Efficacy and Dose-Dependent Safety of Intra-Arterial Delivery of Mesenchymal Stem Cells in a Rodent Stroke Model
et al. (2014) Efficacy and Dose-Dependent Safety of Intra-Arterial Delivery of Mesenchymal Stem Cells in a
Rodent Stroke Model. PLoS ONE 9(5): e93735. doi:10.1371/journal.pone.0093735
Efficacy and Dose-Dependent Safety of Intra-Arterial Delivery of Mesenchymal Stem Cells in a Rodent Stroke Model
Dileep R. Yavagal 0
Baowan Lin 0
Ami P. Raval 0
Philip S. Garza 0
Chuanhui Dong 0
Weizhao Zhao 0
Erika B. Rangel 0
Ian McNiece 0
Tatjana Rundek 0
Ralph L. Sacco 0
Miguel Perez-Pinzon 0
Joshua M. Hare 0
Toru Hosoda, Tokai University, Japan
0 1 Cerebral Vascular Disease Research Laboratories, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida, United States of America, 2 Department of Neurology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida, United States of America, 3 Interdisciplinary Stem Cell Institute, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida, United States of America, 4 The Department of Medicine, Leonard M. Miller School of Medicine, University of Miami , Miami, Florida , United States of America
Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 16106, 56105, 26105, 16105 and 56104) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 16105 and below did not compromise MCA flow and hence an IA MSC dose of 16105 considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 16105 delivered at 24 hours in rodent model of stroke.
Funding: This study was supported by Department of Neurology, University of Miami (DRY); James and Esther King Biomedical Research Program, Florida
Department of Health Grant # 2KN09 (DRY); University of Miami intramural grant: Interdepartmental Research Development Initiative grant 20092010 (DRY) and
the Anderson Family Gift 20102011, 20122014 (DRY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
Stroke is the main cause of long-term disability and the third
leading cause of death in the United States. The public health
burden of stroke continues to be staggering with an estimated cost
of 73.3 billion in 2010 . Despite the approval of intravenous
recombinant tissue plasminogen activator (rtPA) 18 years ago, and
rapid growth in number of endovascular recanalization therapies
for acute ischemic stroke (AIS), their impact on reducing
strokerelated long-term disability is limited [2,3]. Hence, there continues
to be a critical need for novel therapies for AIS. In this regard,
over the last decade, various types of stem cell have been tested in
several pre-clinical studies suggesting improved functional
neurological outcomes after AIS . The leading type of stem cell for
clinical translation in stroke is the mesenchymal stem cell, which is
an adult, non-hematopoietic progenitor cell with capacity to
differentiate into a variety of cell lineages including osteoblasts,
chondrocytes and neuron-like cells .
Mesenchymal stem cells (MSCs) are multipotent
non-hematopoietic stem cells found mainly in the stromal fraction of the bone
marrow as well as in the connective tissue of most organs .
MSCs can be easily isolated from adipose tissue, amniotic fluid,
placenta and umbilical cord, though they are most commonly and
efficiently derived from adult bone marrow. MSCs are an
attractive cell source because they are relatively easy to obtain,
expand and manipulate in vitro . Moreover, adult MSCs do not
confer the risk of tumorgenicity that pluripotent cells carry .
Importantly, MSCs are also immunoprivileged, with low MHC I
and no MHC II antigen expression [12,13]. Their
immunoprivileged nature obviates the need for immunosuppression in
allogeneic administration of MSCs. Thus allogeneic MSCs,
already produced from a healthy donor, can be given as an
offthe-shelf product without delay if needed without the need of
immunosuppression. This feature is especially attractive for future
translation of MSCs into a treatment for ischemic stroke, which is
presents most often without warning and may benefit from early
administration of cell therapy .
There are multiple routes of stem cell delivery to the brain in
AIS. Among these, the intra-arterial (IA) route of stem cell delivery
has a high potential for clinical translation, in view of the
increasing clinical application of endovascular therapy in the
treatment of AIS [6,1720]. Furthermore, IA delivery of stem cells
after AIS is minimally invasive and results in a larger number and
more diffuse distribution of stem cells in and around the infarcted
area when compared to intra-parenchymal,
intra-cerebroventricular and intravenous stem cell delivery . IA delivery avoids
the first pass trapping of stem cells in the lungs and liver seen with
intravenous delivery . Also, a recent study has shown superior
functional and histological outcomes with IA delivery of stem cells
as compared to intravenous administration . However MSCs
range in size from 550 microns and a major limitation to IA
delivery of MSCs is the possibility of compromise of regional
cerebral blood flow (rCBF) owing to those MSCs in the larger size
range of 2050 microns; thus a potentially novel therapeutic
strategy can paradoxically worsen of cerebral ischemia outcome
In the current study, first we hypothesized that compromise of
MCA blood flow during IA delivery of MSCs was dose-dependent
and therefore, lowering the dose of IA MSCs would mitigate rCBF
compromise. The efficacy of IA MSC in focal cerebral ischemia,
with doses lower than 16106 has not been studied. Hence, we also
hypothesized that the lower safe dose we identify, based on our
first hypothesis, would be efficacious in focal cerebral ischemia.
Furthermore, the efficacy of IA MSCs given immediately after
reperfusion (within 60 minutes) as compared to the efficacy when
given at 24-hours is unknown. Hence, we additionally
hypothesized that the efficacy of the MTD of IA MSCs delivered at
24 hours (sub-acute) and 60 minutes (acute) in a rodent cerebral
ischemia model would be the same.
Materials and Methods
All animal procedures were carried out in accordance with the
Guide for the Care and Use of Laboratory Animals published by
the U.S. National Institutes of Health and approved by the Animal
Care and Use Committee of the University of Miami.
Female Sprague-Dawley rats (Charles River Laboratories, Inc.,
Wilmington, MA) weighing 260310 g were used. The animals in
the experimental groups were allocated in a randomized fashion.
Investigators were blinded to dose and treatment group allocation
during surgery and during outcome evaluations.
The animals underwent Reversible Middle cerebral artery
occlusion (rMCAo) following an overnight fast. Anesthesia was
induced with 3% isofluorane and 70% nitrous oxide. Rats were
intubated endotracheally and ventilated mechanically on a
mixture of 10.5% isofluorane, 70% nitrous oxide and a balance
of oxygen. The right femoral artery and vein were catheterized to
permit to monitor blood pressure and to take arterial samples for
blood gas and glucose assessments. Arterial PCO2 and PO2 were
maintained in the normal range by ventilator adjustment. For
immobilization, rats received pancuromium bromide 0.75 mg/kg
i.v. Rectal temperature was measured continuously and
maintained at 3737.5uC by a heating pat under the rats body. Cranial
temperature was separately monitored by a 29-gauge
thermocouple implanted into the right temporalis muscle and was maintained
at 3636.5uC by a warming lamp placed above the rats head
throughout the experiment. Our previous studies have shown that
the cranial temperature 3636.5uC corresponds to a brain
temperature of 36.537uC . Physiological variables (plasma
glucose concentration, pH, PCO2, PO2 and mean arterial blood
pressure (MABP)) were maintained normal before and after
ischemia (Table S1 and S2; presented as supplementary data).
The left scalp was opened and the skull was exposed. A 2-mm
bur hole was drilled on the left sphenoid, 3 mm below the up edge,
0.5 mm anterior and 6 mm lateral to the bregma, leaving the dura
intact. A short Laser Doppler Flowmetry (LDF, Perimed Inc.)
probe was placed above the dura and fixed on to the skull with
glue and dental cement. From this point, the CBF inside the trunk
of the ascending vessel from cortical branch of MCA was
monitored. The LDF signal reading was recorded, starting at
30 min before the suture insertion and continuously throughout
the experiment and 60 min after the cell infusion, using a specific
designed monitoring system.
Reversible Middle cerebral artery occlusion (rMCAo)
The previously well-described suture technique for MCAo was
utilized in this study . The common carotid artery (CCA)
was exposed through a midline incision on the ventral neck and
carefully separated from the surrounding tissue including the
adjacent vagus nerve by blunt dissection using microsurgery.
Under an operation microscope, the branches of external carotid
artery (ECA), superior thyroid and ascending pharyngeal arteries
and further the terminal lingual and maxillary artery branches,
were dissected and coagulated. Two long 50 silk sutures tied the
ECA as distal to carotid bifurcation as possible. Two short (3 cm)
50 silk sutures were tied loosely around the segment of ECA
closed to the bifurcation. The ICA was exposed, and the origin of
the pterygopalatine artery (PPA) was visualized. An intraluminal
3.0 nylon suture, having a spherical and enlarged tip produced by
heating near flame, introduced into the distal segment of ECA via
a small incision on the vessel and advanced into the ECA lumen to
reach the bifurcation. The 2 short silk sutures subsequently were
tied to prevent bleeding. The ECA was cut at the distal segment
between the 2 long-suture notes. The stump of the ECA was held
toward the surgeon; thus the stump and the ICA were on a straight
line. The suture was then turned into ICA with a special care not
enter the PPA. The suture went advance in ICA until the sharp
drop of regional cerebral blood flow confirmed by the LDF. The
length of inserted suture inside the ICA from the bifurcation was
1925 mm. A sudden and sharp decrease in the LDF signal was
interpreted to indicate a successful MCA occlusion. The suture
was gently withdrawn after the 90-min occlusion period. During
the procedure and the 2-h recirculation, blood pressure and the
blood gas were maintained in the normal ranges, cranial and rectal
temperature were maintained at normal levels.
Inclusion and Exclusion Criteria
Rats that had less than 50% drop in LDF signal on suture
insertion and premature mortality during rMCAo were excluded
from the study prior to randomization and allocation to treatment
(1) Safety Study
Forty-three female rats underwent rMCAo. The suture was
gently withdrawn after a 90-minute occlusion period (Figure 1A,
B). At 60 minutes after recirculation, a 1 ml syringe was filled with
the MSCs suspended in phosphate buffered saline (PBS) or PBS
alone. The cell suspension in 0.5 mL PBS or PBS alone (0.5 mL)
was delivered into the ipsilateral IA over 3 minutes. The different
groups of animals received 16106, 56105, 26105, 16105 & 56104
MSCs in PBS or only PBS. Following MSCs or PBS delivery, the
catheter was carefully withdrawn and the ECA was tied. During
the surgical procedure of rMCAo and IA MSCs deliver, we
measure blood flow over ipsilateral MCA using laser Doppler flow
(2) Efficacy Study
In another cohort, 34 female rats underwent 90 minutes of
rMCAo. Twenty-four hours after induction of rMCAo, rats were
assigned to receive IA MSCs (16105) or IA PBS or intravenous
(IV) MSCs 16106. In the IV MSCs group, the cells were given
into the femoral vein at a 10-fold of the IA MSC dose in order to
potentially increase the number of MSCs reaching the injured
brain. In an additional experimental group, IA MSCs at 16105
were given at 60 minutes after rMCAo to study the effect of acute
timing on efficacy. The primary outcome measure was
neurodeficit score and the secondary outcome was infarct volume at 4
Donor rodent MSCs
MSCs used in this study were derived from green fluorescence
protein (GFP) transgenic male Sprague-Dawley rats. At the Cell
Manufacturing Program at the University of Miami, the bone
marrow was harvested from rats and the mononuclear cells
isolated by density gradient centrifugation. The cells were then
cultured at 1 to 56106 cells per ml in 25 ml of alpha MEM plus
20%FCS in T162 cm2 culture flasks at 5%CO2 and 37uC. MSCs
exhibited spindle-shaped morphology and were characterized by
(i) adherence to plastic, (ii) negativity for hematopoietic cell surface
markers CD34 and CD45 (0.160.1% and 0.26.0.1%,
respectively) and positivity for CD73, CD90.2, and CD105 (8865.4%,
99.260.7% and 9563.7%, respectively). The cells were expanded
to passage 6 and then the MSCs were suspended in 10% of
cryopreservant dimethylsulfoxide (DMSO) and frozen in liquid
nitrogen. Prior to injection, the cells were thawed rapidly and
washed to remove (DMSO), and then re-suspended in PBS to the
required cell dose. We evaluated cell viability prior to infusion
using trypan blue technique. Only cell doses at and above 70%
viability were administered in the study.
The presence of hematopoietic cell surface markers was
investigated using fluorescence-activated cell sorting (FACS).
Briefly, a total of 0.5216106 MSCs was used for FACS
characterization. GFP positivity was detected in 7965% of MSCs
in culture. For surface markers, cells were incubated for 1 hour
with FACS buffer (1% bovine serum albumin and 5% FBS diluted
in distilled water) on ice, and subsequently 1 hour with the
primary and secondary antibodies (washed 3 times for 5 minutes
during centrifugation between the primary and secondary and
after the secondary). Each analysis included at least 10,000 events
and was performed on at least three separate cell preparations (BD
FACSAria, University of Miami). All experiments for MSCs
characterization were performed between passages 8 to 11 after
isolation. Cells were incubated with anti-mouse
PE-fluorochromeconjugated antibodies against CD45, CD73, and CD90.2 (BD
Biosciences, San Jose, CA), PE-fluorochrome-conjugated against
CD34 (Santa Cruz Biotechnology, Santa Cruz, CA),
PE/Cy7fluorochrome-conjugated against CD105 (BioLegend, San Diego,
CA), and their respective isotype controls (BD Biosciences, San
Figure 1. Lower doses of IA MSCs mitigate adverse effect IA injection on MCA blood flow. (A) Experimental timeline showing rMCAo for
90 minutes followed by withdrawal of the suture to allow reperfusion. At 60 minutes of reperfusion, IA MSC or vehicle only injection was given,
followed by LDF monitoring for 60 min. (B) Comparison of relative LDFS worsening from baseline to final recording, among de-escalating dose
groups (C) The 16105 dose and placebo has significantly less maximum LDFS worsening as compared to the 16106 dose. The comparisons in B and C
were done using general linear modeling (GLM) to compare mean differences among groups. LDFS = Laser Doppler Flow Signal.
Intra-arterial MCA infusion
A PE-10 polyethylene catheter connected to a 30G needle and a
1-ml syringe was inserted into the stump of left ECA through an
incision on the vessel under the operation microscope and was
tightened to ECA by a 50 silk suture. The catheter went forward
into ICA and avoided PPA, the extracranial branch of ICA, to
approach the skull base . The catheter was filled with
heparinized physiological saline to prevent air bubble and
coagulation. The syringe was filled with MSCs in PBS or PBS
only. Using slow hand injection over 3 minutes, 0.5 ml volume of
the cells in PBS or PBS only were then delivered into the ICA via
the PE-10 catheter.
Histology and detection of MSCs in the brain
Under anesthesia rats were perfused via ascending aorta with
FAM (a mixture of 40% formaldehyde, glacial acetic acid, and
methanol, 1:1:8 by volume) for 20 min following a 2-min initial
perfusion with physical saline. The rat heads were immersed in
FAM for 1 day before the brains were removed. The brains were
placed in FAM at 4uC for at least 1 additional day, and then
coronal brain blocks were embedded in paraffin. All brains were
cut into 10- mm thick sections from 5.5 mm to 27.5 mm from
bregma at 9 standard levels to cover the whole infracted areas.
Sections of the 9 levels were stained with hematoxylin and eosin to
display the infracted areas and to obtain infarct volumes.
In a parallel study, adjacent sections were subjected to
immunohistochemistry (IHC) studies with Vectastain ABC
peroxidase method (Vector Labs, Burlingame, CA). Sections from
levels 28 in the dose group 16105 & 56105 were tested by
GFPIHC to reveal the MSCs immigration inside the brain via injection
through IA. To detect GFP positive MSCs, sections were
incubated in the working dilution of 1:100 of anti-GFP
(SC101525, Santa Cruz Biotech, CA.) for 90 min at room
temperature, and 3, 30-diaminobenzidine (DAB) was used for
visualization of the primary antibody binding. The dark-brown appearance
was considered the specific labeling to indicate the MSCs.
Hematoxylin counterstain was conducted to identify cells and
brain regions. Blocked vessels were defined as: complete filling of
the microvessel caliber with MSCs in axial section or segment of
vessel in sagittal section of a vessel. The absolute total number of
blocked microvessels per section was reported.
Both positive control, to confirm the negative labeling being
truly negative, and negative control, to confirm the positive
staining not false positive, were applied in the present study. The
two negative controls including 1) mouse IgG 1 (X0931, Dako,
Carpenteria, CA) replaced anti-GFP in the female brains of the
present study and 2) anti-GFP applied to male rat brains having
infarction from previous study were performed. Negative labeling
represented in the 2 negative controls supported that the positive
staining were true positive. Anti-glial fibrillary acidic protein
(GFAP, Dako, Carpenteria) was employed in the IHC study as the
positive control to ensure that the reduced sensitivity of reaction
not due to improper technique because positive control slide, i.e.
the known histological slide containing the specific antigen
A standardized neurobehavioral test battery was conducted as
described previously , which includes tests for postural reflex,
sensorimotor integration and proprioception. Total neurological
score ranged from a normal score of 0 to a maximal possible score
Descriptive statistics for physiological continuous variables were
presented as means with standard deviations. For safety analysis,
the primary outcome was the relative change in MCA blood flow
from pre-injection to the final assessment after injection of MSCs
or saline, whereas the secondary outcome was the maximum
relative reduction in blood flow within the 60 minutes after
injection. General linear model (GLM) was used to compare mean
differences among groups in the relative change at the final LDFS
measurement and in the maximum relative reduction in blood
flow. Mixed model was used for analyzing for comparing LDFS
over time between two groups. For efficacy analysis, mixed-effects
model was used to assess differences among groups in neurodeficit
scores based on the repeated measures. Due to skewed
distribution, log transformation was first performed for infarct volume and
ANOVA was used to compare the differences among groups at
week 4 after treatment. All analyses were performed with SAS 9.3
(SAS Institute Inc., Cary, NC) or SPSS 16.0 (SPSS Inc., Chicago,
Lower IA MSC doses mitigate adverse impact on MCA
We first determined the impact of lowering the dose of IA MSC
on MCA blood flow, starting with a dose of 1 million cells, which
was previously shown to severely decrease MCA flow in one-thirds
of animals . In the animals receiving 16106and 56105 MSCs,
there was a considerable decrease in the final post-injection MCA
flow by 32% and 45% respectively, compared to the pre-IA MSC
injection MCA flow (Figure 1B). Lowering the dose below 56105
resulted in mitigation of the adverse effect of IA MSC injection on
MCA flow. At the dose of 26105, the MCA flow compromise was
less as compared to the 56105 dose group, but this was
nonsignificant (LDFS decreased by 9% vs 45%, p = 0.06). At the next
lowered dose level of 16105 MSCs, the MCA flow compromise
was significantly less as compared to 56105 dose group (LDFS
decreased by 0.2% vs 45%, p = 0.01). When we analyzed the
maximum reduction in LDFS among the dose groups, the rats
receiving 16106and 56105 MSCs had a maximum reduction of
LDFS over 50%, whereas those receiving a dose at 16105 MSCs
had a maximum reduction in LDFS of 23%, similar to 20% in
placebo group (Figure 1C). Furthermore, we found that as
compared to the 56105 dose, the 16105 IA MSC dose injection
led to less worsening in LDFS post injection over the course of 60
minutes post injection, becoming significantly improved at 45
minutes and 60 minutes post-injection (Figure 2A). Based on these
results the 16105 IA MSCs dose was utilized for subsequent
studies and defined as maximum tolerated dose (MTD).
Mortality with MCAo and IA MSCs
The mortality rate of the MCAo procedure (premature
mortality) in this study was 13.46%. In the part of the study
evaluating safety of de-escalating doses, the mortality rate after IA
MSC dose administration was: 8.3%, 28.6%, 12.5%, 42.9%,
42.9% and 42.9% in the placebo, 56104, 16105, 26105, 56105
and 16106 IA MSC dose groups respectively.
Impact of MSC Dose on Microvascular Occlusion and
Consistent with the effect on MCA flow, at 35 days post
injection, we found a significantly higher number of brain
microvessels showing complete occlusion with MSCs in rats
Figure 2. Comparison of LDFS changes over time and microvascular occlusion between dose-groups. (A) IA MSC dose of 16105 and IA
placebo have a transient and less MCA LDF worsening during 60 minutes post injection as compared to 56105 dose using mixed model. LDFS =
Laser Doppler Flow Signal. (B) On comparing the total number of microvessels with complete occlusion among the two dose groups, there were a
significantly lower number of complete occlusions in the lower dose group. Mean 6SD, * P,0.05, ANOVA. (C) Representative brain sections from IA
MSC 16105 dose group showing GFP+ MSCs identified by 3, 30-diaminobenzidine (DAB), showing localized complete filling of microvessels at 35
days post-injection, as well as MSCs just outside the vessel wall in brain parenchyma and (D) high power field of representative brain sections from IA
56105 dose group showing single MSC partly inside and partly outside microvessel wall.
receiving IA MSCs at the higher dose of 56105 as compared to the
MTD of 16105(366.5 vs. 185.75, p = 0.047) (Figure 2B). However,
in rats in both dose groups, MSCs were also seen in the brain
parenchyma just outside the microvessels at the level of the
blockage. (Fig. 2C) We noted two sections in one rat from the
larger dose group in which individual MSCs were located across
the vessel wall partly inside the vessel and rest in the adjacent brain
parenchyma, consistent with them being in the process of
Efficacy Study of MTD of IA MSCs
We chose the 16105 MSCs as the dose to use for our efficacy
study of IA MSCs in rMCAo based on the above results showing
that this was the highest dose group that had minimal adverse
effect on MCA LDF post-injection. We compared the efficacy of
IA MSCs given at 24 hours (IA MSC_24 h), at one hour (IA
MSC_1 h), IV MSCs at a ten-fold dose given at 24 hours (IV
MSC_24 h) and IC PBS given at 24 hours (IA PBS_24 h) post
rMCAo. On mixed model analysis we found a significant
interaction between treatment group and time point (p = 0.02),
with no significant difference among the groups at day 1 (p = 0.49)
and most significant difference at 28 days (p,0.0001) after
treatment (Figure 3B). The IA MSC_24 h group had a
significantly lower neurodeficit score (5.8, 95% CI: 4.4 to 7.2) at
28 days after treatment as compared to IA PBS_24 h (10.4, 8.5 to
12.3, p,0.0001), as well as to IV MSC_24 h at dose of 16106
(10.3, 8.8 to 11.7, p = 0.0002), and IA MSC_1 h (9.2, 7.3 to 11.1,
Figure 3. Functional neurologic outcomes are superior in the IA MSC_24 h group. (A) Experimental timeline of the efficacy study showing
90 min rMCAo followed by 24 hour reperfusion except in group receiving IA MSCs at 1 hour reperfusion followed by ND score assessment at
1,7,14,21 & 28 days. (B) On day 1 post rMCAo, the ND scores were not significantly different among groups. The ND score of the 16105 group
progressively decreased over time and at 28 days was significantly lower than the other groups. C, The day 28 ND score was significantly lower in the
IA MSC_24 h group as compared to all the remaining groups. ND = Neurodeficit
p = 0.005) groups. The IA MSC_1 h group showed no significant
difference in functional outcome compared to the IA PBS_24 h
(p = 0.37) and IV MSC_24 h (p = 0.38) groups (Figure 3C).
Also, the mean infarct volume of the group treated with IA
MSC_24 h group (4.6 mm3, 1.2 to 12.72) was significantly lower
as compared to IA PBS_24 h (39.5, 11 to 136.1, p = 0.01) The
groups treated with IV_24 h MSCs and IA MSC_24 h did not
show a significant reduction in their infarct volume as compared to
IA PBS_24 h (Fig. 4A). In order to compare the location of
infarcted brain, we used an infarct topography frequency map,
which showed a significantly decreased volume of infarction in the
penumbral region in the IA MSC_24 h group as compared to the
IA PBS_24 h group (Fig 4B).
The major challenge to the IA method of delivery of MSCs is
the possibility of vascular occlusion in small arterioles and
capillaries owing to the large size of the cells ranging from 15
50 microns . We report a major new finding of a dose response
relationship in safety of IA delivery of allogeneic IA MSCs and
Figure 4. Comparison of infarct volume among treatment groups. (A) Geometric mean infarct volumes are compared among groups after
log transformation to achieve normal distribution. Only the IA MSC_24 h group shows significantly reduced infarct volume as compared to the IA
PBS_24 h. B, Frequency infarct map statistically comparing the location of the mean infarct volume in the IA MSC_24 h and IA PBS_24 h groups using
color- coded representation of the percent of rats showing infarction in each brain region using Fishers exact test with a color-coded
representation of the p-value, the color bar in 1-p format. Displays are in Coronal presentation; middle sections are selected. The IA MSC_24 h
group shows a much reduced infarction frequency, particularly surrounding the core as quantized by the Fisher test.
that lowering of the IA dose overcomes the decrease in blood flow
in the middle cerebral artery after IA MSC injection in a rat model
of acute ischemic stroke. Furthermore, the lower safe dose of
16105 MSCs, when given IA at 24 hours results therapeutic effect
to reduce infarct volume and improve functional status. Together
these findings support further translational investigation to develop
intra-arterial allogeneic MSCs as a novel therapy for AIS in the
first 24 hours after stroke onset.
With the increasing number of catheter-based endovascular
treatments for patients with AIS, intra-arterial delivery of stem
cells has tremendous potential for clinical translation. Intra-arterial
delivery of bone marrow mononuclear cells into the affected MCA
was found to be safe within 37 days in a 20 patients trial of
patients with MCA strokes . The strategy of endovascular
intra-arterial delivery of MSCs has been successfully implemented
in early clinical studies of intra-coronary-delivered MSCs after
acute myocardial infarction [31,32]. Given this tremendous
promise for clinical application, pre-clinical studies addressing
crucial translational hurdles in intra-arterial delivery of stem cells,
including MSCs in stroke, are crucial in the efforts to bring this
therapy to the bedside.
A previous study of IA MSC delivery reported severely reduced
ipsilateral MCA blood flow (8090%) in 35%, and moderately
reduced (1030%) in 47%, of animals after intra-carotid delivery
of 16106 MSCs given 30 minutes after rMCAo . On dose
deescalation, starting at the dose of 16106, we found a similar
reduction in CBF signal on IA MSC delivery at the doses of 16106
and 56105. This adverse effect was somewhat ameliorated at the
26105dose and resolved with further lowering of the dose to
16105 consistent with a dose-response relationship in the safety of
IA delivery of MSCs in rMCAo. In another recent study the safety
of MSC delivery was tested using non-MCAo rats . Lowering
of MSCs dose from 26106 to 16106 dose decreased stroke lesions
on MRI the rodent rMCAo model. Stroke lesions occurred
frequently (12 out of 15 animals) when injecting 26106 MSCs, but
not after lowering the dose to 16106 cells. While the decrease in
dose of MCSs led to better safety, similar to our study, the safety of
the 16106 dose is likely due to the use of non-MCAo rats in that
study. The twice bigger dose 26106 produced frequent
microstrokes mostly in the area of corpus callosum. With regards to
mortality post cell injection, the groups receiving 26105 and
higher doses of MSCs had a mortality rate of 42.9% similar to that
seen in other studies . The 16105 dose group and the PBS
group had a numerically a lower mortality rate (12.5% and 8.3%
respectively) as compared to the higher dose groups although not
statistically significant. Similarly, mortality in 56104 group is
numerically higher than 16105 however, there is no statistically
significant difference between two groups. The surgical procedure
of MCAo is highly invasive and observed mortality could be
confounded by the mortality related to the MCAo in the safety
phase of our study.
Despite approximately three times slower speed of injection as
compared to a previous study we did not see mitigation of the
adverse effect on blood flow in the MCA at the dose of 16106
MSCs, the only dose tested in that study . The speed of cell
injection from our rodent study and others cannot be directly
translated in humans. Apart from the large difference in scale, the
size of microcatheters available for clinical use allow for excellent
blood flow around the microcatheters in the ICA whereas, the PE
10 has a very snug fit in the rodent ICA. Testing of the IA
injection speed into ICA in larger animal species may hence be
The reduction of CBF signal over the MCA at the dose of
56105 is seen shortly after intra-carotid delivery of MSCs at the
higher doses and progressively worsens over 60 minutes
(Figure 2A), suggesting a mechanical obstruction in the MCA,
likely caused by microvascular sludging owing to stacking of
cells in the microvasculature at higher doses. This is supported by
our histology results that showed significantly increased
microvascular occlusion at 35 days at the higher dose as compared to the
lower dose and no evidence for other possible mechanisms for
microvascular occlusion such as thrombosis or spasm. The
occlusion of microvessels might be due to the higher ratio of the
size of some MSCs (some being 1550 microns in size) to the
microvessels size. Along with intravascular MSCs, we found the
vast majority of extravascular MSCs to be in the peri-vascular
regions at this early time point. We found two separate instances in
two separate animal brains of MSCs positioned across the
endothelial lining partly inside a capillary and partly in the
parenchyma, suggestive of transendothelial migration as the
mechanism of transport of these cells into the brain, (Figure 2C,
D) as seen in previous studies of intra-carotid stem cell infusion
In the current study we observed the maximum reduction in
LDFS was 20% following PBS delivery. Most importantly, the
observed reduction in LDFS after PBS delivery occurred shortly
after injection and was transient, lasting for short duration
(,5 min) and reverted back to the baseline. In contrast, at the
higher doses of 56105 and 16106 of IA MSCs, the reduction in
LDFS lasted throughout the period of recording (60 min). Hence,
the observed transient initial reduction with PBS injection is very
unlikely to lead to worsening of ischemic injury
In our study, the animals receiving IA MSCs at the safe dose of
16105 at 24 hours post rMCAo had a clearly superior functional
neurologic outcome at one month as compared to controls
receiving IA saline as well as compared to IV MSCs at the same
time point. In parallel with the superior functional outcome, we
also found the infarct volume to be significantly smaller only in the
IA MSC_24 h group as compared to IA PBS_24 h. This decrease
in infarct volume was found to be in the penumbral area
(Figure 4B) in this group as compared to control suggesting that
MSC-mediated neuroprotection as the likely mechanism of
benefit. This mechanism has been confirmed in other studies that
showed increased concentration of anti-apoptotic factors in the
peri-infarct area in animals treated with MSCs.
While previous studies have shown efficacy of IA autologous
bone marrow mononuclear cells (BMMCs) and fetal-derived
neural stem cells [34,35], we found that allogeneic MSCs given via
the IA route are efficacious in ameliorating neurological deficits in
rodent cerebral ischemia without a need for immunosuppression.
The application of allogeneic MSCs can facilitate rapid
off-theshelf therapy in patients with acute ischemic stroke. We found
that not only of 16105 IA MSCs lead to superior functional
neurological recovery as compared to the control group given IA
PBS, but also as compared to the group given IV MSCs at a
10fold higher dose, indicating a robust relative efficacy of the IA
route of delivery and its superiority to the IV route when MSCs
are administered at 24 hours post rMCAo. Our results are
consistent with the functional neurological benefit previously
reported  with intra-arterial allogeneic MSCs administered at
24 hours post rMCAo. However, the dose of IA MSCs in our
study was 10-fold lower as compared to the previous study. Our
finding of efficacy at the MTD being safer as compared to higher
doses, is novel and supports careful dose escalation studies in
clinical translation of IA MSC therapy. In a previous study
comparing the IA and IV routes of delivery of autologous
BMMCs, efficacy was seen in the IA group but not the IV group
. Our results confirm a similar superiority of the IA route but
add to this previous finding, as we also found a clear superiority of
IA over the IV route on direct comparison of the effect of the two
routes of delivery on functional recovery. Of note, in order to
compensate for this systemic trapping of IV delivered MSCs, we
chose an IV MSC dose that is 10 fold higher than the IA MSC
dose so as to serve as an adequate control to IA MSCs that do not
undergo systemic trapping. The choice of the IV dose was based
on studies showing that IA administration of cells leads to a 7 to 12
times higher number of cells reaching the brain as compared to IV
cell administration [34,36,37].
The optimal timing of IA stem cell delivery after AIS is
unknown. We found that allogeneic IA MSCs given at 24 hours
results in efficacy but not when given at 60 minutes after cerebral
ischemia. The fact that there was no reduction in the infarct
volume in the IA MSC_1 h group indicates that neuroprotection
was not significant when MSCs are given at this hyperacute timing
after rMCAo. This important finding may be a result of higher
concentration of chemoattractant factors for transendothelial
transport of MSCs at the more delayed time point, but we did
not test this hypothesis. Another study has shown results consistent
with the need to delay the administration of IA stem cells after
stroke for efficacy . In the prior study of IA neural stem cells in
a mouse model of hypoxia-ischemia, authors found that increased
phagocytosis of the cells in the post-ischemic brain when
transplanted within 24 hours whereas transplantation at 3 days
led to lower co-localization of MSCs with pan-monocytic marker
Iba-1. These findings suggest that in the hyperacute phase the
environment in the post-ischemic brain tissue may be detrimental
to the administered IA cells and waiting for this inflammatory
response to wane in the first few days is advantageous .
Therefore, while administration of IA stem cells via intra-carotid
catheters immediately after endovascular reperfusion therapy for
ischemic stroke in patients would avoid a second endovascular
procedure to administer the cells a few days later, our data suggest
that this hyperacute phase may not be an optimal treatment time
point for IA cell therapy. Thus early clinical trial designs of IA
allogeneic MSCs in ischemic stroke should consider administration
of the cells in the first few days after stroke and not in conjunction
or immediately following IA thrombolytic therapy.
One of the major limitations of the current study is that we did
not test the possibility of slower IA injection to decrease cell
sludging. However, the finding of efficacy at the MTD in our study
offsets the concern that the higher doses than MTD may be
necessary for benefit. Also, we did not comprehensively study the
mechanism of action of MSCs, as our primary goal was safety and
efficacy of the IA route of MSC delivery. We did not test for time
points beyond 24 hours for IA allogeneic MSC injection.
Although there is evidence for lack of efficacy of IA BMMC, as
beyond 24 hours after rMCAo , the outer limit of the
treatment time window remains to be established for allogeneic IA
MSCs. Additionally, in the present study we monitored the LDF in
the MCA for 60 minutes after IA MSCs delivery. However, effect
of IA MSCs on LDF beyond 60 minutes is not known and remains
to be investigated. Apart from LDF, the information such as
presence of MSCs at perivascular locations and overall MSCs
survival following IA MSCs needs investigation.
The present study used an experimental model of ischemic
stroke in female rats to investigate efficacy of IA MSCs. The MSCs
used for IA delivery were generated from male rats. Our approach
of delivering MSCs derived from male rats in to the female
experimental animals offer an added advantage of localizing
MSCs in the brain by cytogenetic approach after IA or IV delivery
in future. On the other hand, our use of female rats remains a
limitation because it is well known that the circulating ovarian
hormones influence ischemic pathology . Future studies
employing ovarian hormones-deprived (ovariectomized) female
rats are needed to confirm the findings of current study.
In conclusion IA delivery of MSCs at low dose (16105 cells) did
not compromise MCA blood flow in a rat model of MCAo.
Importantly, sub-acute delivery of MSCs at lowered dose
protected brain from ischemic damage. Our study findings outline
the core principles of MSC therapy for ischemic stroke using IA
delivery. A clinical translational impact of these results needs to be
determined in the future.
We thank Drs. Myron Ginsburg, MD and Brant Watson, PhD for their
critical review of the manuscript.
Conceived and designed the experiments: DRY JH. Performed the
experiments: BL. Analyzed the data: PSG AR CD WZ. Contributed
reagents/materials/analysis tools: JH IM ER. Wrote the paper: DRY AR.
Critically read manuscript: TR MAPP RLS JH.
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