Reparative Therapy for Acute

Ischemic Stroke with AllogeneicMesenchymal Stem Cells from Adipose Tissue:

A Safety AssessmentA Phase II Randomized, Double-blind, Placebo-controlled,

Single-center, Pilot Clinical Trial

Exuperio D�ıez-Tejedor, MD, PhD, FAHA, FESO, Mar�ıa Guti�errez-Fern�andez, BS, PhD,

Patricia Mart�ınez-S�anchez, MD, PhD, Berta Rodr�ıguez-Frutos, BS, PhD,

Gerardo Ruiz-Ares, MD, PhD, Manuel Lara Lara, MD,

and Blanca Fuentes Gimeno, MD, PhD

From the Stroke Ce

University Hospital, IdiP

Aut�onoma de Madrid, M

Received February 5, 20

June 15, 2014.

This study was suppo

Policy and Equality (EC1

2694

Background: Few studies have evaluated the possible beneficial effect of the admin-

istration of stem cells in the early stages of stroke. Intravenous administration of allo-

geneic mesenchymal stem cells (MSCs) from adipose tissue in patients with acute

stroke could be a safe therapy for promoting neurovascular unit repair, consequently

supporting better functional recovery. We aim to assess the safety and efficacy of

MSC administration and evaluate its potential as a treatment for cerebral protection

and repair. Materials: A Phase IIa, prospective, randomized, double-blind, placebo-

controlled, single-center, pilot clinical trial. Twenty patients presenting acute ischemic

strokewill be randomized in a 1:1 proportion to treatment with allogeneicMSCs from

adipose tissue or to placebo (or vehicle) administered as a single intravenous dose

within the first 2 weeks after the onset of stroke symptoms. The patients will be fol-

lowed up for 2 years. Primary outcomes for safety analysis: adverse events (AEs)

and serious AEs; neurologic and systemic complications, and tumor development.

Secondary outcomes for efficacy analysis: modified Rankin Scale; NIHSS; infarct

size; and biochemical markers of brain repair (vascular endothelial growth factor,

brain-derived neurotrophic factor, and matrix metalloproteinases 9). Results and

Conclusions: To our knowledge, this is the first, phase II, pilot clinical trial to investi-

gate the safety and efficacy of intravenous administration of allogeneic MSCs from

adipose tissue within the first 2 weeks of stroke. In addition, its results will help us

define the best criteria for a future phase III study. Key Words: Acute ischemic

stroke—stem cells—reparative therapy—clinical trial—safety.

� 2014 by National Stroke Association

Introduction

Cerebrovascular disease is a serious public health prob-

lem. In Spain, it is the leading cause of mortality in

nter, Department of Neurology, La Paz

AZ Health Research Institute, Universidad

adrid, Spain.

14; revision receivedMay 12, 2014; accepted

rted by Spanish Ministry of Health, Social

0-171).

Journal of Stroke and Cerebrovascular Diseases

women and the third in men and the estimated annual

incidence of stroke is 187 cases per 100,000 individuals.1

It also represents the greatest cause of disability.

Address correspondence to Exuperio D�ıez-Tejedor, MD, PhD,

FAHA, FESO, Department of Neurology and Stroke Center, La Paz

University Hospital, IdiPAZ Health Research Institute, Paseo de la

Castellana 261, Madrid 28046, Spain. E-mail: exuperio.diez@salud.

madrid.org.

1052-3057/$ - see front matter

� 2014 by National Stroke Association

http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2014.06.011

, Vol. 23, No. 10 (November-December), 2014: pp 2694-2700

REPARATIVE THERAPY FOR ACUTE ISCHEMIC STROKE 2695

Therefore, strategies for prevention and treatment of

stroke need to be improved to reduce its incidence and

avoid potential sequelae.

Brain repair is a natural process that is activated after

injury and that involves neurogenesis, gliogenesis, oligo-

dendrogenesis, synaptogenesis, and angiogenesis. These

inter-related processes could provide new therapeutic

tools to mitigate the damage caused by stroke.2 The pro-

motion of brain plasticity can be achieved by stimulating

endogenous mechanisms through the use of rehabilitation

and trophic factors or by exogenousmechanisms using cell

therapy to promote tissue repair and functional recovery.

Stem cells are immature cells with the capacity to differ-

entiate into multiple cell types. Various stem cell lines

have been tested in neurologic diseases, and preclinical

data regarding mesenchymal stem cells (MSCs) suggest

that strategies based on these cells may become effective

and safe therapies for a large number of diseases associ-

ated with inflammation and/or tissue damage.3 Progress

in this field has opened a new and promising avenue in

the search for new therapeutic strategies for stroke: the

enhancement of brain plasticity, a therapy that would

complement the current treatment of acute cerebral

infarction.

At present, designing new clinical trials that demon-

strate efficacy is complicated by a number of ethical, tech-

nical, and medical issues,4-6 some of which are related to

discovering a more suitable cell type, route, and time of

administration. In terms of cell type, the safer cells for

use would be autologous because the risk of rejection is

eliminated. However, this therapy’s primary limitation

is that it can only be applied several weeks after the

stroke because it takes time to cultivate the cells. The

administration of allogeneic stem cells could be a good

option because these cells demonstrate a lack of

HLA-class II antigens,7 thus avoiding the risk of rejection.

An important point regarding translational research is

that the optimal route of administration remains unclear.

For the transition to clinical practice, less invasive

methods such as intravenous administration would be

preferable. Several studies have shown that MSCs do

not need to migrate and nest at the injury site to achieve

good functional recovery, which could be related to the

stimulation or secretion of trophic factors.8,9

Another issue for investigation is the proper time win-

dow. Most of the studies using animal models have been

conducted in the later stages after stroke.10 However, in

our opinion, stem cells should be administered during

the acute phase to help inhibit the first steps of the

ischemic cascade and to enhance the mechanisms of pro-

tection and brain repair.

MSC transplantation has been tested on animal models

for the treatment of various neurologic diseases, including

cerebral infarction,11,12 and has produced good results in

terms of structural and functional recovery.10,11,13 The use

of MSCs from adipose tissue has recently gained more

interest because these cells are easier to obtain than

MSCs from bone marrow and are also effective in stroke

models.14,15 With respect to the intravenous

administration of allogeneic MSCs from adipose tissue,

our group has conducted a study on cerebral infarction

in rats in which these allogeneic MSCs were compared

with other bone marrow-derived MSCs. The results

showed that both cell types have similar efficacy in func-

tional recovery with reduced brain damage (cell death)

and increases in cellular proliferation, neurogenesis, oligo-

dendrogenesis, synaptogenesis, and angiogenesis markers

at 14 days after stroke.15

From a clinical standpoint, these cells have been suc-

cessfully transplanted into the brains of stroke patients,

with excellent tolerability and without complications. A

clinical trial that evaluated the safety of the procedure

and the tolerance of the stereotactic implantation of autol-

ogous bone marrow-derived MSCs in 5 patients with

cerebral infarction showed no significant adverse effects

during the 1-year follow-up. Improvements were noted

in the patients’ neurologic condition, but conclusive re-

sults could not be obtained given the small sample

size.16 Regarding other noninvasive routes of administra-

tion, a clinical trial of intravenous administration of autol-

ogous MSCs in 5 patients with severe stroke showed this

treatment to be feasible and safe, with improved neuro-

logic recovery.17 Another study evaluated the long-term

safety and efficacy of intravenous transplantation of

MSCs in 85 patients with cerebral infarction. In this

case, the therapy was found to be safe and recovery

improved, which was correlated with serum levels of

stromal cell-derived factor 1 (SDF-1 alpha).18

There are currently 31 registered worldwide clinical

trials on stem cell therapy for cerebral infarct, using a

variety of cell types, with most of the studies focusing

on the later stages of stroke recovery (between 1 month

and 5 years).19

Thus, few studies to date have evaluated the possible

beneficial effect of MSC administration in the early stages

of stroke, and it would be useful to evaluate its efficacy in

stimulating brain plasticity in this phase. As mentioned

previously, our research group has developed preclinical

studies with positive results, both in terms of safety and

efficacy, in an animal model of cerebral ischemia using

MSCs from bone marrow and adipose tissue.15 For the

transition to clinical practice, we propose the use of

allogeneic MSCs from adipose tissue in acute cerebral

infarction, given that this type of MSC could have a signif-

icant scientific and health impact because they are easier

to obtain than from other sources such as bone marrow.

Regarding safety, prior studies have shown that the intra-

venous infusion of autologous MSCs is feasible and safe,

and results have suggested an improvement in neurologic

recovery in patients with severe stroke. The use of alloge-

neic stem cells could be an alternative because of the

demonstrated lack of HLA-class II expression in

E. D�ıEZ-TEJEDOR ET AL.2696

mesenchymal cells, which means no allergic reactions

neither than immunologic response nor rejection would

be expected. This therapy has not been associated with

cancer development; however, to our knowledge, no

studies are available on the safety of the intravenous

administration of allogeneic MSCs from adipose tissue

in patients within the first days after a stroke.

Hypothesis

The intravenous administration of allogeneic MSCs

from adipose tissue in patients with acute stroke is a

safe therapy that will result in the stimulation of neuro-

genesis, gliogenesis, oligodendrogenesis, synaptogenesis,

and angiogenesis in the injured area, promoting the repair

of the neurovascular unit and repairing cortical and

subcortical brain damage.

Therefore, the administration of MSCs in the acute

phase of stroke, along with the conventional treatment

of cerebral infarction, will significantly improve patient

recovery in the early stages of stroke by repairing

ischemic brain tissue.

Methods

Design

A Phase IIa, pilot, single-center, prospective, random-

ized, double-blind, placebo-controlled clinical trial.

Patients will be included sequentially, and a safety analysis

will be conducted after the inclusion of every 3 patients.

Patient Population: Inclusion and Exclusion Criteria

Patients presenting an acute ischemic stroke within

24 hours of onset.

Inclusion Criteria

(1) Male and female acute ischemic patients,

60-80 years of age, with symptoms of acute cere-

bral infarction less than 12 hours from stroke

onset. If the time of symptom onset is unknown,

this shall refer to the last time the patient was

observed as asymptomatic.

(2) Patients should have been treated within 2 weeks

from the onset of stroke symptoms.

(3) Patients with a measurable focal neurologic deficit

that persists to the time of treatment without

clinically meaningful improvement.

(4) A computed tomography (CT) and/or magnetic

resonance imaging (MRI) scan compatible with

the clinical diagnosis of acute ischemic stroke in

the region of the middle cerebral artery.

(5) A score on the National Institute of Health Stroke

Scale (NIHSS) of 8-20, with at least two of these

points in sections 5 and 6 (motor deficit) at the

time of inclusion.

(6) A prestroke score on the Modified Rankin Scale

(mRS) #1 (no symptoms at all or no significant

disability despite symptoms and able to perform

everyday tasks and activities).

(7) A negative pregnancy test for women of child-

bearing age.

(8) Signed informed consent (after a detailed explana-

tion of the nature and purpose of this study, the

patient or their guardian or legal representative

must give their consent to participate by signing

the informed consent document); or consent from

a relative or caregiver if the patient is unable to pro-

vide meaningful consent (eg, in cases of dysphasia,

confusion, or reduced level of consciousness).

Exclusion Criteria

(1) Comatose patients; patients with a score of 2 or

more on item 1a of the NIHSS related to the degree

of awareness.

(2) Evidence on neuroimaging (CT or MRI) of a brain

tumor, cerebral edema with midline shift and a

clinically significant compression of ventricles, cere-

bellar or brainstem infarction, and intraventricular,

intracerebral, or subarachnoid hemorrhage.

(3) Current drug or alcohol use or dependence that, in

the opinion of the site investigator, would interfere

with adherence to the study requirements.

(4) Active infectious disease, including human immu-

nodeficiency virus, hepatitis B, and hepatitis C.

(5) Pre-existing dementia.

(6) A health status, any clinical condition (eg, short

life expectancy, and coexisting disease) or other

characteristic that precludes appropriate diag-

nosis, treatment, or follow-up in the trial.

(7) Patients who are participating in another clinical

trial.

(8) Inability or unwillingness of the individual or

their legal guardian/representative to provide

written informed consent.

Randomization

Each patient will be sequentially assigned a number as

they enter the study. The patients will be assigned the

study drug according to a randomization schedule based

on the randomization plan. Randomization will be simple

rather than stratified. The study drug will be labeled with

the study number and a unique identification number.

The 2 treatments (allogeneic MSCs and placebo) will be

indistinguishable.

This studywill be double-blind and placebo-controlled.

To maintain the blinding during the course of the study,

the following procedures will be established: the placebo

and the study treatment (allogeneic MSCs) will be sup-

plied in identical vials, and the protocols will be designed

to ensure that the physician evaluating patient safety and

REPARATIVE THERAPY FOR ACUTE ISCHEMIC STROKE 2697

efficacy outcomes (clinical stroke scales: NIHSS, mRS, and

infarct volume), as well as the laboratory analysis, will not

have access to the randomization codes.

Treatment or Intervention

The experimental drug is a solution of MSCs from adi-

pose tissue. The manufacturing process is detailed in the

investigator’s brochure, which is available at the study

center and has been approved by the Spanish Agency of

Medicines and Health Products. In brief, it consists of

the release of the active ingredient composed of MSCs

obtained from the adipose tissue of the same donor

from whom the adipose tissue was previously obtained

for this study. Cells are maintained using the technology

of the cellular in vitro culture until the number of MSCs

is obtained that cover the administration of the product

to the patient, while meeting the required quality control

procedures according to good manufacturing practice

guidelines. The MSC solution is prepared for its final con-

ditioning and is transported to the study center for

administration to the patient.

Thus, once a patient is randomized, there are 3 steps

before the administration of the MSCs to the patient,

which takes approximately 7-10 days:

(1) Phase 1: unfrozen and in vitro maintenance of the

MSCs.

(2) Phase 2: preparation and conditioning of the final

product.

(3) Phase 3: transport of the drug under investigation

from the manufacturer’s laboratory to the study

center.

The patients assigned to the placebo armwill be given a

vehicle solution with the same appearance as the drug

under investigation. The drug will be administered in a

single intravenous dose within the first 2 weeks from

the onset of stroke symptoms. All patients will receive

conventional treatment for ischemic stroke according to

current clinical practice guidelines and pathways.

All study medications will be manufactured, tested, and

released according to current goodmanufacturing practice

guidelines. The study medications will be prepared in

identical vials containing either allogeneic MSCs or pla-

cebo solutions. Regarding the treatment dose, its adminis-

tration, and dispensing, included patients will receive an

intravenous infusion of the study medication (allogeneic

MSCs or placebo) within the first 2 weeks from stroke

onset. The dose will be 1 million units per kilo of weight,

administered at an infusion rate of 4-6 mL/minute. The

study medication must be discontinued if any of the

following conditions occur: a suspected anaphylactic reac-

tion, a severe adverse event, or withdrawal of consent by

either the patient or their representative.

All included patients will be managed according to cur-

rent guidelines20-22 for acute stroke management and the

stroke clinical pathway of the Stroke Center at La Paz

University Hospital.23 They will be admitted to the stroke

unit, and continuous noninvasive monitoring will be pro-

vided during the first days following the stroke until the

physician in charge evaluates the patient’s clinical stabil-

ity and approves a transfer to the neurology ward. This

approval will be independent from the inclusion of the

patient in the study.

Study Schedule

The study will have a total duration of 24 months per

patient and will consist of 9 scheduled visits (Table 1,

Figure 1).

Primary Outcomes

The safety of MSCs from adipose tissue will be assessed

using the following parameters:

(1) Adverse events (AEs), reported spontaneously or

in response to prespecified questions at each study

visit. Serious AEs will be recorded at each visit

during the study period.

(2) Neurologic and systemic complications: deterio-

rating stroke, stroke recurrences, brain edema, sei-

zures, hemorrhagic transformation, respiratory

infections, urinary tract infections, deep venous

thrombosis, pulmonary embolism, and gastroin-

testinal hemorrhage will be recorded at each visit

during the study period.

(3) Tumor developments.

Secondary Outcomes

The efficacy of MSCs from adipose tissue will be

assessed using the following parameters:

(1) mRS, which will be measured on day 7 and at

3 months.

(2) NIHSS, which will be measured during all the

scheduled visits.

(3) Infarct size, which indicates the total volume of the

infarction, measured in neuroimaging (MRI) on

day 7 and at 3 months.

(4) Biochemical markers of brain repair (vascular

endothelial growth factor, brain-derived neurotro-

phic factor, and matrix metalloproteinases 9),

which will be measured at baseline, on day 7 and

at 3 months.

Data Management and Monitoring Committee

(1) Monitoring will be performed by a Clinical

Research Organization. Safety will be carefully

monitored during the trial. An independent data

monitoring committee will monitor the accumu-

lated safety data through periodic reviews of the

unblinded data to identify any safety issues.

Table 1. Flowchart

Flowchart Screening

V1 V2 V3 V4 V5 V6 V7 V8 V9

0 h 2 h 24 h Day 7 Month 3 Month 6 Month 12 Month 18 Month 24

Informed consent signature x

Inclusion and exclusion

criteria review

x

Past medical/surgical history x

Physical examination x x x x x x x x

Pregnancy test in women of

childbearing age

x

NIHSS x x x x x x x x x x

Modified Rankin Scale (prestroke) x x x x x x

Charlson Index x x x

Blood pressure x x x x

Heart rate x x x x

Body temperature x x x x

Oxygen saturation x x x x

Capillary blood glucose x x x x

Transcranial Doppler x

Neuroimaging (brain CT) x

Neuroimaging (brain MRI) x x x

Laboratory assessments

Hematology, coagulation

test, and biochemistry

x x x

Brain repair markers x x x

Recording of adverse events x x x x x x x x x

Concomitant drugs x x x x x x x x x

Abbreviations: CT, computed tomography; MRI, magnetic resonance imaging.

E. D�ıEZ-TEJEDOR ET AL.2698

(2) Collection and processing of safety data and re-

view by independent data monitoring commit-

tee: safety will be carefully monitored during

the trial. An independent data monitoring com-

mittee will monitor the accumulated safety data

through periodic reviews of the unblinded data

to identify any safety issues. The independent

data monitoring committee will provide recom-

mendations as to whether to continue the trial

based on the results of the safety analysis. The

independent committee will follow the re-

commendations listed in the ‘‘Guideline on

Data Monitoring Committees’’ of the European

Medicines Agency (EMEA) (Ref. Doc EMEA/

CHMP/EWP/5872/03).

Sample Size

This pilot trial will include 20 patients, distributed to

each treatment group in a proportion of 1:1. A formal pre-

determination of sample size cannot be calculated because

of the exploratory nature of this pilot study. This sample

size should allow us to obtain sufficient information for

the development of further studies (Phase III).

The estimated recruitment period is 2 years, as it is

feasible to include approximately 10 patients per year,

Figure 1. Schematic diagram(s) of the trial

design.

REPARATIVE THERAPY FOR ACUTE ISCHEMIC STROKE 2699

taking into account the inclusion and exclusion criteria

and the number of acute ischemic stroke admissions to

the stroke unit of the Neurology Department of La Paz

University Hospital (approximately 350 per year).

Statistical Analyses

The demographic and baseline characteristics will be

summarized for each treatment group to assess the

comparability of both groups. For this purpose, the

following variables will be considered: age, gender, race,

smoking and drinking habits, vascular risk factors (eg, hy-

pertension, dyslipidemia, diabetes mellitus, metabolic

syndrome, and heart disease), previous strokes, comorbid-

ity (Charlson Index), prior functional status (mRS), time

from stroke onset, and start of study treatment.

(1) Primary analysis: safety. The safety analysis will

be based on the reports of AEs, clinical and labora-

tory data, and vital signs. The incidence of serious

AEswill be provided, regardless of their classifica-

tion as AEs due to treatment, to their relationship

with the study drug, or to the deaths related to

them.

(2) Secondary analysis: efficacy. For continuous vari-

ables, the following information will be provided:

number of subjects, mean, standard deviation,

median, minimums, maximums, 25% and 75%

quartiles, and changes from baseline to end visit

values. For categorical variables, the frequency

distribution (absolute and relative) and 95% confi-

dence interval, if applicable, will be provided. An

exploratory analysis will be conducted by

comparing both treatment groups using Student

t test or the Mann–Whitney test, according to the

data distribution.

The ‘‘intention to treat’’ population will consist of the

patients for whom basal variables and at least 1 value of

a principal or secondary outcome variable will be avail-

able. If the patient does not attend the final visit, the

data will be replaced by the last available value (Last

Observation Carried Forward). The ‘‘per-protocol’’ popu-

lation will consist of the patients who have begun treat-

ment with the study drug and for whom major

violations of the protocol do not exist.

Study Organization and Funding

The study is investigator initiated, is promoted by the

Fundaci�on de Investigaci�on del Hospital Universitario La

Paz, and is funded by the Spanish Ministry of Health, So-

cial Policy and Equality. It has been approved by the La Paz

University Hospital Ethics Committee and by the Spanish

Agency of Medicines and Health Products. EudraCT:

2011-003551-18. The study is registered at http://www.

clinicaltrials.gov and identified by NCT01678534.

Recruitment is aimed to begin in May 2014.

Study Procedures:

(1) Screening and informed consent

Once the diagnosis of cerebral infarction has been

confirmed and it has been determined that the patient

fulfills the inclusion criteria and meets none of the exclu-

sion criteria, written informed consent will be obtained

from the patient or the patient’s representative before the

performance of any trial-specific procedure. A copy of the

signed informed consent will be given to the participants.

The original signed form will be retained at the study site.

(2) Data management

All data management will follow the principles of the

Spanish law Ley Org�anica de Protecci�on de Datos (LOPD

15/1999), ensuring the confidentiality of all personal data.

In compliance with European regulations/International

Conference of Harmonization Good Clinical Practice

(ICHGCP) Guidelines,24 the investigator and institution

are required to permit authorized representatives of the

company, of the regulatory agency(s), and the IEC/IRB

direct access to review the subject’s original medical re-

cords for verification of study-related procedures and data.

(3) Study schedule

The study will have a total duration of 24 months per

patient and will consist of 9 scheduled visits. The baseline

visit will take place during the first 24 hours of the stroke

and before the first administration of the study drug. The

second visit will take place 2 hours after the inclusion of

the patient in the study and will coincide with the

completion of intravenous treatment. Other visits will

be conducted at 24 hours, 7 days (or hospital discharge

if it occurs before day 7), 3 months, 6 months, 12 months,

18 months, and 24 months, according to the schedule

illustrated in the Table 1 and Figure 1. The efficacy end-

points will be evaluated by an investigator blinded to

the treatment received by the patient.

Discussion

Autologous MSCs have been successfully transplanted

into the brains of stroke patients with excellent tolerance

and no complications. The intravenous infusion of autol-

ogous MSCs has been shown to be feasible and safe and

has improved neurologic recovery in patients with se-

vere stroke. The safety of this therapy at 5 years post-

treatment has been reported. The use of allogeneic

MSCs could be an alternative therapy because it has

been demonstrated that they lack HLA-class II antigens,

and no allergic reactions other than an immunologic

response or rejection would be expected. In addition,

this therapy has not been associated with the develop-

ment of tumors.

To our knowledge, this is the first pilot, phase II, clinical

trial to investigate the safety and efficacy of intravenous

E. D�ıEZ-TEJEDOR ET AL.2700

administration of allogeneic MSCs from adipose tissue

within the first 2 weeks of stroke. In addition, its results

will help us to define the best criteria for a future phase

III study.

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