves, and there may be limited availability ofonor tissue, especially in the cases of larger gaps (Terzist al., 1997). Several alternatives to autografting haveeen explored, falling into three broad categories: syn-hetic (e.g. silicon) (Longo et al., 1983; Lundborg et al.,982; Pfister et al., 2007), biological (acellular muscle,ein, arterial, collagen) (Archibald et al., 1991; Fansa et al.,003; Keilhoff et al., 2005; Suematsu et al., 1988), andioartificial (combining elements of both) (Ansselin et al.,997; Hadlock et al., 2000; Hsu et al., 2009; Yoshitani etl., 2007). While biological material tends to offer superioregeneration than synthetic material, many alternativerafts have failed with increasing gap length (Fansa andeilhoff, 2004; Navarro et al., 1996).

Studies examining the potential of allogenic nerverafting have concluded that without adequate immuno-uppression, host-graft immunorejection limits the useful-ess of this tactic (Mackinnon et al., 2000; Zalewski andulati, 1984). Acellular grafts reduce this risk, as removal

ig. 5. Representative light micrographs of semithin sections from nerveistal to graft repair 8 weeks following surgical manipulation and experi-ental treatment. (A) Autograft. (B) SKP–SC. (C) SC (D) Media. Theutograft group showed numerous large well-myelinated axons in eacheld (arrows) (A). SKP–SC (B) and SC (C) treated groups also demon-trated large numbers of mature axons, while the media-treated controlD) displayed generally fewer, small fibers throughout (arrowheads).000� (Oil Immersion); scale bar�10 �m. For interpretation of the ref-rences to color in this figure legend, the reader is referred to the Webersion of this article.

f live cells reduces immunogenicity of these materials a

Gulati and Cole, 1994; Hare et al., 1993) (Hems andlasby, 1993). However, like artificial conduits, they are

ess effective in supporting regeneration of nerves overonger gaps. This is considered to be a result of the ab-ence of viable cells (Wiberg and Terenghi, 2003), whichormally aid in degenerating the debris and remodeling theerve environment of the graft (Hall, 1986a,b). In addition,Cs provide an ideal trophic milieu for axonal regenerations they produce a number of trophic factors and cytokinesBunge, 1994). Therefore, an “ideal” tissue engineeredrtificial nerve graft could be described as one comprisingscaffold material, trophic factors, seeded cells, and ex-

racellular matrix (Wang et al., 2008).Supplementing various acellular grafts or tubes with

Cs has achieved varying results in other experiments,wing to differences in graft structure, gap size, and injuryodel (Fansa et al., 2001; Frerichs et al., 2002; Guenardt al., 1992). Studies using gap sizes similar to those of theurrent work have demonstrated little effect of adding SCso acellular graft material, owing to the fact that SCs mi-rating from the host stumps are able to cross such shortistances and provide sufficient regeneration despite ad-ition of exogenous cells (Fansa et al., 2001; Fox et al.,005; Frerichs et al., 2002). The ingrowing SC fronts haveeen shown to meet by 14 days in a 10-mm gap (Nishiurat al., 2004). However, when Schwann cells are preventedrom entering the graft, axonal regeneration is poor (Hall,986b). A similar failure in regeneration is observed whenCs must migrate over long distances, such as in the casef large gap models (Lundborg et al., 1982). This suggestshat cell supplementation of acellular grafts may be ofreatest clinical use for large gaps, rather than those withdequate host SC infiltration (Nadim et al., 1990).

The present data confirm previous results that nerve-erived SC supplementation of acellular grafts may notrovide equivalent recovery to autograft levels in all pa-ameters of regeneration. This study, however, presentshe novel finding that SKP–SCs do seem to accomplishhis task. This is a somewhat unexpected finding that weust further pursue. It may be that the SKP–SC representsless mature form of Schwann cell than those derived

rom postnatal nerve, and therefore possess unique char-cteristics that allow them to survive, migrate and supportegenerating axons (Woodhoo et al., 2007). It has beenuggested that SC supplementation of acellular graftsould be improved by genetically engineering SCs to over-roduce growth factors so that transplanted cells would bedvantageous over host cells (Fox et al., 2005). Sinceirect, continuous infusion of growth factors in patients witherve injuries presents somewhat of a technical challenge,lternative strategies for local delivery of neurotrophinsuch as cell infusion are highly warranted (Terenghi, 1999).n this study, we demonstrate that SKP–SCs produceome neurotrophins at levels higher than those of nerve-erived SCs, which may explain their apparent advantage.e are currently exploring whether SKP–SCs continue to

roduce neurotrophins in vivo in order to further elucidatehe mechanisms of these cells’ support of nerve regener-

tion. It is of interest to note that undifferentiated SKP

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S. Walsh et al. / Neuroscience 164 (2009) 1097–11071104

pheres consistently produced the lowest amounts of neu-otrophins, underscoring the importance of exploring andptimizing the ideal stem cell phenotype for therapeuticse (Walsh and Midha, 2009).

A desirable attribute of an ideal nerve guide is that itegrades and integrates into host tissue at a rate suffi-iently slow enough to allow newly regenerating axons to

ig. 6. Morphometric analyses of the regenerating myelinated axonalculated total axon counts in autograft and SKP–SC groups were s

egion containing myelinated axons was higher in cell treated and auutograft and both cell treated groups than media control, with a lowerves from SKP–SC and autograft groups have greater distribution oistogram data). Data represent mean�SEM, * P�0.05; ** P�0.01; Teader is referred to the Web version of this article.

ully traverse the gap (Hems and Glasby, 1993). Freeze l

haw grafts have a relatively rapid revascularization time of–7 days, allowing for infiltration of macrophages. While aertain number of phagocytic cells are important for graftlearance and regeneration, supernumerary macrophagesan result in deleterious activation of T cells and prematurehagocytosis of grafts (Keilhoff et al., 2005). In this case,

sogenic acellular grafted tissue remained stable for at

ion within the distal nerve 8 weeks following graft implantation. (A)ly greater than media control. (B) Cross-sectional area enclosing theroups than media groups. (C) G-ratio was also significantly lower inindicating thicker, more mature myelin. (D) Histogram showing that

ameter fibers relative to media controls (Inset: line graph plotted from. For interpretation of the references to color in this figure legend, the

populatignificanttograft g

er G-ratiof large diukey test

east 8 weeks, long enough for axons to regenerate into

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S. Walsh et al. / Neuroscience 164 (2009) 1097–1107 1105

he distal stump. The fate of transplanted stem cells in theraft is also of great interest in order to fully understand thetility of such therapies. We did not observe excessive

nfiltration of ED-1 positive macrophages in any of the su-gical groups, and furthermore injected SKP cells (DiI pos-tive) were not observed to be engulfed by phagocytic cells,ut were rather spatially isolated (data not shown). We arelso currently exploring the rate of proliferation of SKP–Cs in various nerve injury models.

Acellular grafts containing SKP–SCs possess the de-irable traits of an ideal nerve guide and should be con-idered as a potential therapy in cases where host SCsay fail: for example in the case of large gaps where theigratory ability of SCs is overwhelmed, or in chronicallyenervated nerve where SCs lose their growth-supportivehenotype (Fu and Gordon, 1995). Since the most logicalpproach of supplementing nerve injuries with autologousCs is complicated by their requirement for sacrifice ofealthy nerve tissue, other sources of SC-like cells muste sought. Embryonic neural stem cells or cell lines haveeen employed to repair nerve injuries with demonstrationf regenerative success (Heine et al., 2004; Murakami etl., 2003) but suffer the drawback of being impractical tobtain. On the other hand, adult stem cells have the ad-antage of being available from relatively non-invasive,utologous harvest methods, and are likely the most prom-

sing choice for the majority of clinical nerve injuries. Aumber of investigators have been able to show in vitro or

n vivo SC differentiation from a wide variety of adult stemell sources (Amoh et al., 2005; Dezawa, 2002; Kinghamt al., 2007), further supporting our assertion that precur-or cells derived from skin dermis generate functional SCs:KP–SCs have the added advantage of being easy tobtain from autologous sources and readily expandable initro, making them a strong candidate for cell transplanta-ion in peripheral nerve lesions.

cknowledgments—The authors thank Shahbaz Syed, Joanneorden and Dr. Qing Gui Xu for their technical expertise andssistance with the preparation of this manuscript. We thank Dr.reda Miller and her laboratory personnel for demonstrating the

ig. 7. Electrophysiological recovery 8 weeks following grafting and eCMAP) was significantly greater in the autograph and SKP–SC grouparalleled those of CMAP; however statistical significance was not re

echniques of SKP culture and generation. This research was

upported by a grant from the Canadian Institutes for Healthesearch (MOP 82726) and an NSERC CGSD studentshipranted to S.K.W. The authors have no conflict of interest.

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(Accepted 28 August 2009)(Available online 6 September 2009)