Experimental Neurology 248 (2013) 30–44

Contents lists available at SciVerse ScienceDirect

Experimental Neurology

j ourna l homepage: www.e lsev ie r .com/ locate /yexnr

Demonstrating efficacy in preclinical studies of cellular therapies forspinal cord injury — How much is enough?

Brian K. Kwon a,⁎, Lesley J.J. Soril b, Mark Bacon c, Michael S. Beattie d, Armin Blesch e,Jacqueline C. Bresnahan d, Mary Bartlett Bunge f, Sarah A. Dunlop g, Michael G. Fehlings h, Adam R. Ferguson i,Caitlin E. Hill j, Soheila Karimi-Abdolrezaee k, Paul Lu l, John W. McDonald m, Hans W. Müller n,Martin Oudega o, Ephron S. Rosenzweig l, Paul J. Reier p, Jerry Silver t, Eva Sykova q, Xiao-Ming Xu r,James D. Guest f, Wolfram Tetzlaff s

a ICORD, University of British Columbia, Room 6196, 818 West 10th Avenue, Vancouver, BC, V5Z 1M9, Canadab Rick Hansen Institute, 818 West 10th Avenue, Vancouver, BC, V5Z 1M9, Canadac Spinal Research, Bramley Business Centre, Station Road, Bramley, Guildford, GU5 0AZ, UKd Brain and Spinal Injury Center, Dept. of Neurological Surgery, University of California at San Francisco, 1001 Potrero Ave., Bldg 1, Room 101, San Francisco, CA 94110, USAe Spinal Cord Injury Center, University Hospital Heidelberg, Schlierbacher Landstr. 200A, 69118 Heidelberg, Germanyf The Miami Project to Cure Paralysis, 1095 NW 14th Terrace, Miami, FL 33136, USAg The University of Western Australia, Experimental & Regenerative Neurosciences, School of Animal Biology (M317), 35 Stirling Highway, Nedlands 6009, Australiah Division of Neurosurgery and Spinal Program, University of Toronto, Toronto Western Hospital, Suite 4WW-449, 399 Bathurst St., Toronto, Ontario M5T2S8, Canadai Brain and Spinal Injury Center (BASIC), Department of Neurological Surgery, University of California, San Francisco, San Francisco General Hospital, 1001 Potrero Ave, Bldg. 1, Rm 101,San Francisco, CA 94107, USAj Burke Medical Research Institute, Weill Medical College of Cornell University, 785 Mamoroneck Avenue, White Plains, NY 10605, USAk Regenerative Medicine and Spinal Cord Research Center, University of Manitoba, 629-BMSB, 745 Bannatyne Ave, Winnipeg, MB R3E 0J9, Canadal University of California, San Diego, 9500 Gilman Drive, # 0626, La Jolla, CA 92093-0626, USAm International Center for Spinal Cord Injury, Kennedy Krieger Institute, Johns Hopkins University School of Medicine, 716N. Broadway, Baltimore, MD 21205, USAn University of Düsseldorf, Moorenst. 5, 40225 Düsseldorf, Germanyo Physical Medicine & Rehabilitation, University of Pittsburgh, W1452 BST, 200 Lothrop Street, Pittsburgh, PA 15213, USAp University of Florida College of Medicine, McKnight Brain Institute, 1149 Newell Dr, Gainesville, FL 32611-0015, USAq Institute of Experimental Medicine AS CR, Videnska 1083, 142 20 Prague 4, Czech Republicr Indiana University School of Medicine, 950W. Walnut Street, Indianapolis, IN 46202, USAs ICORD, University of British Columbia, 818 West 10th Avenue, Vancouver, BC V5Z 1M9, Canadat Case Western Reserve University, Dept. of Neuroscience, School of Medicine, 10900 Euclid Avenue, Cleveland, OH, USA

⁎ Corresponding author at: Room 6196, Blusson SpiFax: +1 604 875 5858

E-mail addresses: Brian.Kwon@vch.ca (B.K. Kwon), larmin.blesch@med.uni-heidelberg.de (A. Blesch), JacqueMichael.fehlings@uhn.ca (M.G. Fehlings), Adam.fergusplu@ucsd.edu (P. Lu), mcdonaldj@kennedykrieger.orgephronr@gmail.com (E.S. Rosenzweig), reier@ufl.edu (tetzlaff@icord.org (W. Tetzlaff).

0014-4886/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.expneurol.2013.05.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 April 2013Accepted 21 May 2013Available online 29 May 2013

Keywords:Spinal cord injuryCell transplantation therapiesStem cellClinical trialPreclinical studiesAnimal model

Cellular therapies represent a novel treatment approach for spinal cord injury (SCI), withmany different cellularsubstrates showing promise in preclinical animal models of SCI. Considerable interest therefore exists to trans-late such cellular interventions into human clinical trials. Balanced against the urgency for clinical translationis the desire to establish the robustness of a cellular therapy's efficacy in preclinical studies, thereby optimizingits chances of succeeding in human trials. Uncertainty exists, however, on the extent towhich a therapy needs todemonstrate efficacy in the preclinical setting in order to justify the initiation of a lengthy, expensive, and poten-tially risky clinical trial. The purpose of this initiativewas to seek perspectives on the level of evidence required inexperimental studies of cellular therapies before proceeding with clinical trials of SCI. We conducted a survey of27 SCI researchers actively involved in either preclinical and/or clinical research of cellular interventions for SCI,and then held a focus group meeting to facilitate more in-depth discussion around a number of translationalissues. These included: the use of animalmodels, the use of injurymodels andmechanisms, thewindow for dem-onstrating efficacy, independent replication, defining “relevant, meaningful efficacy” in preclinical studies, andthe expectation of therapeutic benefits for cellular interventions. Here we present the key findings from boththe survey and focus group meeting in order to summarize and underscore the areas of consensus and

nal Cord Centre, Vancouver General Hospital, 818 West 10th Avenue, Vancouver, British Columbia, V5Z 1M9, Canada.

soril@rickhanseninstitute.org (L.J.J. Soril), mark@spinal-research.org (M. Bacon), Michael.beattie@ucsf.edu (M.S. Beattie),line.bresnahan@ucsf.edu (J.C. Bresnahan), MBunge@med.miami.edu (M.B. Bunge), sarah.dunlop@uwa.edu.au (S.A. Dunlop),on@ucsf.edu (A.R. Ferguson), Cah2024@med.cornell.edu (C.E. Hill), karimis@cc.umanitoba.ca (S. Karimi-Abdolrezaee),(J.W. McDonald), Hanswerner.mueller@uni-duesseldorf.de (H.W. Müller), moudega@pitt.edu (M. Oudega),P.J. Reier), sykova@biomed.cas.cz (E. Sykova), Xu26@iupui.edu (X.-M. Xu), jguest@med.miami.edu (J.D. Guest),

rights reserved.

31B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

disagreement amongst the sampled researchers. It is anticipated that the knowledge generated from this initia-tive will help to incite future scientific discussions and expert guidelines towards translation of a cell therapy forpersons with SCI.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Despite considerable progress over the past four decades in themedical, surgical, and rehabilitative care of individuals with spinalcord injury (SCI), treatment options to improve neurologic functionare limited. As a result, SCI remains catastrophic for those affected.Numerous promising therapeutic approaches for SCI have been devel-oped in the experimental setting. A growing number of these areemerging from the laboratory with the hope of being translated intohuman clinical trials (Tetzlaff et al., 2011; Kwon et al., 2011a, 2011b).With this has come much optimism that efficacious treatments forhuman SCI will be established in the not-so-distant future.

The experience of conducting clinical trials on promising experi-mental therapies for SCI has taught the field much about the challengesassociated with successfully translating such novel technologies intoclinically efficacious treatments for human patients. These experienceshave led to important initiatives to identify the obstacles and challengesto successful translation and potentially improve upon the chances ofsucceeding in future trials (Fawcett et al., 2007; Lammertse et al.,2007; Steeves et al., 2007; Tuszynski et al., 2007; Blight and Tuszynski,2006; Steeves et al., 2004; Anderson et al., 2005; Dietrich, 2003;Courtine et al., 2007;Hewson et al., 2013). Such initiatives span the con-tinuum from recommendations around preclinical laboratory studies tothe planning and conduct of clinical trials.

One area of specific interest within this continuum is the issue ofdetermining what preclinical evidence is needed to justify moving apromising experimental therapy towards a lengthy and expensivehuman clinical trial (Blight and Tuszynski, 2006; Dietrich, 2003).This issue would be less problematic and relevant if the clinical evalua-tion of a promising new therapy for SCI was relatively straightforward.Past experience with SCI clinical trials that have either been completedor were terminated midstream has revealed that this is unfortunatelynot the case (Tator, 2006; Steeves and Blight, 2012). Optimally, experi-mental therapies should demonstrate robust efficacy in preclinical stud-ies before considerable time, financial, and personnel resources arecommitted to their clinical testing. Such sentiments have been voicedin the stroke and traumatic brain injury research communities, whichhave both accumulated a much longer history of negative clinical trialsthan has the SCI field (O'Collins et al., 2006; Maas et al., 2012). Further-more, resources are limited in the SCI field, and the decision to initiate aclinical trial for one therapy may mean that a different yet similarlypromising therapy will not have the chance to be tested. Given this‘opportunity cost’, evidence supporting the robustness of a therapy'sefficacy is extremely important. At the same time, neither the SCI re-searchers nor the individuals with SCI wish to have the expectationsfor demonstrating robust preclinical efficacy set at unachievably highlevels, such that novel treatments never progress beyond endlessrounds of animal experiments. Striking a rational balance between de-manding strong preclinical evidence before progressing to human trialsand setting realistic expectations such that such progress is still possibleis complex and challenging, yet necessary (Reier et al., 2012).

Previously we solicited clinical and scientific researchers as well asindividuals living with SCI for their perspectives on the extent towhich a therapy's efficacy should be demonstrated in preclinical studiesin order to justify translation into human clinical trials. In a survey ofover 300members of the research community and over 200 individualswith SCI,we characterized and contrasted a spectrumof opinions on thepreclinical evaluation of novel technologies such as neuroprotectivedrugs or neuro-regenerative cellular therapies for SCI (Kwon et al.,

2010, 2012b). An acknowledged limitation of these previous initiatives,however, was that the opinions garnered were from survey respon-dents with diverse expertise. For example, we previously reported onthe opinions of researchers on the need for large animal studies in thepreclinical development of cellular therapies for SCI. However, a portionof such researcherswould likely have limited experience in this particularline of research, andmight, for instance, be instead focused on developingpharmaceuticals for acute SCI neuroprotection. In order to examine thesetranslational issues in more depth and detail, the perspectives ofresearchers with specific expertise in the field were felt to be desirable.

In the current initiative, we brought together a focus group of SCIresearchers involved in cellular therapies for SCI to specifically addressquestions around what preclinical evidence of efficacy is necessarybefore advancing such cellular therapies to human clinical trials.Given the growing interest from both scientific and public communitiesin cellular transplantation therapies (and stem cell technology in partic-ular), we felt that a specific focus on this treatment approach waswarranted. Our intention was not to provide recommendations orguidelines for the SCI community in the context of a regulatory frame-work. Rather, we sought to paint in a structured manner the currentlandscape of perspectives from researchers who specifically study celltherapies for SCI, so that we may identify points of general consensusas well as areas of disagreement. We contend that this knowledgewould further guide the scientific dialog around rationally translatinga cell transplantation approach for human SCI.

Methods

A focus group meeting supported by the Rick Hansen Institute(http://www.rickhanseninstitute.org/) and hosted by the lead andsenior authors (BK and WT) was held on the evening of October 18,2012 during the Society for Neuroscience Annual Meeting, in NewOrleans, LA, USA. Members from the SCI scientific and clinical com-munity who were already attending the annual SFN conferencewere invited to participate in this side meeting if they were, at thetime, known to be actively conducting preclinical cell therapy re-search or were involved in a clinical trial for a cell therapy in SCI. Inorder to ensure a broad invitation outreach, the accepted abstractson the SFN conference website were also scanned using the searchterms “cellular transplantation” or “cell therapy or therapies” and“spinal cord injury” and persons not already identified, were invitedto attend. Out of a total of 45 invitations, 27 researchers with demon-strated expertise in the field of SCI and cell therapy attended the focusgroup meeting. The remaining 18 researchers declined participationbecause they were either not attending the SFN meeting, or hadscheduling conflicts that would preclude their attendance at thefocus group meeting.

Prior to the focus group meeting, a 42-item questionnaire (includedas a Supplement) was issued via e-mail to those who had confirmedtheir attendance. They were asked to complete and submit it before at-tending themeeting. The pre-meeting questionnaire focused specificallyon the topic of cellular therapies and the demonstration of their efficacyin preclinical SCI studies, rather than on their safety or regulatoryrequirements. For the purposes of the questionnaire, the respondentswere instructed to consider as a basic operational definition of efficacy:“the promotion of a quantifiable functional benefit associated with thecells' activity within the tissue”. The focus on efficacy rather than safetyin the pre-meeting questionnaire and during the focus group meetingwas intentional. We took the approach that the scientific goal of the

Table 1Meeting participants and questionnaire respondents.

Soheila KarimiAbdolrezaee

University of Manitoba Canada

Mark Bacon Spinal Research EnglandMichael Beattie University of California,

San FranciscoUnited States

Armin Blesch University of Heidelberg GermanyJacqueline Bresnahan University of California,

San FranciscoUnited States

Mary Bunge University of Miami United StatesSteven Davies University of Colorado United StatesSarah Dunlop University of Western Australia AustraliaMichael Fehlings University of Toronto CanadaAdam Ferguson University of California,

San FranciscoUnited States

Bob Grossman Methodist NeurologicalInstitute/N.A.C.T.N.

United States

Jim Guest University of Miami United StatesCaitlin Hill Weill Medical College

at Cornell UniversityUnited States

John Houle Drexel University College of Medicine United StatesBrian Kwon University of British Columbia CanadaPaul Lu University of California,

San DiegoUnited States

Oudega Martin University of Pittsburgh United StatesJohn McDonald Kennedy Krieger

Institute & Johns HopkinsUniversity School of Medicine

United States

Marion Murray Drexel University College of Medicine United StatesHans Werner Muller University of Dusseldorf GermanyMasaya Nakamura Keio University JapanPaul Reier University of Florida United StatesEphron Rosenzweig University of California,

San DiegoUnited States

Jerry Silver Case Western University United StatesEva Syková Institute of Experimental

Medicine AS CRCzech Republic

Wolf Tetzlaff University of British Columbia CanadaXiao-Ming Xu Indiana University United States

32 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

research community is to translate treatments that are not merely safe,but also efficacious at improving the neurologic outcome for individualswith SCI. Whereas safety and toxicity are obviously important consider-ations, the requirements for demonstrating safety are largely adjudicatedby regulatory bodies such as the Food and Drug Administration (FDA).

The questionnaire was composed of a series of statements thatasked for respondents' opinions in the following six categories:1) the use of different animal models; 2) the use of different injurymodels and mechanisms; 3) the time window for demonstrating effi-cacy; 4) independent replication; 5) defining “relevant, meaningfulefficacy” in preclinical studies; and 6) expectations of therapeuticbenefit. For themajority of the questions, respondents were asked to in-dicate the extent to which they agreed or disagreed with the providedstatement by respondingwith: 1) Strongly Disagree; 2)Mildly Disagree;3) Neither Agree nor Disagree; 4) Mildly Agree; and 5) Strongly Agree.For the questions that specifically addressed the time window for effica-cy, respondentswere asked to consider a cell transplantation clinical trialwith enrollment at specific times post-injury, and the type of preclinicaldata that would support such a delay in transplantation. For the expecta-tions of therapeutic benefit, respondents were simply asked to indicatewhat chance of success they would expect in human SCI with the givenpreclinical data.

The questionnaire responses were compiled and presented to theparticipants during the focus groupmeeting. Additional questionsweregenerated from the pre-meeting survey and posed to the group usinganonymous audience response system (ARS) keypads (provided byVistacom Information Systems, Inc.), which captured their real-time re-sponses as the discussion evolved. The data generated from both thepre-meeting questionnaire and ARS were collectively used to facilitatethe focus group meeting discussion. The figures illustrate the extent ofagreement or disagreement to the proposed statements, although forsuccinctness in the text, we grouped together thosewho agreed stronglyor mildly as “agreed” and those who disagreed strongly or mildly as“disagreed”.

Results

A total of 27 completed questionnaires were received prior to thefocus group meeting, indicating a response rate of 100% from thosewho confirmed their participation in the meeting. The same 27respondents were also in attendance for the focus group meetingand were each assigned an ARS keypad to respond to questionsposed within the meeting. The meeting attendees were individualsin the SCI scientific or clinical community representing institutionsfrom North America, Europe, and Asia (Table 1). All were involvedin SCI research at a preclinical or clinical level.

Animal models used in preclinical SCI research

Pre-meeting questionnaire responsesThe questionnaire began with an assessment of how the respon-

dents viewed the importance of demonstrating a cell therapy's efficacyin different animalmodels used in SCI research. In particular, their opin-ions regarding the need for rodent, larger animal (e.g. pig, sheep, dog)and primatemodelswere sought. Seventy-eight percent of respondentsfelt that the demonstration of efficacy in rodent studies alone was notsufficient to proceed with a human trial; only 15% of the respondentsopined that such rodent data were sufficient. Sixty-seven percent indi-cated that demonstrating efficacy in large animalmodels was needed toproceedwith human trials, while 22% felt that this was not. Interesting-ly, the responseswere divided on the need for demonstrating efficacy inprimates, with 40% opining that such studies were needed, while 45%indicated that such studies were not (Fig. 1).

The majority of the SCI population incurs cervical injuries and thereis great interest in recruiting individuals with tetraplegia for cellulartransplantation therapies to potentially improve their hand/upper

extremity function. Hence, we asked about the need for demonstratingefficacy in cervical models of SCI. Seventy-four percent agreed that effi-cacy in rodent models of cervical SCI was necessary if considering theenrolment of human individuals with cervical SCI. Respondents weredivided in their opinion about the need for efficacy in primate modelsof cervical SCI, with 46% indicating that these were needed, while 45%opining that they were not (Fig. 2).

A question that arises around the use of animal models is that if onewere to commit the resources to employ a larger animal (mammal) ornon-human primate, what research questions would it be used toanswer? We considered that some might contend that demonstratingefficacy in a larger mammal is not necessary, but that such modelsmight have an important role in evaluating other translationally rele-vant issues, such as biodistribution, cell survival/migration, and dosescale-up. Respondents were divided on this issue for large animalmodels, but the majority (67%) did not agree with the statement thatbiodistribution, cell survival/migration, and dose scale up were neededin non-human primate models (Fig. 2).

ARS questions and group discussionAfter presenting the results of the pre-meeting questionnaire to the

group, we used the ARS keypad systems to acquire opinions regardingthe importance placed on efficacy data in animal models. By questioningthe group about the use of mouse, rodent, large animal, and primatemodels, it was apparent that the group viewed the demonstrationof efficacy in higher order species as ‘stronger evidence’ (Table 2).The respondents were equally divided (50% each) when asked “if theregulatory/funding bodies required a demonstration of efficacy inlarge animals, would this be raising the bar too high?”. Seventy-eightpercent felt that such a requirement for primate studies would be too

Fig. 1. Animal models of SCI. Respondents' expressed their opinions about whether efficacy in rodent models alone would be sufficient to proceed with human trials, or whetherefficacy in large animal or primate models was needed.

33B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

strict and could potentially prevent the advancement of cellular thera-pies into clinical testing.

The groupwas thenposed the scenario of a cell therapy that had beenreportedly efficacious in a variety of rodent models: in different injurymodels (e.g. SCI contusion and compression), in a severe injury model

Fig. 2. Use of animal models of cervical SCI or non-efficacy outcomes. Respondents expreshuman translation. Also, the perceived need for large animal or primate models for outcom

(with BBB less than 9 in controls), and in an independent replicationstudy. Despite these multiple demonstrations of efficacy in the rodentmodel, 44% still felt that efficacy in a large animal model was required,while 41% felt that while efficacy was not needed in a large animalstudy, such a large animal model was still needed to define

sed their opinions about whether efficacy in cervical SCI models was needed prior toes other than efficacy was assessed.

Table 2Audience response system questions related to animal model of SCI.

Yes No

Is efficacy in a rat ‘stronger evidence’ than efficacy in a mouse? 68% 32%Is efficacy in a large animal ‘stronger evidence’ than efficacy in a rat? 89% 11%Is efficacy in a primate ‘stronger evidence’ than in a large animal? 73% 27%If regulatory/funding bodies required efficacy in large animals wouldthis be too strict a bar for the SCI field?

50% 50%

If regulatory/funding bodies required efficacy in primates, would thisbe too strict a bar for the SCI field?

78% 22%

Should a cell therapy with evidence of efficacy in a rodent model ofthoracic SCI be permitted to enroll cervical SCI patients in a trial?

19% 81%

34 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

biodistribution and dose. Only fifteen percent opined that further largeanimal studies would not be needed in such a scenario. When asked ifa cell therapy trial should be allowed to enroll individuals with cervicalSCI if efficacy had only been shown in thoracic SCI, 81% responded ‘no’(Table 2).

Injury models used in preclinical SCI research

A variety of different models are currently used to injure the spinalcord with blunt or penetrating mechanisms. These injury modelsserve different purposes for studies, such as neuroprotection or axonalregeneration of a particular tract. For investigating cellular therapies,researchers have previously utilized partial or complete transection in-juries, and various contusive/compressive injuries that can produceneurologic deficits of incremental severity. While it is recognized thatmost human injuries are non-penetrating, 66% felt that studies of celltherapies that utilized partial or full transection injuries still providedimportant preclinical evidence. Such studies (particularly completetransection injuries) at least provide a setting to unequivocally demon-strate axonal regeneration. Having said that, even if a cell therapy wereto demonstrate efficacy within a full transection SCI model, 93% agreedthat efficacywas still required in a contusion/compression injurymodelbefore proceeding with human trials. As for blunt, non-penetratinginjuries, the majority of respondents did not feel that the differentinjury mechanisms observed in human SCI need to be reproducedpre-clinically. Sixty-one percent opined that evidence of efficacy incontusion or compression model of SCI was sufficient to apply thetreatment in SCI individuals with fracture dislocations or distractivespinal injuries (Fig. 3).

With respect to injury severity, most researchers employ rodentmodels of contusive or compressive SCI that result in partial (and

Fig. 3. Use of partial or complete transection injury models. Respondents expressed

often modest) deficits in hindlimb function. In such models, a therapymay produce ‘statistically significant locomotor improvement’ overcontrol untreated animals that themselves have considerablehindlimb function. In contrast, individuals with SCI who are mostlikely to be recruited to clinical trials of such therapies will have lostall motor function in their lower extremities (ASIA ImpairmentScale [AIS] A or B). Whether the therapeutic effect observed in thepreclinical studies with a partial neurologic deficit would be applicablein human subjects with complete paralysis is unknown. Half of therespondents felt it would not be justified to enroll individuals with AISA SCI based on rodent studies where efficacy was demonstrated butcontrol animals still achieved considerable hindlimb function (e.g. plantarweight support). We tried to distill how severe the locomotor deficitswould need to be to justify enrolling AIS A SCI patients. Eighty-ninepercent felt that if the control animals achieved BBB scores of 12that testing should be done in a more severe injury model before aclinical trial of complete SCI was undertaken. Sixty-six percent feltthe same way if control animals achieved BBB scores of 9. If controlanimals achieved a BBB score of 6, the majority (59%) felt that efficacyin a more severe injury model was not necessary (Fig. 4).

ARS questions and group discussionTo establish how researchers view the role of higher order animal

experiments, we asked the question “should a cell therapy showingefficacy in a rat study where the control animals have a BBB of 9 berequired to be tested in amore severe rat injurymodel before proceedingto further preclinical studies in large animals or primates?”. Themajority(64%) said ‘yes’ to this.

Time window of intervention

Cellular transplantation may theoretically be performed in humansat any time post-injury. While there is much interest in developingtreatments that would be applicable in the ‘chronic’ injury state, in themajority of preclinical studies, the cells are transplanted at relativelyearly time points (most often between 1 and 2 weeks post-injury)(Tetzlaff et al., 2011). Here, we surveyed opinions on what timewindows in animals were reasonable to support intervening inhuman SCI at various time points. In particular, we sought to establishwhat preclinical time windows would justify a chronic SCI interventionin a rodent injury model.

For a cellular therapy to be transplanted into individuals with SCIwithin 1–2 weeks post-injury, the majority of researchers (>80%)felt that the cells should demonstrate efficacy in rodent studies

their opinions about the relevance of efficacy data in transection models of SCI.

Fig. 4. The severity of impairment in rodent models of SCI. Respondents provided their opinion about how severe the impairment should be in rodent models of SCI in order tojustify translation to human subjects with complete paralysis.

35B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

when implanted 1 or 2 weeks post-injury. For a cellular therapy to betransplanted 1–2 months post-injury in humans with SCI, the major-ity (>60%) indicated that efficacy should be demonstrated in rodentstudies with a delay of at least 6 weeks post-injury. For a clinical trialwith transplantation 3–6 months post-injury, 90% indicated that a mini-mum delay of 6 weeks or 3 months in preclinical studies was needed.Finally, for a “chronic” time frame of 12–18 months post-injury inhumans, over 80% recommended a delay between 6 weeks and12 months in preclinical studies (Fig. 5).

We then asked if the requirements for demonstrating efficacy inchronic time points should be less stringent than at sub-acute(1–2 weeks post-injury) time points (Fig. 6). Respondents were ingeneral agreement that the requirements were less stringent, solong as there was still “quantifiable functional benefit” demonstratedin preclinical models of chronic SCI. However, in the absence of suchquantifiable functional benefit in chronic models, most felt that thedemonstration of the same activity of the cells (e.g. survival, integration,remyelination) as observed in sub-acute models was not by itself suffi-cient evidence to proceed.

ARS questions and group discussionThe discussion of the appropriate time window for a cell therapy

intervention in chronic human SCI brought about an interesting discus-sion on what should be considered a chronic SCI in animal models.

From a recent review of the literature that built upon the workconducted for a systematic review of experimental cell therapies in SCI(Tetzlaff et al., 2011), we found that there was large discrepancy inwhat was considered a chronic SCI model. Specifically, from the 28 pub-lications surveyed that reported performing cell transplantation in achronic SCI model, there was a large range in time windows reportedfrom 21 days to 10 months (Table 3).

On the issue of chronicity, we asked if there was an absoluteminimum delay in intervention that should be evaluated if consideringa cell therapy for individuals with chronic SCI? 54% of the respondentsindicated 3 months, while 25% indicated 6 weeks. No participant indi-cated that 2 or 4 weeks post-injurywas enough of a delay to rationalizeconsidering transplantation in a chronic patient population (Fig. 7).

Independent replication of efficacy

It was acknowledged that the independent replication of cellulartherapy studies was complicated by the challenges associated withestablishing comparable cell batches and variations in the deliveryprocess, and potential intellectual property issues. Despite this, weasked a series of questions about the need for replicating preclinicalstudies of cellular therapies. For preclinical studies involving rodentmodels, there was strong agreement amongst almost all respondents(89%) that independent replication was necessary before proceeding

Fig. 5. Time windows for delayed transplantation. The respondents were asked the question “if a cellular therapy is going to enroll SCI patients in the given time windowspost-injury, the animal studies should demonstrate efficacy with a delay in its administration of how long after injury?”.

36 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

with human trials. Similarly, respondents strongly agreed (81%) thatthe efficacy of a cell therapy being developed by a commercial partyshould be independently replicated. Interestingly, 89% of respondentsalso agreed that if two independent laboratories demonstrated effica-cy of the same cellular therapy but in different animal or injurymodels, this was still considered a meaningful “replication” of thetherapy's efficacy (Fig. 8).

Fig. 6. Efficacy in models of chronic SCI. The respondents provided their expectations for effineeded to be demonstrated in the chronic setting, but the extent to which efficacy was dpost-injury).

We additionally sought the perspectives of researchers on negativereplication studies that were deemed to be otherwise carefullyperformed, methodologically sound, and unbiased. First, there wasstrong agreement (92%) that such negative replication studies were asimportant in considering the efficacy of cellular therapies as a positivestudy. There was, however, considerable diversity in how such negativeresults were interpreted. Respondents were divided in their opinions

cacy in the chronic transplantation paradigm. There was an agreement that efficacy isemonstrated did not need to be as great as in the sub-acute time frame (1–2 weeks

37B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

aboutwhether the experimental conditions of the index studywere nottruly replicated, or whether the cellular therapy was simply noteffective (Fig. 9).

ARS questions and group discussionWe posed to the group the question of whether an expectation of

independent replication of rat studies represented too high a bar toachieve for the SCI field. Ninety two percent indicated that if regulatory/funding agencies required independent replication of efficacy in ratstudies (not replication of large animal or primate studies) that thiswould not set the bar too high for the SCI field. We then asked “shoulda company be required by regulatory/funding agencies to provide its

able 3reclinical studies of transplantation in “chronic” SCI. The literature reveals considerable variation in the length of delay post-injury that is utilized to assess the effect of “chronic”ansplantation.

Publication Intervention Animal/injury model Cell transplantation“chronicity”

Houle and Reier,J. Comparative Neurology, 1988

Rat fetal spinal cord tissue Rat lumbar level hemisection 7 weeks

Houle and Reier,Neuroscience Letters, 1989

Rat fetal spinal cord tissue Rat lumbar hemisection or complete transection 11 weeks

Lu et al.Brain, 2002

Olfactory ensheathing glial cells (OEGs) Rat T10 complete transection 4 weeks

Hains et al.Neuroscience, 2003

Neural stem/progenitor cell Rat T13 Hemisection 4 weeks

Zurita and Vaquero,Neuroreport, 2004

Bone marrow stromal cells (BMSCs) Rat T6–T8 (?) Contusion, Weight drop 3 months

Barakat et al.Cell Transplant, 2005

Schwann cells/OEGs Rat T8 contusion 2 months

Keirstead et al.J Neuroscience, 2005

Human embryonic stem cells (ESCs) Rat T9/T10 contusion 10 months

de Haro et al.Neuroscience Letters, 2005

BMSC Rat T7 contusion 3 months

Deumens et al.J NeuroscienceResearch, 2006

OEGs and olfactory nerve fibroblast(ONF)-biomatrix complexes

Rat T11–T12 dorsal hemisection 4 weeks

Steward et al.Experimental Neurology, 2006

OEGs Rat T10 Complete Transection 30 days

Karimi-Abdolrezaee et al.J Neuroscience, 2006

Neural stem/progenitor cells Rat T7 clip compression 8 weeks (56 d)

Pfeifer et al.Regenerative Med, 2006

Neural stem/progenitor cells Rat C3 dorsal column transection 8 weeks

Bakshi et al.J Neurotrauma, 2006

BMSC Rat “midthoracic” contusion 27 days

Vaquero et al.Neuroscience Letters, 2006

BMSC Rat, thoracic (?) contusion, weight drop 3 months

Zurita and Vaquero,Neuroscience Letters, 2006

BMSC Rat T6–T8 contusion, weight drop 3 months

Lopez-Vales et al.Glia, 2007

OEGs Rat complete T8 transection 45 days

Parr et al.J Neurotrauma, 2007

Neural stem/progenitor cells Rat T8–T9 clip compression 28 days

Lu et al.Exp Neurology, 2007

BMSC (NT-3 expressing) Rat, C3 dorsal column transection 6 weeks

Zurita et al.Transplantation, 2008

Autologous BMSC Pig T12–T13 compression injury 3 months

Munoz-Quiles et al.J Neuro Exp Neurol, 2009

OEGs Rat complete T8–T9 transection 4 months

Karimi-Abdolrezaee et al.J Neurosci, 2010

Neural stem/progenitor cells + ChABC Rodent T7 clip compression 7 weeks(ChABC at 6 weeks)

Kusano et al.Biochem Bio Res Com, 2010

Neural stem/progenitor cells (excreting NT-3) Rodent T9 clip compression 6 weeks

Salazar et al.PLoS One, 2010

(Human) Neural stem/progenitor cells Mouse T9 contusion 30 days

Hejcl et al.Stem Cell Dev, 2010

BMSCs + hydrogel based on 2-hydroxypropylMethacrylamide

Rat T8–T9 Balloon compression 5 weeks

Nishida et al.Am J Vet Res, 2011

BMSC (at least 1 month post-decompressive surgery) Dog contusion (level?) 1 to 3 months

de Almeida et al.J Neurotrauma, 2011

(Human) Dental Pulp cells Mouse, T9 Clip compression 28 days

Cusimano et al.Brain, 2012

Neural stem/progenitor cells Mouse T12 contusion 21 days

Zhang et al.Brain Res, 2012

OEGs + tail nerve electrical stimulation (TANES) Rat T10 contusion 6 weeks

TPtr

cells to an independent laboratory for testing?”. Seventy-three percentresponded ‘yes’. A discussion about this ensued, inwhich it was pointedout that companies typically acquire (or are established from) cellulartherapy technologies that have been shown to be promising in indepen-dent scientific laboratories. Under such circumstances, the companywould typically do its own internal replication of the data, which –

while not available for peer review –would encourage it to proceed to-wards translation. After this discussion and the caveat that a companywould internally replicate the initial findings of an independent labora-tory, we re-posed this question of whether a company should berequired by regulatory/funding agencies to provide its cells to an inde-pendent laboratory for testing. In this case, 61% responded ‘no’.

Fig. 7. Minimum post-injury delay in preclinical studies of cell transplantation. Theparticipants were asked “What is the absolute minimum delay in intervention thatshould be evaluated if considering a cell therapy for a truly chronic SCI population?”.

38 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

Defining clinically meaningful efficacy in rodent preclinical studies

We provided the following operational definition of ‘efficacy’ forrespondents to utilize when answering the survey questions: “thepromotion of quantifiable functional benefit associated with thecells' activity within the tissue”. Previously, a grading scale for acuteneuroprotective therapies had been developed which articulated out-comes considered to be ‘clinically meaningful’ and assigned points tothese (Kwon et al., 2011c). Here, we sought to determine what re-searchers viewed to be ‘clinically meaningful’ in preclinical cellulartherapy experiments.

In experiments using rodent models of thoracic SCI, 81% of respon-dents agreed that specific improvements in locomotor recovery were

Notes to Table 3:Houle, J.D., Reier, P.J., 1988. Transplantation of fetal spinal cord tissue into the chronically iRegrowth of calcitonin gene-related peptide (CGRP) immunoreactive axons from the chroni253–258.Lu, J., Feron, F., Mackay-Sim, A., Waite, P.M., 2002. Olfactory ensheathing cells promot14–21.Hains, B.C., Johnson, K.M., Eaton,M.J., Willis,W.D., Hulsebosch, C.E., 2003. Serotonergic nspinal hemisection in rat. Neuroscience 116, 1097–1110.Zurita, M., Vaquero, J., 2004. FuncNeuroreport 15, 1105–1108.Barakat, D.J., Gaglani, S.M., Neravetla, S.R., Sanchez, A.R., Andrade, Cand axon growth support of glia transplanted into the chronically contused spinal cord. Cell TraSteward, O., 2005. Human embryonic stem cell-derived oligodendrocyte progenitor cell tr4694–4705.de Haro, J., Zurita, M., Ayllon, L., Vaquero, J., 2005. Detection of 111In-oxine-labeleparaplegic rats. Neurosci. Lett. 377, 7–11.Deumens, R., Koopmans, G.C., Honig, W.M.,corticospinal axons do not cross large spinal lesion gaps after a multifactorial transplantatioJ. Neurosci. Res. 83, 811–820.Steward, O., Sharp, K., Selvan, G., Hadden, A., Hofstadter,M., Au, E., Rolamina propria following complete spinal cord transection in rats. Exp. Neurol. 198, 483–499.Karitransplantation of adult neural precursor cells promotes remyelination and functional neurologicaM., Aigner, L., Bogdahn, U., Weidner, N., 2006. Autologous adult rodent neural progenitor cell tranjured spinal cord. Regen. Med. 1, 255–266.Bakshi, A., Barshinger, A.L., Swanger, S.A., Madhavani,stromal cells in spinal cord contusion: a novelmethod forminimally invasive cell transplantationbonemarrow stromal cells in chronic paraplegic rats: systemic or local administration? Neurosciof chronic paraplegic rats: functional andmorphological outcome oneyear after transplantation. Nplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord tranof adult rat spinal cord stem/progenitor cells for spinal cord injury. J. Neurotrauma 24, 835–845chronic spinal cord injury. Exp. Neurol. 203, 8–21.Zurita, M., Vaquero, J., Bonilla, C., Santos, M., Deogous transplantation of bonemarrow stromal cells. Transplantation 86, 845–853.Munoz-Quilesolfactory bulb ensheathing glia and feasibility for autologous therapy. J. Neuropathol. Exp. Neuro2010. Synergistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and gJ. Neurosci. 30, 1657–1676.Kusano, K., Enomoto, M., Hirai, T., Tsoulfas, P., Sotome, S., Shinomiyamyelination and partial hindlimb recovery in the chronic phase after spinal cord injury. BiocheB.J., Anderson, A.J., 2010. Human neural stem cells differentiate and promote locomotor recoverA., Sedy, J., Kapcalova, M., Toro, D.A., Amemori, T., Lesny, P., Likavcanova-Masinova, K., KrumbHPMA-RGD hydrogels seeded with mesenchymal stem cells improve functional outcome in chrH., Kitamura, M., Hatoya, S., Sugiura, K., Suzuki, Y., Ide, C., Inaba, T., 2011. Evaluation of transplaof chronic spinal cord injury in dogs. Am. J. Vet. Res. 72, 1118–1123.de Almeida, F.M., Marques, SS.K., Martinez, A.M., 2011. Human dental pulp cells: a new source of cell therapy in amousemodeBrambilla, E., Donega,M., Alfaro-Cervello, C., Snider, S., Salani, G., Pucci, F., Comi,G., Garcia-Verduginstruct phagocytes and reduce secondary tissue damage in the injured spinal cord. Brain 135, 44combined with scar ablation and neural transplantation promotes locomotor recovery in rats w

‘clinically meaningful’, but this depended upon the extent of recoveryin control animals, and needed to be associated with improvements inhistologic outcomes such as cell survival, integration, remyelination,neuroprotection, or facilitation of axonal growth/sprouting. In otherwords, merely demonstrating a ‘statistically significant’ improvementin locomotor recovery was not by itself sufficient. The majority of re-spondents (89%) also viewed other sensorimotor recovery outcomes,pain outcomes, and autonomic outcomes to be clinically meaningfulin such thoracic SCI models. For cervical models of SCI, respondentswere in agreement that motor recovery in the forelimb and othernon-motor behavioral outcomes were clinically meaningful, so long asthey were accompanied by improvements in other non-behavioral out-comemeasures (i.e. cell survival, integration, remyelination, or facilita-tion of axonal growth/sprouting). Demonstrating a dose responsewhere increasing efficacy was related to increased dose or survival ofcells was also considered to be ‘clinically meaningful’ by approximately60% of respondents (Fig. 10).

Expectations of therapeutic benefit

The questionnaire and focus group meeting concentrated on theconcept of efficacy. While early human trials are focused on safety, re-searchers ultimately hope that these cellular therapies will indeedimprove the function of individuals with SCI. In a survey of individ-uals with SCI, we observed that many have high expectations forfunctional benefit if they were to participate in a clinical trial of a‘stem cell therapy’ (Kwon et al., 2012a). Here, we sought to determinehow scientific experts consider the chances that a cellular therapywould promote meaningful functional benefit to clinical trial partici-pants, based on the extent of preclinical evidence that was available.Respondents were asked to estimate the chances of successfully dem-onstrating the clinical efficacy of a cellular therapy entering into clinicaltrial (with the term ‘efficacy’ being broadly and operationally defined as

njured adult rat spinal cord. J. Comp. Neurol. 269, 535–547.Houle, J.D., Reier, P.J., 1989.cally injured rat spinal cord into fetal spinal cord tissue transplants. Neurosci. Lett. 103,e locomotor recovery after delayed transplantation into transected spinal cord. Brain 125,eural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons aftertional recovery in chronic paraplegia after bone marrow stromal cells transplantation..M., Pressman, Y., Puzis, R., Garg, M.S., Bunge,M.B., Pearse, D.D., 2005. Survival, integration,nsplant. 14, 225–240.Keirstead, H.S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K.,ansplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25,d bone marrow stromal cells after intravenous or intralesional administration in chronicMaquet, V., Jerome, R., Steinbusch, H.W., Joosten, E.A., 2006. Chronically injuredn strategy using olfactory ensheathing cell/olfactory nervefibroblast-biomatrix bridges.skams, J., 2006. A re-assessment of the consequences of delayed transplantation of olfactorymi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Morshead, C.M., Fehlings, M.G., 2006. Delayedl recovery after spinal cord injury. J. Neurosci. 26, 3377–3389.Pfeifer, K., Vroemen,M., Caioni,splantation represents a feasible strategy to promote structural repair in the chronically in-V., Shumsky, J.S., Neuhuber, B., Fischer, I., 2006. Lumbar puncture delivery of bone marrow. J. Neurotrauma 23, 55–65.Vaquero, J., Zurita, M., Oya, S., Santos, M., 2006. Cell therapy using. Lett. 398, 129–134.Zurita, M., Vaquero, J., 2006. Bonemarrow stromal cells can achieve cureeurosci. Lett. 402, 51–56.Lopez-Vales, R., Fores, J., Navarro, X., Verdu, E., 2007. Chronic trans-section in the rat. Glia 55, 303–311.Parr, A.M., Kulbatski, I., Tator, C.H., 2007. Transplantation.Lu, P., Jones, L.L., Tuszynski, M.H., 2007. Axon regeneration through scars and into sites ofHaro, J., Oya, S., Aguayo, C., 2008. Functional recovery of chronic paraplegic pigs after autol-, C., Santos-Benito, F.F., Llamusi, M.B., Ramon-Cueto, A., 2009. Chronic spinal injury repair byl. 68, 1294–1308.Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Schut, D., Fehlings, M.G.,rowth factors promote functional repair and plasticity of the chronically injured spinal cord., K., Okawa, A., 2010. Transplanted neural progenitor cells expressing mutant NT3 promotem. Biophys. Res. Commun. 393, 812–817.Salazar, D.L., Uchida, N., Hamers, F.P., Cummings,y in an early chronic spinal cord injury NOD-scid mouse model. PLoS One 5, e12272.Hejcl,holcova, E., Pradny, M., Michalek, J., Burian, M., Hajek, M., Jendelova, P., Sykova, E., 2010.onic spinal cord injury. Stem Cells Dev. 19, 1535–1546.Nishida, H., Nakayama, M., Tanaka,ntation of autologous bone marrow stromal cells into the cerebrospinal fluid for treatment.A., Ramalho Bdos, S., Rodrigues, R.F., Cadilhe, D.V., Furtado, D., Kerkis, I., Pereira, L.V., Rehen,l of compressive spinal cord injury. J. Neurotrauma 28, 1939–1949.Cusimano,M., Biziato, D.,o, J.M., De Palma,M.,Martino, G., Pluchino, S., 2012. Transplanted neural stem/precursor cells7–460.Zhang, S.X., Huang, F., Gates,M., Holmberg, E.G., 2012. Tail nerve electrical stimulationith chronically contused spinal cord. Brain Res. 1456, 22–35.

Fig. 8. Independent replication. The respondents provided their opinion on the need for independent replication prior to human translation.

39B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

previously). Respondentswere told to assume that the cells had fulfilledall the necessary safety requirements, that subjects would receive theanticipated therapeutic ‘dose’ of cells, that the time window of thepreclinical study was comparable to the clinical study.

In general, the demonstration of efficacy in large animal models orprimatemodels increased the perceived chances of success over rodentmodels alone. For a cellular therapy that had been reportedly efficaciousin a rodent contusive or compressive SCI model where control animalsachieved a BBB score of 12, respondents indicated that a thoracic AIS Apatient would have 0–55% chance of functional recovery (median 10%).The addition of efficacy in a large animal model or primate modelincreased the perception that functional recovery would occur to2–80% (median 20%) and 5–85% (median 27.5%), respectively. Withthe confirmation of biodistribution and dose scale up in a large animalmodel, respondents described the chances of functional recovery tobe between 0 and 90% (median 25%) for thoracic AIS A subjects(Fig. 11).

Interestingly, for a cellular therapy shown to be efficacious in a rodentmodel of contusive or compressive SCI, the addition of a negative inde-pendent replication study did not markedly change the respondents'view on the chances that the cell would promote recovery in humanSCI. Respondents still perceived the chances of functional recovery forthoracic AIS A subjects to be in the range of 0–45% (median 10%). In con-trast, in the presence of positive independent replication of efficacy theperceived range in chance of functional recovery increased to 0–85%(median 25%).

Fig. 9. Interpreting ‘negative’ replication studies. The respondents we

Discussion

Howmuch scientific evidence that a cellular therapy ‘works’ in preclinicalmodels of spinal cord injury is enough before proceeding with a humanclinical trial? This is a complex question. Given that no cellular therapyhas emerged from the full gamut of clinical testing with convincing evi-dence of efficacy in human SCI, an empirically-based answer to this ques-tion does not exist. In the absence of data or firsthand experience, theopinions of stakeholders in the SCI field are relevant for establishing thecurrent landscape and providing perspectives for the field in guidingthe translation of cellular therapies. With the breadth of stakeholders inthe SCI field, one would expect a spectrum of opinions for the questionof “how much is enough?”. On one hand, the devastating effects of SCIand the untreatable nature of the neurologic impairment provides goodreasons to aggressively move therapies forward that appear promising,particularly if they have an acceptable safety profile. On the other hand,the time and money required to complete a clinical trial and the surgicalinvasiveness of cellular transplantation provides good reason to“thoroughly investigate and establish the robustness” of a promisingtherapy in preclinical studies. This might mitigate the risk of expendingmassive resources on an ineffective agent. The current ambiguity overwhat it means to “thoroughly investigate and establish the robustness”of a promising cellular therapy in preclinical studies is why we under-took this initiative. Here, we sought to capture the opinions of individ-uals with specific expertise in cellular therapies for SCI, particularlythose who study cellular therapies in the laboratory setting.

re asked about how they interpret ‘negative’ replication studies.

Fig. 10. Clinically meaningful demonstrations of “efficacy” in animal studies. The respondents were asked to provide their opinions on what defines efficacy for a cell therapy inexperimental SCI.

40 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

One of the key objectives of this initiative was to identify areaswhere consensus existed and where it was lacking. All of the datafrom the pre-meeting questionnaire and the in-meeting ARS votingare presented in this paper, but a summary of the questions and is-sues where there was greater than 60% agreement is presented inTable 4.

Animal models of SCI

We first addressed the use of rodent and higher-order animalmodels for preclinical testing. An important consideration to ac-knowledge at the outset of this discussion is that animal models ofSCI are inherently an imperfect approximation to human biologyand none may fully predict efficacy in humans. This having beensaid, over 50% of the respondents ‘strongly disagreed’ with the state-ment that efficacy in rodent studies was sufficient to move forwardwith human trials. This is noteworthy given that the overwhelmingmajority of preclinical evaluation of cellular therapies has beenobtained from rodentmodels. In fairness, the availability of large animaland primate models of SCI to conduct such testing has historically beenlimited, and even currently, the capital and operational costs ofemploying such models are prohibitive for many. The respondentswere in favor of demonstrating efficacy in large animal studies, butthere was no consensus on the need for primates: 40% agreed for theneed for primatemodels and 44% agreed for the need for primate cervi-cal models if enrolling individuals with cervical SCI. Interestingly, in ourprevious survey of approximately 320 individuals from the SCI researchcommunity, a considerably higher proportion (74%) advocated for theneed for demonstrating efficacy in primate models (Kwon et al.,2010).We speculate that themuch lower percentage in our current ini-tiative reflects the greater degree of specialization amongst the partici-pating researchers, who were more conscious of the challenges andlimitations and conducting SCI research in non-human primates. Fur-ther questioning is warranted to discern the specific reasons behind

these perceptions of evaluating cellular therapies in non-humanprimate SCI models. For example, if cost were not an issue, wouldthere be greater support for such studies in the preclinical pathwaytowards translation? As pointed out by others, while such models areexpensive, they pale in comparison to the cost of conducting a clinicaltrial (Courtine et al., 2007). How aware are researchers of thenon-human primate models that are currently available and their ad-vantages, limitations, and costs? Are the currently available primatemodels considered to be adequate testing grounds for such therapies,and what questions are best addressed in them? Going into greaterdepth on these issues would help to clarify the perceived role ofnon-humanprimatemodels in the preclinical evaluation of SCI therapies.

Injury models and injury mechanisms

Similar to the discourse on animal models, the discussion of injurymodels acknowledges the fact that modeling the heterogeneous natureof human SCI in animals is a challenging and imperfect science. Forexample, performing a standard experimental contusion injury gener-ally involves an open laminectomy and impact upon the dorsal aspectof the cord, which obviously differs from the closed injuries that occurin patients. With regard to injury models, a high proportion of respon-dents (66%) indicated that efficacy in partial or full transection modelswere important preclinical evidence for cell therapies. Almost everyone(93%), however was in agreement that further studies in compressive/contusive SCI models were needed before proceeding with clinicaltrials.

Given this perspective, it is interesting to reflect on the autologousactivated macrophage treatment for subacute human SCI, which wasreported last year to have not improved neurologic recovery in thePhase 2 “ProCord” multicenter randomized clinical trial (Lammertseet al., 2012). This technology entered clinical trials after showing efficacyin a full transection model of thoracic SCI in 1998 (Rapalino et al., 1998),and to the authors' knowledge had not been tested in a contusion/

Fig. 11. Expectations of functional benefit for human SCI. The respondents provided anestimate of the chances of an individualwith a complete thoracic SCI (blue) or complete cer-vical SCI (red) achieving functional benefit for a cell therapy with a given demonstration ofefficacy. In essence, we asked “Given this preclinical data that is considered to be ‘promis-ing’… what do you think a patient's chances of gaining any meaningful function backwould be if theywere to receive these cells?”The lengths of the blue and red boxes representthe interquartile (IQR) ranges (the distance between the 25th and 75th percentiles). The ◊symbol inside the box represents the group mean, whereas the horizontal line representsthe group median. The vertical error bars on the top and bottom of the box extend to thegroup minimum and maximum values. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

Preclinical evidence available:A. Efficacy in a rodent contusion/compression thoracic SCI model (controls with BBB of 12).B. Efficacy in a rodent contusion/compression thoracic SCI model (controls with BBB of 12).C. Efficacy in a rodent contusion/compression thoracic SCI model (controls with BBB of 12)

and a rodent cervical SCI model.D. Efficacy in a rodent contusion/compression thoracic SCI model (controls with BBB of 12)

and a rodent cervical SCI model.E. Efficacy in a rodent and in a pig contusion or compression thoracic SCI model.F. Efficacy in a rodent contusion/compression thoracic model and confirmation of

biodistribution and dose scale up in a pig model of thoracic SCI.G. Efficacy in a rodent contusion/compression thoracic model and a primate model of

cervical SCI.H. Efficacy in a rodent contusion/compression thoracic model and a primate model of

cervical SCI.I. Efficacy in a rodent contusion or compression thoracic SCImodel (controlswith BBBof 12)

with a positive independent replication of efficacy.J. Efficacy in a rodent contusion or compression thoracic SCImodel (control with BBB of 12)

with a negative independent replication study.

41B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

compression injury model prior to the initiation of the clinical trial.With the benefit of hindsight, it is indeed hard to be critical of the inves-tigators for deciding to pursue the translation of this cellular therapywith the information they had at the time, given that the SCI field hasevolved considerably over the past decade and a half. At the currenttime, there is stronger support for following up promising resultsfrom transection models with further study in contusion/compressionmodels of SCI. More recently, promising results were published fromthe 1 week post-injury transplantation of human neural stem cellswithin a fibrin matrix/growth factor ‘cocktail’ into a rodent model ofcomplete T3 transection (Lu et al., 2012). Amongst two human celltypes tested in this study, onewas a gift from the company NeuralStem(NSI-566RSC, from NeuralStem Inc., Rockville, MD). On January 14,2013, theUnited States FDAgrantedNeuralStem Inc. approval to initiatea Phase 1 clinical trial in chronic thoracic SCI (http://www.prnewswire.com/news-releases/neuralstem-receives-fda-approval-to-commence-spinal-cord-injury-trial-186768041. html). At the time of this writing,results from the use of these stem cells in a contusion or compressionmodel of SCI or in a chronic injury setting were not available in thepeer-reviewed literature. The cells, however, have been transplantedinto the spinal cord in an FDA-approved Phase 1 clinical trial ofamyotrophic lateral sclerosis. The announcement of the SCI clinical

trial on NeuralStem's website is accompanied by references to thepromising results of Tuszynski and colleagues in the rodent completeT3 transection SCI model (Lu et al., 2012). Of note, the authors of thisstudy oppose NeuralStem's decision to initiate a human SCI trial for anumber of reasons. The clinical transplantation protocol lacks thegrowth factor cocktail and fibrin matrix used in the rodent SCI study,and this they fear will limit cell survival. Also, they desire furtherpreclinical data in a blunt injury model and in a chronic injurytimeframe. The authors fear that negative clinical results will limit futureinterest in this otherwise potentially promising therapy.

The other aspect of injury modeling that was recognized to be anissue of uncertainty was that of injury severity, particularly in rodentmodels of SCI where the BBB score is typically used to describehindlimb locomotor recovery. In such models, ‘control’ untreatedanimals may recover sufficiently to achieve plantar weight supporton their hindlimbs, and the ‘treated’ animals might achieve consistentstepping or coordination, for example. In essence, this amounts totaking a rodent that ‘barely walks’ with some impairment to a rodentthat ‘walks a bit better’. For example, in Keirstead et al.'s initial reportof Geron's human embryonic stem cell (ESC) oligodendrocyte pro-genitor technology (Keirstead et al., 2005), the animals treated 7 dpost-injury with the cellular therapy improved to a BBB score ofaround 16, but the control DMEM-treated animals achieved BBBscores of approximately 12 to 13. Extrapolating this therapeutic effectin rodents with an ‘incomplete’ spinal cord injury to humans withcomplete motor paralysis is difficult. Given that individuals with AISA SCI are more likely to be the ones first enrolled in such clinical trials,the respondents feel that it is necessary to test cells with more severemodels of contusive or compressive SCI than are currently employed.The series of questions that addressed this issue suggested that therespondents felt it necessary to demonstrate the efficacy of a cellulartherapy in a severe injury model (with control animals achieving aBBB score of 6) in order to anticipate an effect in human subjectswith complete SCI.

Timing of intervention

With regard to the timing of intervention, we specifically soughtto determine how participants viewed the preclinical testing of cellsthat would be administered in the chronic SCI condition in humans.Relatively little has been published in preclinical models of ‘chronic’injury. As shown in Table 3, the scientific community uses quite vari-able definitions of ‘chronicity’ in animal experiments. There are impor-tant advantages of testing cells in clinical trials of chronic SCI. A morestable baseline neurologic status may be present and the probabilityof further spontaneous neurologic recovery is more predictable,allowing an assessment of the cellular therapy's effect in a smallercohort of subjects. For the chronic time point (with individuals enrolledover 18 months post-injury), the respondents felt that a delay of inter-vention of at least 3 monthswaswarranted in preclinical studies. Fewerthan 20% of respondents felt that 6 weeks was a sufficient delay. How-ever, for clinical trial enrollment in the 1–6 month range, a 6 weekdelay in transplantation in preclinical studies received more support.Perhaps most informative was the fact that no respondent felt that itwould be justified to transplant a cell therapy into an individual witha chronic SCI if the cell's efficacy had been demonstrated only at7–14 days post-injury in a preclinical study. This is relevant, for exam-ple, to the NeuralStem Inc. decision to test their cell transplants in achronic SCI population when the highly-touted preclinical efficacydata was generated with a one week post-injury transplantation. Atthe time of this writing, we are not aware of any published preclinicalstudies of this technology with a more delayed transplantation. Abouthalf of the respondents felt that 3 months was an ‘absolute minimum’

delay in preclinical studies to justify a chronic human intervention;about a quarter advocated for a 6 week delay. These datawould supportthat – at the very least – scientists could come to some consensus

Table 4Summary of topics where consensus existed (>60% agreement).

% ofrespondents

Demonstrating efficacy in a rodent model of SCI alone is not sufficient to proceed with human SCI trials. 78%Demonstrating efficacy in large animal models of SCI is necessary to proceed with human SCI trials 67%Efficacy in rodent models of cervical SCI should be demonstrated before transplanting the cells into human individuals with cervical SCI. 74%Partial or full transection models of SCI provide important preclinical evidence for the efficacy of a cell therapy.However, efficacy still needs to be demonstrated in contusion/compression SCI models even if efficacy is shown in partial or full transection models.

66%93%

If efficacy is demonstrated in a rodent SCI model in which control animals achieve a BBB score of 12, the cellular therapy should be tested in a more severeinjury model before proceeding to human trials.

89%

If efficacy is demonstrated in a rodent SCI model in which control animals achieve a BBB score of 9, the cellular therapy should be tested in a more severe injurymodel before proceeding to human trials.

66%

To substantiate cell transplantation in individuals with chronic SCI, the cells should be tested in animal models with a delay of at least 6 weeks in rodents. 100%When testing a cell therapy in a preclinical model of chronic SCI, the efficacy that is demonstrated does not need to equal that seen in subacute models, so longas it is still ‘quantifiable functional benefit associated with the cells’ activity within the tissue.However, merely demonstrating the activity of the cells within the tissue without the functional benefits is not sufficient.

74%80%

Promising cellular therapies in animal models should be independentlyreplicated;However, the injury model need not be the same.

89%89%

A carefully conducted negative replication study is as important in considering the efficacy of a therapy as a positive study 92%Improvement in either motor or non-motor function following thoracic orcervical injury is clinically meaningful and thus both motor and non-motorfunctions should be examined in an effort to determine cell transplantationefficacy.

81–89%

42 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

around an operational definition of ‘chronicity’ in animal models thatcould then be broadly applied.

Independent replication

As for independent replication, the majority of respondents weresupportive of the need to demonstrate the efficacy of cellular therapiesin independent laboratories. The expectations for replication wereslightly different between a cellular therapy developed by a scientificlaboratory and one that was being developed by a commercial entity.There was discussion at the meeting around whether there was a‘double standard’ applied for replication, with a ‘lower bar’ set forcommercial entities. Comments were raised around the likelihoodthat a company would perform its own ‘internal replication’ of resultsgenerated in an independent scientific laboratory. The example ofGeron was put forth, with the contention that they possessed a greatdeal more efficacy data on its technology than was available. Whilethis may be true, it is difficult for the scientific community to interpretthe robustness of a particular therapy if important efficacy data are pro-prietary and its existence is inferred or loosely referred to, withoutbeing available in the peer-reviewed literature. Despite this, afterdiscussing the issue, the majority (61%) were willing to state that acompany would not need to subject its cells to an independent replica-tion study if they had done their own internal ‘due diligence’ studiesthat would not necessarily be made available to the public but wouldbe available to regulatory authorities (e.g. FDA in the US).

We also attempted to discern how respondents viewed negativereplication studies. A series of replication studies in SCI have beenperformed to date, and almost all have failed to replicate the efficacyseen in the index study (Steward et al., 2012). Somemight interpret thefailure to replicate the efficacy reported in the index study to simplymean that the treatment in question was not effective. However, suchfailure has perhaps more commonly invoked the rationalization thatthe replication study failed to reproduce the conditions of the indexstudy. The responses to our questions on this were very mixed, andperhaps we were unable to capture the essence of this complex issueadequately with this ‘multiple-choice’ question. Nonetheless, the re-spondents were in strong agreement that a carefully performed andmethodologically sound study that was negative was as important toconsider as a positive study. During the discussion there was strongsupport for the notion that well-conducted negative studies were im-portant, and that both researchers should be encouraged to submit

such results and reviewers/editors should not dismiss such replicationstudies as being “not novel enough”.

Expectation of therapeutic benefit

This statement was tested in our subsequent question aroundexpectations of therapeutic benefit. In this, the respondents indicatedthat a positive replication study would increase the potential fortherapeutic benefit in a clinical trial (amedian of 10% increased to ame-dian of 25% chance of recovery); however, after a negative replicationstudy, the chance of therapeutic benefit remained virtually unchanged(i.e. a median of 10% chance of recovery remained unchanged). Thisreveals that the expert respondents were not immune to a degree of‘positive bias’ in their interpretation of negative and positive replicationresults. While the respondents tend to interpret a positive replicationstudy as data that bolster the robustness of the therapy in question, itappears that they are less certain about how to factor a negative replica-tion study into the estimation of a therapy's efficacy. This raises ques-tions about the design and conduct of replication studies — for if thescientific community is unprepared to severely question the robustnessof a cellular therapy's efficacy based on the results of a well-conductedyet negative replication study, it begs to question what the role of suchstudies actually is. Discounting the results of a replication study due to afailure to reproduce all of the exact experimental conditions of theindex study may be scientifically justified, but this argument overlooksthe harsh reality that human SCI occurs with variability that far exceedsthe typical experimental conditions (Noble et al., 2011).

Setting the bar for preclinical evidence

In previous work that has addressed this question of ‘how muchevidence is enough?’, we have frequently encountered the sentimentthat ‘you don't want to set the bar too high for preclinical studies ornothing will ever get translated into humans’. Conceptually, weacknowledge and understand this concern. However, if it is too higha bar to expect that a therapy is robust enough to demonstrate effica-cy in more than a single rodent model of SCI conducted under tightlycontrolled experimental conditions, then it is hard to imagine howthat therapy is going to demonstrate efficacy in a clinical trial popula-tion that is vastly more heterogeneous. In short, while objections tomaking it ‘too hard’ to demonstrate sufficient efficacy in preclinicalstudies are reasonable, we should also not delude ourselves into

43B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

thinking that the task of demonstrating efficacy in humans will some-how be easier. The past experience in SCI and the much longer historyof clinical trials in stroke and TBI that failed to show efficacy would re-mind researchers of the daunting task of clinically validating promisingnew treatments. If nothing else, this experience of negative stroke andTBI clinical trials simply highlights the need to carefully consider the ef-ficacy of what goes into clinical trial. In looking back at the manyneuroprotective agents that failed to show efficacy in clinical trials,the stroke community has questioned whether the preclinical data onsome of those agents were robust enough to justify their translationinto human trials (O'Collins et al., 2006). We in the SCI communityshould heed this experience.

That having been said, as concern has been voiced about creating‘too high a bar’, we used the audience response system to seek whatresearchers felt would be ‘too high a bar’ for preclinical studies. Forexample, the majority felt that at a minimum, a 3 month delay intransplantation should be applied in preclinical studies in order tosubstantiate a clinical trial of chronic SCI individuals. Ninety-two percentfelt that the expectation of independently replicating the efficacy of acellular therapy in a rodent model would not be too high a bar. With re-spect to larger animal models, 50% felt that the expectation of efficacy inlarge animal models would be too high a bar, as did 78% around the useof primatemodels. This indeed conflicts with the 67% that felt that effica-cy in large animalmodelswas needed to proceed. This discrepancy likelyreflects the scientific sentiment that we researchers would rather policeour own field through the process of scientific exchange and peer reviewthan have regulatory or funding agencies impose ‘must-have’ demon-strations of efficacy.

To that point, we reject the notion that regulatory bodies like theFDA would impose rules based on our data for what efficacy mustbe shown in preclinical studies prior to proceeding with clinical trials.With a sample size of 27 researchers and a multiple-choice survey, wecontend that it would be inappropriate to create such standards forthe field, and it was clearly not our intention to try to establishthem. Rather, our goal was to illustrate the spectrum of opinionsaround the issue of ‘what is enough’ to justify translation to humantrials, and to demonstrate where agreement existed and where itdid not. While this might seem unambitious, we contend that thisprocess of painting the landscape of opinions is an important exercisefor the field. We all have our differing opinions and our underlyingbiases, and an awareness of these is essential for establishing a ratio-nal framework for translation. A good example of this is the issue ofanimal models. For those with a strong and vocal opinion that efficacyin a rodent model is sufficient to proceed with clinical trials, it isworth noting that a large proportion of researchers (arguably, alarge majority) who also have considerable expertise in the fieldfeel strongly that efficacy in rodent models does not provide adequatejustification to proceed into human trials. For those who advocate theuse of primate models and heavily invested in these expensivemodels, it is worth noting that many feel that such models are notneeded for demonstrating efficacy or, for that matter, biodistribution,cell survival, or dose scale up. And for those promoting the use oflarge animal models and similarly invested in them, it is worth not-ing that some view rodent models to be totally sufficient and are ac-tually proceeding with clinical trials based solely on these, andothers likely view large animal models to be inadequate in lieu ofthe biological similarities between humans and primates. Having abalanced perspective on these issues is ultimately needed in orderto rationally decide how to move forward with novel therapeuticsin SCI.

Translational science ultimately depends on the process of com-munication, whereby progress is made through the principles ofpeer-review and open dialog. Our intention was to add a perspectiveto the research culture within which novel therapies are being devel-oped for spinal cord injury. We explicitly did not intend to create aregulatory framework for how therapies should be validated in

preclinical studies en route to clinical translation, and would objectto regulatory agencies utilizing these results to determine what couldand could not move forward. It is readily apparent that a diversity ofopinion exists about when a cell therapy is ‘ready’ for translation, andthere is no ‘correct’ pathway that has been empirically established(Fehlings and Vawda, 2011). As such, criticism leveled at past decisionsto move forward with clinical trials is not warranted — it would beunfair to harshly judge such past decisions armed with the knowledgethat has emerged with progress in the SCI field, and the luxury ofhindsight.

Having gone through this exercise, it is natural to considerwhat would be reasonable next steps. Similar to what we observedin the questionnaire, there are likely to be many different opinionsabout how to move this discussion forward. Our previous forayinto asking these types of questions about preclinical evidence forneuroprotection SCI therapies has sparked other analogous initiativessuch as that being led by the Center for Neuronal Regeneration(CNR, http://www.cnr.de) in Europe (H.W. Mueller, personal com-munication, http://www.neurologie.uni-duesseldorf.de/priv-mueller/AGMueller/Events_files/ 1stCNRFlyer.pdf) and by AO Spine (Tatoret al., 2012); such added perspectives and methodologies are stronglyencouraged for cell therapies. Our current questionnaire and focusgroup meeting covered 5 main topics (animal models, injury models,independent replication, timing of intervention, and clinicallymeaning-ful efficacy) and the discussion around each revealed that we couldhave spent the entire effort on the complexities of a single topic. Furtherdialog that focused specifically on the issue of which animal models touse and for what research questions would be of practical value forthe field, as would a more in-depth discussion of the different injurymodels and how to best represent the heterogeneity of human SCI.This could guide the establishment of a consortiumof scientific researchlaboratories to facilitate replication studies or the additional evaluationof promising therapies in different injury paradigms. Ultimately,garnering such collaborative efforts for late-stage preclinical studieswill promote the improved conduct and reporting of translationallyimportant findings. Further discussions would also benefit from theinvolvement of commercial entities and regulatory bodies, as they obvi-ously have a considerable influence on how such therapies are translatedinto clinical trials.

Wedonot have conclusions from this initiative to radically change thecourse of how preclinical SCI studies are conducted or how therapies arechosen to move forward into clinical trials. However, by encouraging themany stakeholders within the research community to consider the ques-tion of ‘what preclinical evidence is enough?’ and by providing a perspec-tive of a collective group of experts in the field of cellular therapies, wehope to nudge and steer thefield forward towards increasing the chancesof bringing effective therapies to clinical trials.

Acknowledgments

We would like to thank all of the SCI researchers for completingthe survey, participating in the focus group meeting, contributed tothis manuscript. We wish to also thank Jae Lee and Elaine Chan fortheir assistance in preparing this manuscript. Support for the focusgroup meeting was generously provided by the Rick Hansen Institute.BKK is the Canada Research Chair in Spinal Cord injury and a MichaelSmith Foundation for Health Research Career Scholar. WT is the RickHansen Man in Motion Chair in Spinal Cord Injury Research. BKK andLJJS consult for the Rick Hansen Institute.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.expneurol.2013.05.012.

44 B.K. Kwon et al. / Experimental Neurology 248 (2013) 30–44

References

Anderson, D.K., Beattie, M., Blesch, A., Bresnahan, J., Bunge, M., Dietrich, D., Dietz, V.,Dobkin, B., Fawcett, J., Fehlings, M., Fischer, I., Grossman, R., Guest, J., Hagg, T.,Hall, E.D., Houle, J., Kleitman, N., McDonald, J., Murray, M., Privat, A., Reier, P.,Steeves, J., Steward, O., Tetzlaff, W., Tuszynski, M.H., Waxman, S.G., Whittemore,S., Wolpaw, J., Young, W., Zheng, B., 2005. Recommended guidelines for studiesof human subjects with spinal cord injury. Spinal Cord 43 (8), 453–458.

Blight, A.R., Tuszynski, M.H., 2006. Clinical trials in spinal cord injury. J Neurotrauma 23(3–4), 586–593.

Courtine, G., Bunge, M.B., Fawcett, J.W., Grossman, R.G., Kaas, J.H., Lemon, R., Maier, I.,Martin, J., Nudo, R.J., Ramon-Cueto, A., Rouiller, E.M., Schnell, L., Wannier, T.,Schwab, M.E., Edgerton, V.R., 2007. Can experiments in nonhuman primates expeditethe translation of treatments for spinal cord injury in humans? Nat. Med. 13 (5),561–566.

Dietrich, W.D., 2003. Confirming an experimental therapy prior to transfer to humans:what is the ideal? J. Rehabil. Res. Dev. 40 (4 Suppl. 1), 63–69.

Fawcett, J.W., Curt, A., Steeves, J.D., Coleman, W.P., Tuszynski, M.H., Lammertse, D.,Bartlett, P.F., Blight, A.R., Dietz, V., Ditunno, J., Dobkin, B.H., Havton, L.A., Ellaway,P.H., Fehlings, M.G., Privat, A., Grossman, R., Guest, J.D., Kleitman, N., Nakamura, M.,Gaviria, M., Short, D., 2007. Guidelines for the conduct of clinical trials for spinalcord injury as developed by the ICCP panel: spontaneous recovery after spinal cordinjury and statistical power needed for therapeutic clinical trials. Spinal Cord 45(3), 190–205.

Fehlings, M.G., Vawda, R., 2011. Cellular treatments for spinal cord injury: the time isright for clinical trials. Neurotherapeutics 8 (4), 704–720.

Hewson, S.M., Fehlings, L.N., Messih, M., Fehlings, M.G., 2013. The challenges of trans-lating stem cells for spinal cord injury and related disorders: what are the barriersand opportunities? Expert. Rev. Neurother. 13 (2), 143–150.

Keirstead, H.S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K., Steward, O., 2005.Human embryonic stem cell-derived oligodendrocyte progenitor cell transplantsremyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25 (19),4694–4705.

Kwon, B.K., Hillyer, J., Tetzlaff, W., 2010. Translational research in spinal cord injury: asurvey of opinion from the SCI community. J. Neurotrauma 27 (1), 21–33.

Kwon, B.K., Okon, E., Hillyer, J., Mann, C., Baptiste, D., Weaver, L.C., Fehlings, M.G., Tetzlaff,W., 2011a. A systematic review of non-invasive pharmacologic neuroprotectivetreatments for acute spinal cord injury. J. Neurotrauma 28 (8), 1545–1588.

Kwon, B.K., Okon, E.B., Plunet, W., Baptiste, D., Fouad, K., Hillyer, J., Weaver, L.C.,Fehlings, M.G., Tetzlaff, W., 2011b. A systematic review of directly applied biologictherapies for acute spinal cord injury. J. Neurotrauma 28 (8), 1589–1610.

Kwon, B.K., Okon, E.B., Tsai, E., Beattie, M.S., Bresnahan, J.C., Magnuson, D.K., Reier, P.J.,McTigue, D.M., Popovich, P.G., Blight, A.R., Oudega, M., Guest, J.D., Weaver, L.C.,Fehlings, M.G., Tetzlaff, W., 2011c. A grading system to evaluate objectively thestrength of pre-clinical data of acute neuroprotective therapies for clinical translationin spinal cord injury. J. Neurotrauma 28 (8), 1525–1543.

Kwon, B.K., Ghag, A., Dvorak, M.F., Tetzlaff, W., Illes, J., 2012a. Expectations of benefit andtolerance to risk of individuals with spinal cord injury regarding potential participa-tion in clinical trials. J. Neurotrauma 29 (18), 2727–2737.

Kwon, B.K., Ghag, A., Reichl, L., Dvorak, M.F., Illes, J., Tetzlaff, W., 2012b. Opinions on the pre-clinical evaluation of novel therapies for spinal cord injury: a comparison between re-searchers and spinal cord-injured individuals. J. Neurotrauma 29 (14), 2367–2374.

Lammertse, D., Tuszynski, M.H., Steeves, J.D., Curt, A., Fawcett, J.W., Rask, C., Ditunno,J.F., Fehlings, M.G., Guest, J.D., Ellaway, P.H., Kleitman, N., Blight, A.R., Dobkin,B.H., Grossman, R., Katoh, H., Privat, A., Kalichman, M., International Campaignfor Cures of Spinal Cord Injury, P, 2007. Guidelines for the conduct of clinical trials

for spinal cord injury as developed by the ICCP panel: clinical trial design. SpinalCord 45 (3), 232–242.

Lammertse, D.P., Jones, L.A., Charlifue, S.B., Kirshblum, S.C., Apple, D.F., Ragnarsson, K.T.,Falci, S.P., Heary, R.F., Choudhri, T.F., Jenkins, A.L., Betz, R.R., Poonian, D., Cuthbert,J.P., Jha, A., Snyder, D.A., Knoller, N., 2012. Autologous incubatedmacrophage therapyin acute, complete spinal cord injury: results of the phase 2 randomized controlledmulticenter trial. Spinal Cord 50 (9), 661–671.

Lu, P., Wang, Y., Graham, L., McHale, K., Gao, M., Wu, D., Brock, J., Blesch, A.,Rosenzweig, E.S., Havton, L.A., Zheng, B., Conner, J.M., Marsala, M., Tuszynski,M.H., 2012. Long-distance growth and connectivity of neural stem cells after se-vere spinal cord injury. Cell 150 (6), 1264–1273.

Maas, A.I., Menon, D.K., Lingsma, H.F., Pineda, J.A., Sandel, M.E., Manley, G.T., 2012. Re-orientation of clinical research in traumatic brain injury: report of an internationalworkshop on comparative effectiveness research. J. Neurotrauma 29 (1), 32–46.

Noble, M., Mayer-Proschel, M., Davies, J.E., Davies, S.J., Proschel, C., 2011. Cell therapiesfor the central nervous system: how do we identify the best candidates? Curr.Opin. Neurol. 24 (6), 570–576.

O'Collins, V.E., Macleod, M.R., Donnan, G.A., Horky, L.L., van der Worp, B.H., Howells,D.W., 2006. 1,026 experimental treatments in acute stroke. Ann. Neurol. 59 (3),467–477.

Rapalino, O., Lazarov-Spiegler, O., Agranov, E., Velan, G.J., Yoles, E., Fraidakis, M.,Solomon, A., Gepstein, R., Katz, A., Belkin, M., Hadani, M., Schwartz, M., 1998. Implanta-tion of stimulated homologous macrophages results in partial recovery of paraplegicrats. Nat. Med. 4 (7), 814–821.

Reier, P.J., Lane, M.A., Hall, E.D., Teng, Y.D., Howland, D.R., 2012. Translational spinalcord injury research: preclinical guidelines and challenges. Handb. 109, 411–433.

Steeves, J., Blight, A., 2012. Spinal cord injury clinical trials translational process, reviewof past and proposed acute trials with reference to recommended trial guidelines.Handb. Clin. Neurol. 109, 386–397.

Steeves, J., Fawcett, J., Tuszynski, M., 2004. Report of international clinical trials workshopon spinal cord injury February 20–21, 2004, Vancouver, Canada. Spinal Cord 42 (10),591–597.

Steeves, J.D., Lammertse, D., Curt, A., Fawcett, J.W., Tuszynski, M.H., Ditunno, J.F.,Ellaway, P.H., Fehlings, M.G., Guest, J.D., Kleitman, N., Bartlett, P.F., Blight, A.R., Dietz,V., Dobkin, B.H., Grossman, R., Short, D., Nakamura, M., Coleman, W.P., Gaviria, M.,Privat, A., International Campaign for Cures of Spinal Cord Injury, P., 2007. Guidelinesfor the conduct of clinical trials for spinal cord injury (SCI) as developed by the ICCPpanel: clinical trial outcome measures. Spinal Cord 45 (3), 206–221.

Steward, O., Popovich, P.G., Dietrich, W.D., Kleitman, N., 2012. Replication and repro-ducibility in spinal cord injury research. Exp. Neurol. 233 (2), 597–605.

Tator, C.H., 2006. Review of treatment trials in human spinal cord injury: issues, difficulties,and recommendations. Neurosurgery 59 (5), 957–982 (discussion 982–957).

Tator, C.H., Hashimoto, R., Raich, A., Norvell, D., Fehlings, M.G., Harrop, J.S., Guest, J.,Aarabi, B., Grossman, R.G., 2012. Translational potential of preclinical trials ofneuroprotection through pharmacotherapy for spinal cord injury. J. Neurosurg.Spine 17 (1 Suppl.), 157–229.

Tetzlaff, W., Okon, E.B., Karimi-Abdolrezaee, S., Hill, C.E., Sparling, J.S., Plemel, J.R.,Plunet, W.T., Tsai, E.C., Baptiste, D., Smithson, L.J., Kawaja, M.D., Fehlings, M.G.,Kwon, B.K., 2011. A systematic review of cellular transplantation therapies for spinalcord injury. J. Neurotrauma 28 (8), 1611–1682.

Tuszynski, M.H., Steeves, J.D., Fawcett, J.W., Lammertse, D., Kalichman, M., Rask, C.,Curt, A., Ditunno, J.F., Fehlings, M.G., Guest, J.D., Ellaway, P.H., Kleitman, N.,Bartlett, P.F., Blight, A.R., Dietz, V., Dobkin, B.H., Grossman, R., Privat, A.,International Campaign for Cures of Spinal Cord Injury, 2007. Guidelines for theconduct of clinical trials for spinal cord injury as developed by the ICCP Panel: clinicaltrial inclusion/exclusion criteria and ethics. Spinal Cord 45 (3), 222–231.