The FASEB Journal • Review

The potential of stem cell therapy for stroke: is PISCESthe sign?

Helen K. Smith and Felicity N. E. Gavins1

Wolfson Neuroscience Laboratories, Department of Medicine, Imperial College London, London, UK

ABSTRACT Substantial developments in the fieldof stem cell research point toward novel therapiesfor the treatment of diseases such as stroke. Thisreview covers the establishment of tissue damage instroke and the status of current therapies. We evalu-ate stem cell therapy with respect to other treat-ments, including clinical, preclinical, and failed, andprovide a comprehensive account of stem cell clinicaltrials for stroke therapy currently underway. Finally,we describe mechanisms through which stem cellsimprove outcome in experimental stroke as well aspotential pitfalls this basic research has identified.—Smith, H. K., and Gavins, F. N. E. The potential ofstem cell therapy for stroke: is PISCES the sign?FASEB J. 26, 2239–2252 (2012). www.fasebj.org

Key Words: ischemic stroke � cell transplantation � cytokines� in vivo imaging

In November 2010, a man in his 60s was the first toreceive a novel therapy to treat his severe and unyield-ing neurological deficits caused by a stroke 18 mopreviously. He was the first patient to be operated on inthe Pilot Investigation of Stem Cells in Stroke (PISCES).PISCES is a phase I clinical trial involving the injectionof fetal stem cells into infarcted brain regions of 12patients, 6-24 mo after ischemic stroke. Although pri-marily concerned with assessing the safety of ReNeuron’sReN001 neural stem cell line for clinical use, the trial isbased on accumulated evidence, which allows the ten-tative proposition that this treatment may be successful.This review will cover evidence that supports this andother promising trials, the mechanisms through whichstem cells appear to provide benefit, and the practicaladvantages and disadvantages of stem cell treatment forstroke in the clinic.

ISCHEMIC STROKE: ETIOLOGY ANDPREVALENCE

Stroke is the third leading cause of death in the UnitedKingdom, behind heart disease and cancer, and claims15 million lives worldwide each year, 70,000 of those inthe United Kingdom (1, 2). Despite this high mortality,76% of those having strokes survive, often with moder-ate or severe disabilities. The subsequent cost is feltwidely: the direct annual expenditure on stroke in theUnited Kingdom is estimated to be £4 billion (5.5% ofthe total UK expenditure on health care), and thecumulative costs (which include “indirect” care: special-ist care and income support) to be close to £9 billion(3). Treatment with “clot-busting” tissue plasminogenactivator (tPA) administered within 3 h of the onset ofischemia is currently the only accepted treatment forischemic stroke. The demand for alternative, moreflexible, and clinically relevant therapies is clear fromthese figures and is reflected in the enormous body ofwork targeting the zenith of stroke research—a sal-vaged penumbra.

The vast majority of strokes (85%) are ischemic innature and are characterized by the obstruction of majorvessels or arteriole ends, preventing or reducing bloodflow to the brain (4). The causes of the obstructions aremost commonly a thrombus or embolus from the heart,myocardial infarction, or trauma. Each of these will pro-duce an ischemic core that will become the focus of(currently) irreversible damage (Fig. 1). The core regionis encapsulated by cells whose survival is dependent onmultiple factors; this region is termed the penumbra, andthe mechanisms influencing its condition are discussed inthe next section.

MECHANISMS OF TISSUE DAMAGE INISCHEMIC STROKE

Pathological changes develop in stroke as reducedoxygen and variably reduced glucose supplies (withrespect to proximity to the ischemic core) result in

1 Correspondence: Wolfson Neuroscience Laboratories,Imperial College Faculty of Medicine, Hammersmith Hospi-tal Campus, Burlington Danes Bldg., Du Cane Road, LondonW12 0NN, UK. E-mail: f.gavins@imperial.ac.uk

doi: 10.1096/fj.11-195719

Abbreviations: Ang1, angiopoietin 1; BDNF, brain-derivedneurotrophic factor; CAM, cell adhesion molecule; GDNF,glial cell line-derived neurotrophic factor; GFP, green fluo-rescent protein; IGF, insulin-like growth factor; IL, interleu-kin; iPSC, induced pluripotent stem cell; I/R, ischemia/reperfusion; MCA, middle cerebral artery; MCAO, middlecerebral artery occlusion; MCP-1, monocyte chemotactic pro-tein-1; MI, myocardial infarction; MN, mononuclear; MSC,mesenchymal stem cell; NMDA, N-methyl-d-aspartate SDF1,stromal-derived factor 1; SGZ, subgranular zone; Shh, sonichedgehog; TGF-�, transforming growth factor �; tPA, tissueplasminogen activator; SVZ, subventricular zone

22390892-6638/12/0026-2239 © FASEB

necrotic and/or apoptotic cell death by a range ofmechanisms (5). In summary, disruption of neuronalion channels produces unmediated release of excit-atory glutamate (5, 6), leading to immense calciuminflux through N-methyl-d-aspartate (NMDA) receptorsand voltage-dependent calcium channels. In addition,

single-strand DNA breaks may cause rapid activation ofthe nuclear protein poly(ADP-ribose) polymerase, re-ducing intracellular concentrations of its substrate(NAD�), thereby slowing cellular mechanisms of en-ergy production (7, 8).

Early restoration of blood flow is crucial to save as much

Figure 1. Proposed sources and routes of administration for stem cell therapy in ischemic stroke. Also shown is the commonposition of human ischemic stroke (MCA) and the location of the penumbra with respect to the ischemic core.

2240 Vol. 26 June 2012 SMITH AND GAVINSThe FASEB Journal � www.fasebj.org

cerebral tissue as possible. Despite this, reperfusion itselfis thought to compound tissue damage and worsen pa-tient recovery, as demonstrated in several animal models(9–11). Ischemia/reperfusion (I/R) injury is character-ized by inflammatory processes: blood returning to aninfarcted brain region carries a flurry of polymorphonu-clear (PMN) and mononuclear (MN) leukocytes set torespond to gradients of chemotactic agents, such asinterleukin (IL)-8 and monocyte chemotactic protein-1(MCP-1). When they reach the microvasculature of anischemic region, these leukocytes extravasate into thebrain parenchyma in response to cell adhesion molecule(CAM) expression on vessel endothelia (the mechanismsof the inflammatory response are to be considered care-fully: CAMs will be referred to later in conjunction withstem cell navigation). Here leukocytes typically releasereactive oxygen species, matrix metalloproteinases(MMPs), and further inflammatory mediators. The de-ployment of such highly reactive components is primarilydesigned to tackle pathogens, and their presence inexcess during a sterile immune response destroys tissue.Inflammation can eventually be resolved through endoge-nous anti-inflammatory mediators, such as glucocorticoid-induced calcium and phospholipid-binding protein,annexin A1 (AnxA1), and eicosanoid-derived lipoxinA4 (LXA4). These promote nonphlogistic apoptosis andphagocytosis of leukocytes already within the tissue and therelease of anti-inflammatory cytokines, such as IL-6 andIL-10. Endogenous resolution of inflammation is now ac-cepted as an active rather than passive process, modulationof which may itself have therapeutic value (12, 13).

Throughout subsequent weeks and months, necroticcells are cleared and scar tissue forms. The peri-infarctcortex appears to provide a neurovascular recess intowhich new neurons and vasculature are encouraged togrow (14). Endogenous neurogenesis (see ProposedMechanisms of Functional Recovery after Stem Cell Ther-apy below) is increased as newborn cells migrate towardthe infarct, differentiate into a requisite phenotype, andassociate with remodeling vasculature [neurogenesis andangiogenesis are inextricably linked through productionof stromal-derived factor 1 (SDF1) and angiopoietin 1(Ang1) by the new blood vessels] (15). There is noevidence that this neurogenesis occurring at this stageprovides any functional improvement; many neurons inthe neurovascular recess either die or remain undifferen-tiated. The implication from cell transplantation studies isthat the benefit of these cells is their neuro/immuno-modulatory activity through paracrine mechanisms.

This dynamic stream of pathological events throughstroke suggests possible opportunities for interveningtherapeutically, increasing cell survival, and aiding re-covery from stroke. It is therefore reasonable to ask whyeffective therapeutic strategies actually in existence arenot correspondingly abundant.

CURRENT STROKE THERAPIES

More accurately, this heading should read “currentstroke therapy,” because success with the translation of

potential stroke therapies from laboratory to clinic hasso far been nonexistent, with the single exception oftPA. The good news is that the transforming patholog-ical processes throughout stroke and during subse-quent weeks, months, and years present several oppor-tunities for intervention as the disease progresses andalso suggest that the most effective treatment may be acombined therapeutic approach. Research to date hasobserved and tried to compensate for several of thepathological processes described in the previous sec-tion, and more than a thousand basic studies havereached clinical trial stage (16). A few key examples aretrials with NMDA antagonists (17) and GABA potenti-ators (18), designed to block initial excitotoxic celldeath; thrombin inhibitors, with the aim of secondaryprevention (19); and ICAM-1 inhibitors (20), targetingthe recruitment of leukocytes to endothelial walls dur-ing inflammation. As alluded to above, not one wassuccessful. The reasons for these failures have beencovered elsewhere in detail (21, 22), but the crux of theproblem seems to be that results obtained using animalmodels are not sufficiently representative of results inthe clinic, the necessary temporal restrictions on treat-ment administration in basic research do not practicallylend themselves to the clinic, and there is poor obser-vation of inclusion criteria outlined after animal stud-ies, once a drug has reached trial stage. The resoundingmessage from each of these concerns is that new studiesand therapies should be designed with flexibility inmind to provide the best outcomes for the greatestnumber of patients.

On these bases, how do studies show that stem cellsmight be effective in clinical trials? Do the data fromanimal experiments give a true indication of what can beexpected from trials? How does the prospect of stem celltherapy compare with previous failed attempts at a treat-ment for stroke—will it be more inclusive than thecurrent gold standard, tPA? The following sections con-tain evidence from the last decade, which addresses thesequestions and suggests how stem cells might figure infuture stroke therapies. In addition, Table 1 covers clini-cal trials currently putting this evidence into practice.

EVIDENCE FOR EFFECTIVE STEM CELLTHERAPY IN STROKE AND CARDIOVASCULARDISEASES

Funding for research involving stem cells from the NIH isexpected to reach $2.4 billion (of approximately $31billion available) in 2011 (23, 24). The California Institutefor Regenerative Medicine has recently awarded 14 re-search groups based in California $230 million in thehope that each will submit a new drug for phase I clinicaltrial within 4 yr (25). In the United Kingdom, funding forstem cell therapy over the last 4 yr has quadrupled, withprojected spending on stem cell research in 2011 at£72–88 million (26). There is confidence, therefore, thatfetal, embryonic, adult (including induced pluripotent),and nonhuman stem cells have great potential for use in

2241STEM CELL THERAPY FOR STROKE

TABLE 1. Current clinical trials involving stem cells as stroke therapy

Title of trial Inclusion criteria Intervention Outcome/status

Intravenous stem cellsafter ischemicstroke (ISIS)

Acute carotid ischemic stroke(�14 d); NIHSS 2–24; 18–85yr; M � F

Intravenous injection of autologousMSCs �6 wk after stroke

Recruiting(phase IIa)

Efficacy study of CD34stem cell in chronicstroke

Ischemic stroke in MCA territory(6–60 mo); NIHSS 9–20; 35–70y; M � F

Intracerebral implantation of 2–8 �106/patient autologous CD34� cells(hematopoietic progenitors), plusconventional stroke therapya

Completed(phase II)

Autologous bonemarrow stem cellsin middle cerebralartery acute stroketreatment

Acute ischemic stroke in MCAterritory (5–9 d); NIHSS �8;18–80 yr; M � F

Intra-arterial implantation of autologousCD34� cells via MCA

Completed

A study of allogeneicmesenchymal bonemarrow cells insubjects withischemic stroke

Ischemic stroke (�6 mo); NIHSS6–20; �18 yr; M � F

Intravenous injection of 0.5–1.5 � 106/kg autologous MSCs �6 wk afterstroke

Recruiting(phase I/II)

Autologous bonemarrow stem cellsin ischemic stroke

Acute total anterior circulationischemic stroke (�7 d); NIHSSsevere; 30–80 yr; M � F

Intra-arterial implantation of autologousCD34� cells via MCA

Unknown(phase I/II)

The variation ofmovement relatedcortical potential,cortico-corticalinhibition, andmotor evokedpotential inintracerebralimplantation ofautologousperipheral bloodstem cells (CD34)in old ischemicstroke

Ischemic stroke in MCA territory(0.5–5 yr); NIHSS 9–20; �30yr; M � F

Stem cell therapy plus granulocytecolony-stimulating factor (no furtherdetails available)

Recruiting

Pilot investigation ofstem cells in stroke

Unilateral ischemic stroke (0.5–5yr); NIHSS �6; 60–85 yr; Monly

Intracerebral injection of 2/5/10/20 �106/patient ReNeuron CTX0E03neural stem cells

Recruiting(phase I)

A study of modifiedstem cells in sableischemic stroke

Ischemic stroke in subcorticalregion of MCA orlenticulostriate artery (with orwithout cortical involvement;6–24 mo); NIHSS �7; 18–75yr; M � F

Intracerebral implantation of 2.5/5/10 �106/patient modified stromal cells(SB623)

Recruiting(phase I/IIa)

fMRI in monitoringintracerebral stemcell implantationfor chronic strokepatients

Ischemic stroke in MCA territory(0.5–3 yr); NIHSS 9–20; 35–75yr; M � F

Intracerebral implantation ofhematopoietic stem cells;remyelination, increased perfusion,and cortical activity in penumbraobserved using fMRI

Active, notrecruiting

Intravenousautologous bonemarrow-derivedstem cells therapyfor patients withacute ischemicstroke

Ischemic stroke (7–30 d);NIHSS�7; 18–80 yr; M � F

Intravenous injection of 30–500 � 106

autologous bone marrow-derivedmononuclear cells

Completed(phase II)

Study of purifiedumbilical cordblood CD34� stemcell on chronicischemic stroke

Ischemic stroke (6–60 mo);NIHSS 5–15; 35–70 yr; M � F

Intercerebral implantation of allogenicCD34� stem cells produced fromStemCyte umbilical cord blood

Not yet recruiting

The clinical trialresearch of stemcell transplantationtreats cerebralischemia

Hemorrhagic or ischemic stroke(�24 h); apoplexy and“obvious clinical symptoms”;40–65 yr; M � F

Intravenous injection of umbilical cordmesenchymal stem cells (d 10–21 afterhemorrhage and d 7–14 after ischemia);second transplant through lumbarpuncture 7 d after initial transplant

Recruiting(phase II)

(continued on next page)

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developing new therapies. The pluripotency and prolifer-ative nature of stem cells (reviewed extensively elsewhere;ref. 27) mean that much of this funding is focused on thedevelopment of techniques to tackle cardiovascular dis-ease and stroke, both conditions requiring compensationfor necrotic tissue. The potential in stem cell therapy isnot only large, but broad; several possible applications arebacked by accumulating evidence of their benefit in

humans. These include stimulation of endogenous stemcells, exogenous production of implantable structuresfrom host tissue, cell therapy through introduction of newstem cells into the host, and the provision of tissue fordrug development (which one presumes will provide asuperior alternative to the animal tissue it would replace).Evidence for the former three potential therapies isdiscussed below.

TABLE 1. (continued)

Title of trial Inclusion criteria Intervention Outcome/status

Autologous bonemarrow stromal celland endothelialprogenitor celltransplantation inischemic stroke

Ischemic stroke in MCA territory(7 d); NIHSS �7; 18–80 yr;M � F

2 times intravenous transplantation of2.5 � 106 bone marrow stromal cellsor endothelial progenitor cells/kg

Recruiting (phaseI/II)

Intravenousautologousmesenchymal stemcells transplantationto treat middlecerebral arteryinfarct

Ischemic stroke in MCA territory;NIHSS 10–30; 30–70 yr; M � F

Intravenous autologous bone marrow-derived mesenchymal stem cells

Recruiting(phase II)

Study to assess thesafety and effects ofautologous adipose-derived stromal cellsin patients afterstroke

Hemorrhagic or ischemic stroke;NIHSS �8; 18–80 yr; M � F

Intracarotid or intravenousadministration of autologous adipose-derived stromal cells (cells isolatedand delivered within 1 h)

Recruiting (phaseI/II)

Study to examine theeffects of MultiStemin ischemic stroke

Cortical ischemic stroke (1–2 d);“moderate to moderatelysevere”; 18–79 yr; M � F

Infusion of MultiStem Recruiting(phase II)

Safety/feasibility ofautologousmononuclear bonemarrow cells instroke patients

Ischemic stroke (24–72 h);NIHSS 6–15(R)/18(L); 18–80yr; M � F

Intravenous injection of autologousmononuclear bone marrow cells

Recruiting(phase I)

Ex vivo cultured adultallogenic MSCs inischemic cerebralstroke

Ischemic stroke (10 d); modifiedRankin scale �4; 20–80 yr;M � F

Intravenous injection of 2 ml/kgallogenic MSCs

Not yet recruiting(phase I/II)

Implantation ofolfactoryensheathing cells(OECs)

Ischemic stroke in MCA territory(6–60 mo); NIHSS 5–15; 35–70yr; M � F

Autologous OECs cultured beforetreatment; intracerebral implantationof 2–8 � 106/patient OECs

Recruiting(phase I)

Study of autologousstem celltransplantation forpatients withischemic stroke

Ischemic stroke in MCA territory(3–90 d); NIHSS 4–20; 18–75yr; M � F

Intra-arterial or intravenous injection of5006/patient autologous bone marrowstem cells

Completed

Study of ALD-401 viaintracarotidinfusion in ischemicstroke subjects

Ischemic stroke in MCA territory(11–17 d); NIHSS �4; 30–75yr; M � F

Intracarotid injection of ALD-401autologous bone marrow stem cells

Recruiting(phase II)

Study of humanplacenta-derivedcells (PDA001) toevaluate the safetyand effectiveness forpatients withischemic stroke

Ischemic stroke in MCA orposterior communicating arteryterritory (no info for recency);NIHSS 6–20; 18–80 yr; M � F

Intravenous injection of 2 lots of 2 or8 � 108/patient, or 1 lot of 2 � 106/patient PDA001 cells

Not yet recruiting(phase IIa)

All information collated from stem cell therapy for stroke trials registered on ClinicalTrials.gov. L, left; R, right; fMRI, functional MRI; M,male; F, female. Inclusion criteria were type of stroke (and days since occurrence of stroke), National Institutes of Health Stroke Score (NIHSS),age of participants, and sex of participants. aConventional therapy: antiplatelet treatment and management in a stroke facility.

2243STEM CELL THERAPY FOR STROKE

Many groups focus on applying appropriate growth/migration stimulants to cells already present in the hostthrough gene therapy. This is a reasonable approachthat is reviewed elsewhere in detail (28, 29), although apromising example is embryonic morphogen sonichedgehog (Shh), having been shown to increase vascu-logenesis in a mouse model of peripheral limb isch-emia, improving function (30). This is supported byadditional work suggesting a beneficial role for Shhafter ischemia (31–36).

The use of biological scaffolds and stem cells fororgan or tissue generation for implants is incrementallybecoming closer to reality (mesmerizing advancesrange from de-/recellularized livers to suitably ethereal“ghost hearts”; refs. 37, 38). On the assertion that thereis little chance that these technologies will apply tobrain regeneration en masse, at least for the time being,the focus in this section will be on cerebrovascular celltherapy.

The accumulation of evidence for the potential ofcell therapy in stroke has relied on the use of animalmodels of the disease. Animal models have providedhuge insights into the possibilities of stem cell therapy,although they are not without limitations. Fewer than10 models of focal stroke exist in animals (39), the mostcommonly used being middle cerebral artery occlusion(MCAO), because of its reproducibility and similarity tothe human condition (40, 41). Rodent studies fre-quently (although not always) use rats in preference tomice; the narrow apertures of the cerebrovasculature inmice render the rat model technically more amenable,but mice are used in genetic studies requiring, forexample, knockout animals. MCAO involves passing asuture up the internal carotid artery until the origin ofthe middle cerebral artery (MCA) is met (42). Thecirculation supplying cerebral tissue not supplied bythe MCA is fed by the circle of Willis throughout anischemic period (often 1 h in duration), while thesuture remains inserted. An infarct is produced, consis-tently and unilaterally, in the region of cortex suppliedby the occluded MCA. Predominantly using this model,researchers have investigated cell therapy in animalswith some key variables: the use of cells of exogenousvs. endogenous origin, the route of administration andcell type used (if an exogenous cell source; see Table 2for cell types), and the growth factors used in associa-tion (if an endogenous cell source).

Fetal neural stem cells are able to form new neuronsand improve behavioral outcomes in humans (43) andin animal models (44) after brain injury. Despite somepositive results, ethical issues shrouding work involvingfetal and embryonic stem cells (45), as well as regula-tory considerations for potentially tumorigenic embry-onic stem cells, have shifted the bulk of work toward theuse of alternatives (or finding them; see Fig. 1 forcurrent sources used for the generation of stem cells).The method of inducing pluripotency in human fibro-blasts (46, 47), the use of adult stem cells and themanipulation of endogenous stem cells are popularalternatives (summarized in Table 2).

It has recently been discovered that by reprogram-ming fibroblasts with a combination of 4 transcriptionfactors [either OCT4, SOX2, c-MYC, and KLF4 (46) orOCT4, SOX2, NANOG, and LIN28 (48)], it is possibleto induce pluripotency in cells with no requirement forfetal or embryonic tissue. Induced pluripotent stemcells (iPSCs) have recently been used in animal studiesas stroke treatments. Jiang et al. (49) transfected humanfibroblasts with the latter combination of transcriptionfactors and injected them into the into the contralat-eral and ipsilateral peri-infarct regions of female ratsafter MCAO. The group discovered that these cellsmigrated to injured areas of the brain and showedsome differentiation into neuron-like cells. The lesionvolumes were reduced significantly, and, crucially, re-covery of sensorimotor function was twice as successfulin iPSC treatment groups vs. controls. These resultswere reflected by earlier work from Chen et al. (50),who found that implants promoted neural differentia-tion as well as immunomodulation. Kawai et al. (51)found that implantation of iPSCs after MCAO in miceresulted in tumorigenesis; the observed simultaneousincrease in dividing neuroblasts led the researchers toconclude that the therapy still has potential, should thetumorigenesis be controlled. However, to date therehave been no studies using neural precursors originat-ing from iPSCs that definitively overcome such tumor-igenic potential, which limits their therapeutic applica-tion.

The ethical and immunological implications of theuse of autologous, adult stem cells has made suitablesources highly sought after. Adult bone marrow stromalcells contain mesenchymal stem cells (MSCs), whichmay be able to differentiate into neural cells (52, 53). Ithas been observed that beyond characteristic differen-tiation into bone cartilage and smooth and skeletalmuscle, MSCs are able to transdifferentiate into skin,liver, and brain cells (including neurons and glia), as,conversely, neural cells may transdifferentiate intoblood cells (54). (The potential of hematopoietic bonemarrow-derived cells is broad enough that they may beof additional therapeutic use in the treatment of vascu-lar diseases, which would be of benefit in stroke pre-vention; ref. 55). MSCs grafted via stereotactic intrace-rebral injections into brains after MCAO reducedinfarct size and improved somatosensory function inrats (56, 57). Transdifferentiation (also referred to as“plasticity”) of MSCs and hematopoietic stem cells toneural precursors as a mechanism for observed recov-ery after MSC transplantation in experimental stroke isregarded as a possibility by some authors. The conceptis, however, largely contentious: it seems more likelythat the “bystander effect ” (see below), through whichstem cells exert various neuroprotective functions, isthe mechanism through which MSCs provide benefit.In this rat model, recovery was extended to improvemotor function when the stem cells were preincubatedwith nerve growth factor or previously transfected withthe brain-derived neurotrophic factor (BDNF) gene.Other groups have also shown that cytokines, including

2244 Vol. 26 June 2012 SMITH AND GAVINSThe FASEB Journal � www.fasebj.org

TABLE 2. Current clinical trials involving stem cells as stroke therapy

Cell type/sourcePotential for

differentiation Relative benefits/problems with useUse in experimental

strokeUse in human

trials

Embryonic/blastocysts(4–5 d afterfertilization)

Pluripotent (able toproduce neuronalprogenitors); mostundifferentiatedstem cells availablefor potential clinicaluse

Ethically controversial; potentiallytumorigenic—canpredifferentiate in vitro toreduce tumor risk; others findpredifferentiation makes nodifference with homologoustransplantation (vs.xenotransplantation; ref. 101)

Hemorrhagic (102)and ischemic(103); bothshowed improvedfunctionaloutcome

No use to datein stroketrials; onephase I trialin spinalcord injury

Fetal Pluripotent (able toproduce neuronalprogenitors)

Safety profile superior toembryonic cells; must beobtained from human fetaltissue therefore limited supply;cell lines overcome the latter(e.g., ReNeuron ReN001)

Hemorrhagic(104)and ischemic(44); bothshowed improvedfunctionaloutcome

One phase Iclinical trial(so farsuccessfullycompleted);ischemicstroke

Mesenchymal(stromalcells)/bonemarrow; autologous

Multipotent;osteoblasts,adipocytes,chondrocytes, andmyocytes; alsoproduce neuron-likecells (possiblynonfunctionaltherapeutically)

Autologous therefore free ofimmunological and ethicalissues; mechanism of actionambiguous: are likely toprovide support throughneurotrophic support,immunomodulation andangiogenesis rather than directreplacement of neurons;retrieval from bone marrow(stromal)/adipose tissue(through liposuction andreadministered in just 1 h(MultiStem trial)/extractedfrom teeth (several sources)during dental therapies/fromdeciduous teeth; dental cellsvery easily retrieved; adiposetissue may provide morefavorable source than bonemarrow as safer and easieraccess (110)

Hemorrhagic (105)and ischemic(106, 107); bothshowed improvedfunctionaloutcome

Several phaseI/II safetytrials (inprogress orcompleted;see Table 1);ischemicstroke

Mesenchymal-like/adipose tissue;autologous

Multipotent;osteoblasts,adipocytes,chondrocytes andmyocytes,endothelium,hematopoietic cells,hepatocytes andneuronal cells

Hemorrhagic (108)and ischemic(109); bothshowed improvedfunctionaloutcome

One phaseI/II(recruiting);hemorrhagicandischemicstroke

Mesenchymal-like/dental tissue (111);autologous

Multipotent;osteoblasts,adipocytes,chondrocytes; alsoproduce neural-likecells (possiblynon-functional)

Ischemic (112);showed improvedfunctionaloutcome

None

Mesenchymal-like/hematopoietic/bone marrow/umbilical cord blood;autologous

Multipotent; umbilicalcord blood mostundifferentiatedMSCs available forclinical use

Autologous therefore free ofimmunological and ethicalissues; may be retrieved non-invasively from umbilical; cordblood

Hemorrhagic (113)and ischemic(114); bothshowed improvedoutcome

One phaseI/II(recruiting),cells frombonemarrow; onephase I(recruiting);cells fromumbilicalcord bloodtransplantedin ischemicstroke

Induced pluripotent/skin (predominantlyfibroblasts)

Pluripotent Autologous therefore free ofimmunological and ethicalissues; less costly than othertypes; however, low replicationrates and early senescenceimpede viability; tumorigenicpotential (103)

Three rodentstudies of usingMCAO (ischemicstroke; refs. 49–51); all improvedoutcome; onedemonstratedteratomaformation (51)

None; currentiPSC trialsinvolve theirproductionfrom humantissue only

Induced pluripotent/oral gingiva

2245STEM CELL THERAPY FOR STROKE

BDNF (58) and glial cell line-derived neurotrophicfactor (GDNF; ref. 59), and peptide survivin (60),improve functional recovery when transfected intoMSCs before implantation. Having established thatthese cells are able to cross the blood-brain barrier(61), studies were conducted using intravenous admin-istration of MSCs (62, 63). Improved motor functionwas comparable to that observed after intracerebraladministration, but recovery in somatosensory functionwas �3 times less effective (�90% improvement inintracerebral groups vs. 25% in intravenous groups). Ina study comparing intravenous and intracarotid admin-istration of MSCs, increased cell proliferation andnumber of blood vessels and decreased cell death andimproved behavioral outcomes were all recorded,whereas there was no significant reduction in infarctvolume (64). In terms of translation of research intoclinical applications, close attention should be paid tostudies comparing intravenous/intracarotid entryroutes for stem cells rather than a direct injection intothe brain. Although such studies frequently favor arte-rial administration (65), any benefit of cell therapy inhuman stroke is dependent on the practical viability ofthe route of delivery vs. efficacy (Fig. 1).

Although reduced infarct size seems to be indicativeof improved recovery from experimentally imposedischemic episodes, the mechanisms through whichstem cell therapy aids this improvement are not entirelyclear. These are discussed in the next section.

PROPOSED MECHANISMS OF FUNCTIONALRECOVERY AFTER STEM CELL THERAPY

Subsequent to an injury, the body is inclined to scar,have the phagocytes pick up the pieces, and move on.This is the best method for avoiding infection, but notfor instances in which rescuing tissue is vital to theproper functioning of the body. After the emergencyclear-up after cerebral ischemia (and in apparent rec-ognition of the brain as a vital organ), the body initiatesits own repair strategy: neurogenesis in the adult brainis regulated by pathological as well as physiologicalevents. The subgranular zone (SGZ) of the dentategyrus and the rostral subventricular zone (SVZ) of thelateral ventricles each contain progenitor cells that area perpetual source of new neurons. Neural progenitorsfrom the SVZ are destined for the olfactory bulb,whereas those of the SGZ migrate into the neighboringgranule layer of the dentate gyrus.

Several studies have shown that in cerebral infarctsthere is increased cellular proliferation (66, 67) andthat increasing this proliferation [using surprisingpharmacological approaches, including antidepres-sants (68), estrogen (69), and sildenafil (Viagra; ref.70)] aids recovery. In addition to neurogenesis, addi-tional means through which the brain may be able torepair itself include synaptogenesis and axonal branch-ing (71), adapting the use of existing neuronal circuits(without the generation of new neurons or the repair

of those injured; ref. 72), and “protective inflamma-tion” (protective function of MN cells; ref. 73).

Working to enhance endogenous activity of the bodyto mend it is not a new concept. Nevertheless, on thebasis that the body is frequently so adept at repairingitself, it is probably a sensible one. (For instance, incountering the excessive immune response in I/Rinjury after stroke, promising work aims to boost en-dogenous anti-inflammatory mechanisms rather thandirectly antagonize inflammation; refs, 9, 21). Theroute of neural progenitors from the SVZ is meticu-lously planned, so the question is, can we promotemovement of these cells or injected cells to regions theyare needed in after cell death? If not, or in addition tothis, what are the properties of stem cells that haveproduced such promising preclinical results?

The premise behind the initial studies into thetherapeutic potential of stem cells was their supposedability to replace lost neuronal circuits. Further workhas shown that although this is partly the case, thefunctional recovery associated with stem cell implanta-tion does not correlate with the relatively minimalrewiring that evolves from exogenously introducedcells. In fact, the majority of recovery seen experimen-tally with stem cells is attributed to angiogenesis andgrowth factor secretion, and the benefit this affords toendogenous cells (Fig. 2). This was neatly demonstratedby Loffredo et al. (74) in work that strongly suggests thisis true of the infarcted heart: the group used femalemice engineered to express green fluorescent protein(GFP) in cardiomyocytes after a pulse of 4-hydroxyta-moxifen. After receiving a pulse, mice were subjected tomyocardial infarction (MI) and then were transplantedwith c-kit expressing bone marrow-derived progenitors(or control cells) from male mouse donors. After MI,the population of GFP-positive cells was diluted byendogenously derived cardiomyocytes and to a muchgreater extent in groups receiving c-kit-positive cells.Although the generation of new neural networks in thisway would be ideal, this is far less likely to be practicalin the brain, where the intricacies of specific intercel-lular connections are more complex than those of theheart. The bystander effect is a mechanism throughwhich transplanted cells aid tissue recovery, without thedirect replacement of neurons; cells promote “thera-peutic plasticity” (75), the functional compensation fora lesion, which is widely substantiated experimentally inseveral neurological diseases, including stroke (76).The bystander effect provides various means throughwhich transplanted cells are able to deliver neuropro-tective functions, including neurotrophic support, im-munomodulation, and angiogenesis. The effect wasinitially described as a feature of neural stem cells buthas since been used to explain the therapeutic effectexerted in brain disorders by other stem cells (thosewith a very low capacity for neural transdifferentiation;refs. 77, 78). The bystander effect of MSCs in strokeformed the basis for a phase I clinical trial in which asingle intravenous infusion of autologous MSCs wasadministered to a cohort of 12 patients during the

2246 Vol. 26 June 2012 SMITH AND GAVINSThe FASEB Journal � www.fasebj.org

subacute or chronic phase of stroke (36–133 d afterstroke onset; ref. 79). Although this is a small sample,modest amounts of efficacy were observed in one-thirdof patients; this result, notably, correlated inversely withthe time interval between stroke onset and treatment.

Fundamental to healthy brain function is a sufficientblood supply through extensive vascularization. Theup-regulation of vascular growth factors seen in strokemodels (particularly once applied with a range ofpotential stem cell therapies) therefore provides one ofthe best explanations for improved outcomes. Morespecifically, a neurogenic/vasculogenic region formsclose to the infarct (15). Formation of this region alongwith mediation is both an endogenous artifact and asignificant means through which stem cells promoterecovery.

For this to occur, cells have to arrive at the correctlocation. Chemokine-driven migration enables stemcells to gather in the peri-infarct region (preventedfrom entering the ischemic core, possibly as a result ofanoxia-induced tissue changes, such as extracellularacidosis; ref. 80), where they contribute to a regenera-tive environment (Fig. 2). Evidence indicates that localinflammation attracts stem cells to the infarct (81, 82).Paradoxically, immunosuppression promotes migra-tion and survival of endogenous stem cells (83, 84).The neurogenic/vasculogenic region holds an assort-ment of cytokines, including BDNF, GDNF, SDF1,Ang1, and vascular endothelial growth factor (VEGF)(34–36, 58). Other factors implicated are basic fibro-blast growth factor (bFGF; ref. 62), transforminggrowth factor � (TGF-�; ref. 44), insulin-like growthfactor (IGF), and epidermal growth factor (EGF; ref.63). In the studies in which up-regulation of thesefactors has been observed, transplanted cells have im-proved, provided, or induced the means for thesefactors to occur; interestingly, one group showed thatafter human MSC transplantation into a postischemic

rat, IGF-I was the only human-derived cytokine de-tected in the core and ischemic border zone 3 d afterMCAO. The subsequent up-regulation of growth fac-tors of rat origin (which were increased compared withcontrols) suggests an inductive role for IGF in thismodel (63). It is also likely that transplanted cellsimprove the inflammatory status of the infarct, that is,enhance neuroprotection, again by changing the na-ture of the ischemic region. I/R injury in the acutestages of stroke (see Mechanisms of Tissue Damage inIschemic Stroke above) develops through a combina-tion of processes that stem cells appear to mediate tosome extent. Lui et al. (85) showed that transplantedMSCs after MCAO in rats caused increased expressionof anti-inflammatory cytokine IL-10 and decreased ex-pression of (generally) proinflammatory tumor necro-sis factor � (TNF-�). Another group show increasedmicroglia/macrophage activation after MSC transplan-tation in the same model (44).

If the therapeutic benefit of stem cells relies on theirpurported ability to induce growth factors, mediateinflammation, and possibly proliferate, what are poten-tial difficulties with their use in humans? The risk ofoncogenesis where any dividing cells (or promotion ofcell division) are concerned is well established, but thisis not the only factor to be considered.

PITFALLS AND PROVISOS

Concerns with stem cells include ethical difficultieswhen dealing with embryonic or fetal tissue, potentialoncogenesis or teratogenesis, and cytokine-relatedcomplications. How they will be traced in humans and(rather importantly) whether they will work are addi-tional concerns.

The increasing use of autologous MSCs and thepotential of iPSCs alleviate ethical pressure to reduce or

Figure 2. Stem cell migration toward infarct andsubsequent mechanisms of brain recovery. Pur-ple arrows indicate sources and routes taken bystem cells (SCs). SDF1, stromal cell-derivedgrowth factor 1; HGF, hepatocyte growth factor;MCP-1, monocyte chemotactic protein 1; MIP-1�, macrophage inflammatory protein 1�;FGF2, fibroblast growth factor 2; EGF, epider-mal growth factor; HSP27, heat shock protein27; COX2, cyclooxygenase 2; ICV, intracerebro-ventricular.

2247STEM CELL THERAPY FOR STROKE

prevent the use of embryonic or fetal stem cells. This isa desirable transition, and there may be even moreadvantage in it than first appears: adult stem cells seemto have less oncogenic potential than fetal or embry-onic stem cells (86). Possible links between oncogenesisand stem cell therapy have been reviewed elsewhere indetail (87); therefore, only two examples reflectingconcerns for malignancy of stem cells in humans will begiven here as caveats. Each has been mentioned inprevious sections for positive preclinical results. Thefirst refers to the reported ability of Shh to increasevasculogenesis and functional outcome in various mod-els of ischemia (30–33). Zhao et al. (88) indicated thatthe hedgehog pathway is responsible for maintenanceand proliferation of stem cells of chronic myeloidleukemia; notably, they demonstrated that ablatingSmoothened (a gene from the pathway), increasessusceptibility to the disease. Second, signaling throughTGF-� (one of the cytokines responsible for maintain-ing the neurogenic/vasculogenic environment suitablefor tissue renewal in the brain after injury) has a keyrole in the epithelial-mesenchymal-like differentiationinvolved in the development of breast cancer (89).

There are possible conflicting interests between stemcell and anti-inflammatory stroke therapies. MCP-1 andmacrophage inflammatory protein 1� attract both (po-tentially therapeutic) stem cells (82) and (potentiallydamaging) leukocytes (90) to areas of damage. Re-solvins are eicosanoid-derived mediators of anti-inflam-mation, which is partly effective through down-regula-tion of endothelial VCAM-1 (91); yet neural precursorcells demonstrate spontaneous adhesion enabledthrough expression the �4 subunit of VLA-4 (65, 92),the ligand for VCAM-1. The partial reliance of stemcells on inflammatory markers for successful migrationmust be considered by groups pursuing anti-inflamma-tory and cell-based stroke therapies: might the optimalconditions for one form of therapy oppose those of theother?

Irrespective of stem cell origin or mode of entry,tracking cells in vivo through clinical trials will benecessary. Tracking a relatively small number of cells,each essentially transplanted as a separate entity, isgoing to be difficult. In basic research, it is possible toobserve cell locations effectively using bioluminescence(93), which requires bone marrow from a transgenic,luciferase-expressing mouse; stumbling blocks for thisexact method to be applied in the clinic are ratherobvious. In addition, although in theory donor cellscould be genetically modified ex vivo to express lu-ciferase, the enzymes generate only visible light (400–700 nm); this has very high absorption/scatter in vivo,which is highly impractical for visualizing deep cellpenetration in humans.

More viable options involve the use of magneticresonance imaging (MRI; refs. 94, 95) and (less com-monly) positron emission tomography (PET; ref. 96).Contrast agents must be nontoxic and allow for thedetection of a single cell; they must not alter thegenetics of the stem cell or become too dilute as cells

divide, and they would preferably enable repeatedviewing of a patient without repeated administration(97). The scanners themselves pose other technicaldifficulties; e.g., with PET, there may be uptake of thetracer by nonspecific tissue, and patients with pacemak-ers and implantable devices cannot undergo MRI (un-doubtedly a problem considering the cohort of patientslikely to be in receipt of stem cell therapy for stroke).Tamaki et al. (98) have used a different approach. Thegroup marked human CNS stem cells with reportergenes (enhanced GFP), introduced by lentiviral vec-tors. Using fluorescence and antibodies for GFP, theywere able to identify migration and differentiation ofthe stem cells in a mouse model.

Finally, the success with cell therapy is not universal.Parr et al. (99) showed that MSCs and neural stem/progenitor cells (NSPCs) implanted in injured ratspinal cord caused no recovery of function and noneural differentiation (although about half of the trans-planted NSPCs develop into astrocytes). The research-ers suggest this could be the result of standard lesionsizes, which were impossibly large for the therapy to beof benefit. In addition, it has been shown elsewherethat stem cells fail to differentiate into neurons whentransplanted into spinal cord, which is, of course, notthe brain. Regardless, the situation is a reminder thatnegative results provide important information to beconsidered when clinical trials are designed.

CONCLUSIONS

The pathophysiological process of stroke involves tem-porally executed interactions between numerous celltypes, including neurons themselves, glia, endothelia,endogenous progenitors and cells of the immune sys-tem. A combination of therapies is ultimately likely tobe of greatest benefit in stroke patients: clot dispersion,followed by mediation of a subsequent inflammatoryresponse and neurogenesis/vasculogenesis, backed bysolid imaging.

Before a cell-based therapy is set for translation, it isessential to have information on each aspect of thepathogenesis, in particular how the endogenous CAMsand cytokines expressed during inflammation may beinvolved in stem cells homing in on and dividing theappropriate region. It is important to identify molecu-lar pathways responsible for directing stem cells toareas of ischemia and to learn how the cells, once in thebrain parenchyma, may release factors that interferewith the immune response. The preferred and mostfeasible source of the cells, whether or not cells shouldbe genetically modified, and best route of administra-tion into the patient must all be considered, as well asthe optimal time (should one exist) to administer celltherapy.

The use of stem cells as a treatment has beenwidespread since the early 1970s, when bone marrowgrafts containing hematopoietic stem cells began to beused for management of acute myeloid leukemia (100).

2248 Vol. 26 June 2012 SMITH AND GAVINSThe FASEB Journal � www.fasebj.org

Their use as treatment for brain damage after stroke toaccommodate for the losses in neuronal tissue is new,and efficacy has yet to be proven in humans. Despitethe minimal increased development of new neuronsdirectly arising from exogenous pluripotent cells, vas-culogenesis and the production of a tissue-regenerativemicroenvironment around an infarct suggest a positivefuture for stem cells as a therapy for stroke. Thepossibility that this treatment may not be so temporallyrestrictive as tPA is highly encouraging and could tosome extent provoke a shift in the dogma surroundinginfarcted neurons after stroke: what’s gone is notnecessarily gone.

F.G. is funded by the Biotechnology and Biological Sci-ences Research Council (BBSRC). H.K.S. is funded by aBBSRC Integrative Mammalian Biology Fund studentship.

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Received for publication October 4, 2011.Accepted for publication February 28, 2012.

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