Delayed Administration of Human Umbilical Tissue

Delayed Administration of Human Umbilical Tissue-Derived
Cells Improved Neurological Functional Recovery in a
Rodent Model of Focal Ischemia
Li Zhang, MD; Yi Li, MD; Chunling Zhang, MD; Michael Chopp, PhD;
Anna Gosiewska, PhD; Klaudyne Hong, PhD
Background and Purpose—The short time window required by neuroprotective strategies for successful treatment of
patients with ischemic stroke precludes treatment for most. However, clinical therapies based on neuroregeneration
might extend this therapeutic time window and thus address a significant unmet need. Human umbilical tissue-derived
cells have shown great potential as neuroregenerative candidates for stroke treatment.
Methods—The effectiveness of intravenous administration of human umbilical tissue-derived cells was tested in a rodent
middle cerebral artery stroke model in a dose escalation study (doses tested: 3⫻105, 1⫻106, 3⫻x106, or 1⫻107
cells/injection) followed by a time-of-administration study (time after stroke: Day 1, Day 7, Day 30, and Day 90 at a
dose of 5⫻106 cells/injection). Controls were phosphate-buffered saline injections and human bone marrow-derived
mesenchymal stromal cell injections. Post-treatment outcome tools included the modified neurological severity score
and the adhesive removal tests. Histology was performed on all cases to evaluate synaptogenesis, neurogenesis,
angiogenesis, and cell apoptosis.
Results—Statistically significant improvements of human umbilical tissue-derived cell treatment versus phosphatebuffered saline in modified neurological severity scores and adhesive test results were observed for doses ⱖ3⫻106 cells
up to 30 days poststroke. At doses ⱖ3⫻106, histological evaluations confirmed enhanced synaptogenesis, vessel
density, and reduced apoptosis in the ischemic boundary zone and increased proliferation of progenitor cells in the
subventricular zone of human umbilical tissue-derived cell-treated animals versus phosphate-buffered saline controls.
Conclusions—These results indicate effectiveness of intravenous administration of human umbilical tissue-derived cells
in a rodent stroke model compared with phosphate-buffered saline control and warrant further investigation for possible
use in humans. (Stroke. 2011;42:1437-1444.)
Key Words: cerebral infarct 䡲 human umbilical cord 䡲 stroke recovery 䡲 synaptogenesis
S
troke is a leading cause of death and disability worldwide. Unfortunately, treatment of ischemic stroke remains 1 of the most challenging areas in medicine today.
Over the last 20 years, cell-based therapies have emerged as
a potential approach in the treatment of stroke.1–3 Transplantation of stem cells derived from fetal tissues as well as
progenitor cells from bone marrow and human umbilical cord
blood promoted neuronal survival, tissue repair, and, most
importantly, recovery after experimental stroke.3– 6 However,
limited stem and progenitor cell sources along with ethical
and safety concerns associated with the use of embryonic
stem cells have spurred significant interest in the search for
alternative stem cell sources for stroke treatment.
Human umbilical cord tissue contains stem cells and is a
candidate source of cells for restorative therapy in stroke.7,8
Previous studies indicated that human umbilical tissue-
derived cells (hUTC) secrete several neurotrophic factors
such as brain-derived neurotrophic factor, fibroblast growth
factor 2, and interleukin 6, all of which have the capacity
to govern neurorestoration.9 –12 However, the efficacy of
systemically-administered hUTC to restore brain function in
rodent stroke models has not been evaluated. In the present
study, we tested whether intravenous administration of hUTC
improves neurological functional recovery in rats after stroke.
The effect of hUTC was compared with that of human bone
marrow-derived mesenchymal stromal cells (hMSCs), which,
when administered intravenously, have been shown to improve functional outcomes in rats after stroke.13,14
Methods
All experimental procedures were approved by the Institutional
Animal Care and Use Committee of Henry Ford Hospital. All
Received October 7, 2010; final revision received December 8, 2010; accepted December 23, 2010.
From the Department of Neurology (L.Z., Y.L., C.Z., M.C.), Henry Ford Hospital, Detroit, MI; the Department of Physics (M.C.), Oakland University,
Rochester, MI; and Advanced Technologies and Regenerative Medicine, LLC (A.G., K.H.), c/o Ethicon, Inc, Somerville, NJ.
The online-only Data Supplement is available at http://stroke.ahajournals.org/cgi/content/full/STROKEAHA.110.593129/DC1.
Correspondence to Michael Chopp, PhD, Henry Ford Hospital, 2799 W Grand Boulevard, Detroit, MI 48202. E-mail michael.chopp@gmail.com
© 2011 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.110.593129
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outcome measurements were performed by observers blinded to
the treatments.
Animal Model
Male Wistar rats (n⫽8 per cohort) weighing 270 to 300 g were
subjected to 2 hours transient focal ischemia.15 Briefly, rats were
anesthetized with 3.5% isoflurane and maintained with 1.0% to 2.0%
isoflurane in 70% N2O and 30% O2 using a face mask. Rectal
temperature was maintained at 37°C throughout the surgical procedure using a feedback-regulated water heating system. The right
common carotid artery, external carotid artery, and internal carotid
artery were exposed. A 4-0 monofilament nylon suture (18.5 to
19.5 mm), determined by the animal weight, with its tip rounded by
heating near a flame, was advanced from the external carotid artery
into the lumen of the internal carotid artery until it blocked the origin
of the middle cerebral artery (MCA). Two hours after MCA
occlusion, animals were reanesthetized with isoflurane and reperfusion was achieved by withdrawal of the suture until the tip cleared
the lumen of the internal carotid artery.
Isolation and Preparation of hUTC and hMSCs
Human umbilical tissue-derived cells were isolated and cultured as
described elsewhere.16 These cells are derived from the umbilical
cord of a single donor and do not exhibit key hematopoietic markers
such as CD34. In addition, these cells express HLA-ABC but do not
express HLA-DR, DP, DQ and do not express markers for antigenpresenting cells CD80 and CD86. Briefly, human umbilical cords
were obtained with donor consent after live births from the National
Disease Research Interchange (Philadelphia, PA). Tissues were
minced and enzymatically digested in a cocktail of 50 U/mL
collagenase containing 500 U/mL Dispase and 5 U/mL hyaluronidase (Sigma, St Louis, MO). After almost complete digestion, the
cell suspension was passed through a 70-␮m filter and centrifuged at
350 g. The cell pellets were washed in Dulbecco minimal essential
medium and seeded at 5000 cells/cm2 in T-flasks (Corning, Corning,
NY) in Dulbecco minimal essential medium– high glucose, 15%
(vol/vol) defined fetal bovine serum (HyClone, Logan, UT),
␤-mercaptoethanol (Sigma)⫹100 U/mL penicillin, and 100 ␮g/mL
streptomycin (Invitrogen). Cells were expanded in culture under
standard conditions in atmospheric oxygen with 5% carbon dioxide
at 37°C. When cells reached approximately 70% confluence, they
were passaged using trypsin/ethylenediaminetetraacetic acid (Gibco,
Grand Island, NY) every 3 to 4 days and reseeded at a density of
5000 cells/cm2 onto gelatin-coated flasks. Cells were harvested after
10 passages (approximately 20 population doublings) and cryopreserved in growth medium containing 10% (vol/vol) dimethyl sulfoxide (Sigma) in a programmable rate freezer (Thermo Forma, Marietta, OH). Cells were stored in a unit maintaining a phase of nitrogen
vapor with a small amount of liquid nitrogen at approximately
⫺120°C until thawing for cell characterization or for administration
in the study. Cell viability was reconfirmed on thawing and only
samples meeting viability requirements were injected into animals.
Thawed cell samples were immunophenotyped before use to ensure
similar immunophenotypes as reported elsewhere,16 and cells were
characterized to ensure safety and identity. Identity was evaluated by
flow cytometry for the expression of several cell surface markers.
Antibodies were purchased from BD Biosciences (Franklin Lakes,
NJ). Antibodies used in the analysis were IgG1-FITC, IgG-PE FITC,
CD10, CD13, CD31, CD34, CD44, CD45, CD 73, CD117, CD141,
HLA-DR, DP, DQ, HLA-ABC, and PDGF-Ra.
Cell karyotype analysis was also performed to confirm that
the cells contained the expected complement of human chromosomes. Cell pellets were evaluated by polymerase chain reaction for
the presence of viral pathogens: HIV-1, HIV-2, cytomegalovirus,
hepatitis B virus, hepatitis C virus, human T-lymphocytic virus, and
Epstein-Barr virus.
Cells were prepared for injection into the MCA-injured animals
immediately before delivery. Cell vials were thawed in a 37°C water
bath and then washed with phosphate-buffered saline (PBS). Aliquots of the cell suspension were removed and mixed with Trypan
blue and counted with a hematocytometer. The cell concentration in
the cell suspension was adjusted to the appropriate density with PBS
(3⫻105, 1⫻106, 3⫻106, 5⫻106, or 1⫻107 in 2 mL PBS) for
infusion.
Human mesenchymal stem cells were cultured according to
instructions provided by the supplier (Cambrex Corp, East Rutherford, NJ).
Experimental Protocol
Rats subjected to MCA occlusion were randomly allocated to the
following groups.
Dose Response
Six groups of 8 animals each were included in the dose–response
study. All treatments were performed intravenously in 2 mL PBS, 24
hours after MCA occlusion: Group 1, hUTC at 3⫻105 cells per
injection; Group 2, hUTC at 1⫻106 cells per injection; Group 3,
hUTC at 3⫻106 cells per injection; Group 4, hUTC at 1⫻107 cells
per injection; Group 5, 2 mL of PBS control only; and Group 6,
hMSC at 3⫻106 cells per injection. For identification of cell
proliferation, rats received daily intraperitoneal injections of bromodeoxyuridine (BrdU, 100 mg/kg; Sigma), a thymidine analog,
starting at 24 hours after stroke and subsequently for 14 consecutive
days. All animals were euthanized at Day 60 poststroke.
Timing of Administration
Five groups of 8 animals each were included in this study based on
the time of administration. Ideally, a PBS control group would have
been needed for every time point evaluated. However, to limit as
much as possible the number of animals euthanized in this study, the
following experimental design was selected: Group 7, 5⫻106 hUTC
cells administered on Day 1 poststroke; Group 8, 5⫻106 hUTC cells
administered on Day 7 poststroke; Group 9, 5⫻106 hUTC cells
administered on Day 30 poststroke; Group 10, 5⫻106 hUTC
cells administered on Day 90 poststroke; and Group 11, 2 mL of PBS
only administered on Day 30 poststroke, which served as a control
group.
Functional Outcome
Modified Neurological Severity Score
The modified neurological severity score is a composite of motor,
sensory, reflex, and balance tests.17 Neurological function was
graded on a scale of 0 to 18 (normal score, 0; maximal deficit score,
18). Animals were tested weekly for up to 60 days and biweekly
thereafter.
Adhesive Removal Test
An adhesive removal test was used to measure somatosensory
deficits.18 The mean time (seconds) required to remove stimuli from
the left limb was recorded. Animals were tested 1 day before MCA
occlusion and weekly thereafter starting 1 day after stroke onset.
To increase the sensitivity of the test, rats were challenged with
half-sized stimuli starting 70 days after stroke onset. The half-sized
stimuli tests were conducted biweekly thereafter.
Measurements of Infarct Volume
Rats were euthanized 60 (dose–response study) or 140 days (timing
of administration study) after stroke. Infarct volume was measured
on 7 hematoxylin and eosin-stained coronal sections using a Global
Laboratory Image analysis program (Data Translation, Marlboro,
MA), as previously described.19 The infarct volume is presented as a
percentage of total contralateral hemisphere volume.19
Immunohistochemistry
Immunohistochemical studies were performed on coronal sections
corresponding to bregma ⫺1.0 to 1.0 mm obtained from each
experimental animal. To identify proliferating cells, monoclonal
antibody against BrdU (Dako) was used at a titer of 1:100. Double
stainings were performed to visualize cellular colocalization of BrdU
with neuron-specific marker NeuN (Chemicon) at a dilution of 1:500
and with glial fibrillary acidic protein (Dako), an astrocyte marker at
a dilution of 1:10 000. For a morphological analysis of vessels, a
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Zhang et al
polyclonal antibody against Von Willebrand factor (vWF; Dako) was
used at a titer of 1:400. To detect presynaptic plasticity and
synaptogenesis, a monoclonal antibody against synaptophysin
(Chemicon) was used at a titer of 1:500. To detect human cells, a
monoclonal antibody against human mitochondria (HM; Chemicon)
was used at a titer of 1:100. To detect neuroblasts, a polyclonal
antibody against doublecortin (DCX; Santa Cruz) was used at a titer
of 1:200. For single labeling of BrdU, vWF, synaptophysin, HM, and
DCX, adjacent sections were incubated with the primary antibodies
at 4°C. Sections were sequentially incubated with the appropriate
biotinylated secondary antibodies (goat antimouse IgG for BrdU,
synaptophysin, and HM; goat antirabbit IgG for vWF; and horse
antigoat IgG for DCX). The signal was detected by avidin– biotin
complex (Vector Laboratories) before staining with diaminobenzidine and hydrogen peroxide. Double immunofluorescent labeling of
BrdU with NeuN and BrdU with glial fibrillary acidic protein were
done in sections that were incubated with secondary antibodies
conjugated with fluorescein isothiocyanate (Calbiochem) or cyanine
(Jackson Immunoresearch). The terminal deoxynucleotidyl transferase–mediated dUTP-biotinic end labeling method (in situ Apoptosis Detection Kit; Chemicon) was used to assess apoptotic cells
according to the procedures provided by the manufacturer.
The adult subventricular zone (SVZ) contains self-renewing multipotent neural progenitors, which are capable of generating all 3
neural lineages.20 –22 To examine whether administration of hUTC
enhanced endogenous neurogenesis, proliferated SVZ cells identified by BrdU-positive cells and neuroblasts detected by DCXimmunoreactive cells were measured at 60 days after stroke onset.
The numbers of BrdU-immunoreactive cells within the ipsilateral
lateral ventricle wall were counted from each rat and divided by the
ipsilateral lateral ventricle wall area to determine the cell density.
The DCX-immunoreactive area within the ipsilateral lateral ventricle
wall was measured and presented as percentage of area.
Synaptic plasticity in the ischemic penumbra region contributes to
functional recovery after stroke.23 To examine whether administration of hUTC enhanced synaptogenesis, synaptophysin immunoreactivity was measured 60 days after stroke onset. Specifically, 8
fields of view from the ischemic boundary zone were digitized using
a 40⫻ objective from a MicroComputer Imaging Device (Imaging
Research, St Catherines, Canada) system. The numbers of vWFimmunoreactive vessels and the positive area of synaptophysin in the
ischemic boundary zone were measured throughout each field of
view. Data are presented as density of vWF-immunoreactive vessels
and percentage of synaptophysin-immunoreactive areas, respectively. To quantify apoptotic cells and human cells, numbers of
terminal deoxynucleotidyl transferase–mediated dUTP-biotinic end
labeling- and HM-positive cells throughout the ipsilateral hemisphere were counted. Data are presented as the total number of
ipsilateral terminal deoxynucleotidyl transferase–mediated dUTPbiotinic end labeling- and HM-positive cells per section.
Statistical Analyses
Behavioral data were evaluated for normality and the nonparametric analysis was considered if data did not follow a normal
distribution. A 1-way analysis of variance was conducted to test
the treatment effect on each outcome between the control and all
dose groups at each time point. Histology data were evaluated
using Student t test. A treatment effect was detected if probability
value was ⬍0.05.
Results
A total of 97 animals underwent MCA occlusion, of which 9
died before completion of the study and were therefore
excluded from the analyses. Of these 9 animals, 6 died
immediately poststroke and were not subjected to treatment.
Two animals died at Day 8 poststroke from Group 2– dose–
response study (treated with hUTC at a dose of 1⫻106
cells/injection) and 1 at Day 38 post stroke from Group
6 – dose–response study (treated with hMSC at a dose of
Effectiveness of hUTC in a Rodent Stroke Model
1439
3⫻106 cells/injections). All animals that died during the
study were replaced such that the final count of animals per
group was 8.
Neurological Functional Recovery
The dose–response study indicated that treatment with hUTC
at doses of 3⫻106 and 1⫻107 cells/injection, but not at doses
of 3⫻105 and 1⫻106 cells/injection, significantly improved
the performance on adhesive removal test and modified
neurological severity score compared with the PBS-treated
rats starting at Day 14. This statistically significant difference
persisted up to Day 60 poststroke. Treatment with hMSC
exhibited a similar effect on neurological functional improvement as compared with hUTC at doses of 3⫻106 and 1⫻107
cells/injection (Figure 1).
The time of administration study indicated that hUTC
administered 1 day after stroke onset significantly improved neurological function as measured by adhesive
removal test and modified neurological severity score as
compared with PBS treatment. This statistical significance
was first seen at Day 7 and was maintained up to day 140
poststroke. Administration of hUTC at Day 7 or 30
significantly improved the performance on modified neurological severity score starting at Days 28 and 70,
respectively, as compared with treatment with PBS. This
statistical difference was maintained up to Day 140 poststroke. In addition, significant improvements on adhesive
removal test were detected in rats treated with hUTC at
Day 7 or 30, starting from Day 49 through 140, compared
with PBS-treated rats. Administration of hUTC on Day 90
poststroke significantly improved performance on the adhesive removal test as observed on Day 140 but failed to
improve the modified neurological severity score score
compared with PBS-treated rats. Our data thus indicate
that delayed administration of hUTC up to 30 days
improved neurological functional recovery (Figure 1).
Infarct Volume
No significant reduction of infarct volume was detected in
rats treated with hUTC regardless of dose and time of
administration when compared with rats treated with PBS.
In addition, treatment with hMSC did not significantly
reduce the lesion volume compared with PBS-treated rats
(Table).
Detection of Human Cells
Few HM-immunoreactive cells were detected within the
ipsilateral hemisphere of rats treated either with hUTC or
hMSC. Specifically, HM-positive cells ranged from 1.4⫾0.9
to 2.9⫾1.6 cells/section in hUTC-treated animals, with no
correlation to dose, and reached 2.9⫾1.5 cells/section in
hMSC-treated animals. The number of HM-immunoreactive
cells did not differ between the experimental groups. No
HM-immunoreactive human cells were found in PBS-treated
rats 90 days after stroke.
SVZ Cell Proliferation
Treatment with hMSC and with hUTC at doses of 3⫻106 and
1⫻107 cells/injection but not at doses of 3⫻105 and 1⫻106
cells/injection significantly increased the number of ipsilat-
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Figure 1. Adhesive removal test and modified neurological severity score for all
groups up to 60 days poststroke for the
dose–response study and 140 days poststroke for the time-of-administration study.
All animals in the dose–response study
were treated on Day 1 at doses indicated
on the graph. Animals in the hUTC groups
of the time-of-administration study were
treated with 5⫻106 cells/injection at times
indicated on the graph. Before the stroke,
there was no significant difference in neurological function measured by adhesive
removal test, as indicated on A, C, and
modified neurological severity scores for
normal, preischemic rats were equal to 0⫾0
for all animals (not shown on graphs). A,
Adhesive removal test of rats in the dose–
response study; (B) modified neurological
severity score of rats in the dose–response
study; (C) adhesive removal test of rats in
the time-of-administration study; and (D)
modified neurological severity score of rats
in the time-of-administration study. *P⬍0.05
as compared with the phosphate-buffered
saline-treated group. hUTC indicates
human umbilical tissue-derived cells.
eral BrdU-immunoreactive cells in the SVZ compared with
PBS-treated rats. Treatment with hUTC thus dosedependently increased SVZ cell proliferation in rats after
stroke (Figure 2A–B). In addition, treatment with hUTC at a
dose of 3⫻106 cells/injection significantly increased the
DCX-immunoreactive area in the ipsilateral SVZ (Figure
2C–D) compared with that of PBS-treated rats when the
treatment was initiated at 24 hours after stroke onset, indicating that hUTC treatment enhanced neuroblast concentration in the ischemic brain.
Double-staining immunohistochemistry revealed that
fewer than 5% of BrdU-positive cells were reactive for NeuN
and glial fibrillary acidic protein across groups. Treatment
with hUTC at doses of 3⫻106 did not significantly increase
Table.
the proportion of BrdU/NeuN (2.7%⫾0.5% versus
2.1%⫾0.5%) or BrdU/glial fibrillary acidic protein
(3.3%⫾0.5% versus 4.1%⫾0.8%) double-labeled cells compared with PBS-treated rats 60 days after stroke onset.
Treatments and Lesion Volumes for All Groups
Treatments
Lesion Volume (%
of Contralateral
Hemisphere)
Group 1
hUTC at 3⫻105 at Day 1 after MCA
34.9⫾2.2
Group 2
hUTC at 1⫻106 at Day 1 after MCA
34.3⫾2.3
Groups
(n⫽8/Group)
6
Group 3
hUTC at 3⫻10 at Day 1 after MCA
Group 4
hUTC at 1⫻107 at Day 1 after MCA
33.8⫾2.1
Group 5
PBS at Day 1 after MCA
34.9⫾2.6
Group 6
hMSC at 3⫻106 at Day 1 after MCA
34.6⫾2.2
6
33.2⫾1.9
Group 7
hUTC at 5⫻10 at Day 1 after MCA
31.3⫾2.6
Group 8
hUTC at 5⫻106 at Day 7 after MCA
33.0⫾2.4
Group 9
hUTC at 5⫻106 at Day 30 after MCA
31.3⫾2.1
Group 10
hUTC at 5⫻106 at Day 90 after MCA
34.0⫾2.5
Group 11
PBS at Day 30 after MCA
33.0⫾2.4
hUTC indicates human umbilical tissue-derived cells; MCA, middle cerebral
artery; hMSC, human bone marrow-derived mesenchymal stromal cells; PBS,
phosphate-buffered saline.
Figure 2. A, Representative micrographs showing BrdU immunoreactivity at the ipsilateral SVZ acquired from rats treated with PBS
(top panel) and hUTC at a dose of 3⫻106 cells (bottom panel); (B)
quantitative analysis of BrdU-positive cell density (number of cells/
mm2) in the ipsilateral and contralateral ventricle walls; (C) representative micrographs of DCX immunoreactivity at ipsilateral hemispheres from rats treated with PBS (left panel) and hUTC at a dose
of 3⫻106 cells (right panel); (D) quantitative analysis of DCX
expression in the SVZ. Bars⫽50 ␮m. *P⬍0.05 as compared with
the PBS-treated group. BrdU indicates bromodeoxyuridine; SVZ,
subventricular zone; PBS, phosphate-buffered saline; hUTC,
human umbilical tissue-derived cells; DCX, doublecortin.
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Zhang et al
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Figure 3. A, B, Micrographs of synaptophysin
immunoreactivity in representative rats treated with
PBS (A) or hUTC at a dose of 3⫻106 cells (B); (C)
quantitative analysis of synaptophysin expression
in the ischemic boundary zone of rats from the
dose-escalation study. The amount of synaptophysin is expressed as percentage area coverage vs
overall area. Bar⫽100 ␮m. *P⬍0.05 as compared
with the PBS-treated group. PBS indicates
phosphate-buffered saline; hUTC, human umbilical
tissue-derived cells.
Synaptogenesis
Administration of hUTC at doses of 3⫻106 and 1⫻107
cells/injection, but not at the lower doses of 3⫻105 and
1⫻106 cells/injection, as well as treatment with hMSC
significantly increased synaptophysin expression in the ischemic boundary area compared with that observed in animals
treated with PBS (Figure 3).
Vascular Density at the Ischemic Boundary Zone
Treatment with hUTC at doses of 3⫻106 and 1⫻107 cells/
injection, but not at the lower doses of 3⫻105 and 1⫻106
cells/injection, as well as treatment with hMSC significantly
increased vWF-immunoreactive vascular density in the ischemic boundary area compared with that observed in PBStreated rats at 60 days after stroke onset (Figure 4).
Apoptotic Cell Death
Scattered terminal deoxynucleotidyl transferase–mediated
dUTP-biotinic end labeling-positive cells were present
throughout the ipsilateral hemisphere. These cells were primarily located in the ischemic boundary area 60 days after
stroke onset. Treatment with hUTC at doses of 3⫻106 and
1⫻107, but not at the lower doses of 3⫻105 and 1⫻106, as
well as treatment with hMSC significantly reduced the
number of terminal deoxynucleotidyl transferase–mediated
dUTP-biotinic end labeling-positive cells compared with that
observed in PBS-treated rats. Treatment with hUTC thus
reduced apoptotic cell death in a dose-dependent manner
(Figure 5).
Discussion
Intravenous administration of hUTC in a rodent MCA model
24 hours after stroke dose-dependently promoted neurological functional recovery, which persisted at least 2 months
after stroke onset. Significant functional recovery was also
observed when treatment was initiated at Day 7 or 30 but not
at Day 90 after stroke onset. Treatment with hUTC enhanced
SVZ cell proliferation, synaptogenesis, and vessel density
and reduced apoptotic cell death in the ischemic boundary
area, which likely contributed to the improvement of neurological functional recovery in rats after stroke.
Prior research indicates that intracerebral implantation of
human umbilical cord-derived mesenchymal stem cells at 2
weeks after stroke onset reduces ischemic brain damage and
neurological functional deficits in rats, indicating a neuroprotective effect of these cells in experimental stroke.7 In the
present study, intravenous administration of hUTC improved
neurological function recovery without the reduction of
lesion volume compared with PBS-treated rats, thus suggesting that the improvement of neurological function observed
here was derived from neurorestorative effects in the ischemic boundary area, resulting in neurogenesis, synaptogenesis,
and angiogenesis. These regenerative processes have previously been shown to contribute to the functional recovery
after ischemic insult.24 –27 Neurogenesis is initiated with
neural progenitor cell proliferation after stroke.28 Treatment
with hUTC significantly increased the number of BrdUincorporating cells and the DCX-immunoreactive area in the
ipsilateral SVZ suggesting that hUTC treatment enhanced
Figure 4. A, B, Micrographs of vWF immunoreactivity at ischemic boundary zone (IBZ) acquired
from representative rats treated with PBS (A) or
hUTC at a dose of 3⫻106 cells (B); (C) quantitative
data of vWF-positive vessel density in the IBZ of
rats from the dose-escalation study. Bar⫽50 ␮m.
*P⬍0.05 as compared with the PBS-treated group.
vWF indicates von Willebrand factor; PBS,
phosphate-buffered saline; hUTC, human umbilical
tissue-derived cells.
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Figure 5. A, B, Micrographs of terminal deoxynucleotidyl transferase–mediated dUTP-biotinic end
labeling (TUNEL) staining at the ischemic boundary
zone (IBZ) acquired from representative rats
treated with PBS (A) or hUTC at a dose of 3⫻106
cells (B); (C) quantitative analysis of the density of
TUNEL-positive cells (absolute number of cells per
section) in the IBZ of rats from the dose-escalation
study. Bar⫽50 ␮m. *P⬍0.05 as compared with the
PBS-treated group. PBS indicates phosphatebuffered saline; hUTC, human umbilical tissuederived cells.
endogenous neurogenesis. Neuronal differentiation was further evaluated here by the presence, albeit in small number, of
BrdU-immunoreactive cells that also coexpressed NeuN, a
neuronal marker. In our study, there was no difference in
BrdU–NeuN coexpression results between hUTC- versus
PBS-treated animals. This may be due to the fact that the
BrdU pulse labeling methodology used here only allowed
detection of cell proliferation within the first 2 weeks poststroke and thus did not detect neuronal differentiation at later
stages. Based on these results, and despite the positive DCX
results, the effect of hUTC on neuronal differentiation remains to be determined. Therefore, the data associating
neurogenesis with functional recovery should be interpreted
with caution. In the present study, treatment with hUTC
increased synaptophysin immunoreactivity at the ischemic
boundary area, suggesting that hUTC treatment enhanced
synaptogenesis. As for angiogenesis, a physiological repair
process that naturally occurs in the ischemic brain,29 the
increased vWF-immunoreactive vessel density in the rats
treated with hUTC indicated that hUTC modulated the
vascular system. Collectively, our data therefore indicated
that intravenous administration of hUTC promoted endogenous neural progenitor proliferation, synaptogenesis, and
increased vessel density, which may explain how hUTC also
improved functional outcomes in the rats. Although multiple
prior studies have reported that implantation of exogenous
stem/progenitor cells from multiple sources can enhance
neurogenesis, synaptogenesis, and angiogenesis,13,25,30 –32 our
study is the first to suggest a similar outcome after intravenous administration of hUTC.
Apoptotic cell death has been identified in normal brain
throughout life and plays a critical role in maintaining tissue
homeostasis.33,34 Apoptosis is a common feature in neurogenic regions during development and in the adult brain, in
which the steady production of newly generated cells is
largely eliminated through an apoptotic mechanism.33 We
have previously demonstrated that stroke-induced apoptotic
cell death peaked at 1 to 2 days poststroke and persisted for
at least 4 weeks after stroke onset.35 In the present study,
hUTC treatment was associated with reduced apoptotic cell
death at 2 months after stroke onset compared with controls,
suggesting that hUTC treatment not only enhances SVZ cell
proliferation, but also reduces the apoptotic cell death in the
damaged brain tissue. Prior research has shown that, although
endogenous neurogenesis occurs in response to ischemic
injury, the majority (approximately 80%) of newly generated
cells die after stroke, indicating that apoptosis contributes
significantly to the fate of newly generated cells.31,36 Thus, it
is reasonable to speculate that by reducing apoptotic cell
death, hUTC treatment may improve the survival of newly
generated cells, an effect that might be linked to the functional recovery observed in the animals after the stroke. The
effects of intravenous hUTC treatment on the survival of
newly generated cells after stroke warrant further
investigation.
Previous studies have indicated that hMSCs are multipotent cells that, when implanted directly into the ischemic rat
brain, can integrate into the parenchymal brain tissue and
express neuronal markers 3 weeks after intracerebral transplantation.7,37 However, in the present study and after intravenous administration of hUTC, few human cells were
detected in the ischemic brain 2 months after treatment,
suggesting that cells were eliminated from the host brain over
time.
In support of this hypothesis is the fact that hUTC were
previously shown to elicit a host tissue immune response,
especially under inflammatory conditions.38 Stroke triggers
an inflammatory response, which contributes significantly to
the progression of brain damage.39 Thus, it is possible that
transplanted cells were scavenged by the host immune system. In the hemiparkinsonian rats, the engrafted human
umbilical cord matrix cells disappeared 6 weeks after transplantation, which is consistent with our finding.7,37 Therefore,
although hMSCs have the potential to differentiate into
parenchymal cells, the limited number of human cells observed in the present study suggests that neural transdifferentiation and fusion are unlikely the predominant mechanisms of action of intravenous administration of hUTC in
stroke.
Considering that functional recovery was observed in our
study, along with angiogenesis, synaptogenesis, and reduction of apoptotic cell death, it can be hypothesized that the
mechanism of action of intravenous hUTC administration is
linked to expression of neurotrophic factors from the cells.
The hUTC secrete several neurotrophic factors such as
brain-derived neurotrophic factor and fibroblast growth factor
Downloaded from http://stroke.ahajournals.org/ by guest on March 5, 2016
Zhang et al
2, all of which have the capacity to induce neurogenesis,
angiogenesis, and synaptogenesis after stroke.40,41 In addition, the antiapoptotic signaling pathway is activated by
neurotrophic factors.42 Thus, although not investigated directly here, the enhancement of host brain plasticity and
suppression of apoptosis observed in the present study are
likely attributable to the paracrine neurotrophic action of
hUTC. This hypothesis is supported by others who showed
that administration of hMSCs increased neurotrophic factor
expression, which in turn is known to augment host brain
plasticity and reduce apoptotic cell death in the experimental
stroke.14
Effectiveness of hUTC in a Rodent Stroke Model
10.
11.
12.
13.
14.
Summary
The intravenous administration of hUTC augmented endogenous neurorestorative responses and neurological functional
recovery with a time of administration window up to 30 days
after stroke onset in the rat MCA occlusion model. Intravenous administration of hUTC thus represents a novel treatment for further evaluation with a potential broad time of
administration window after the treatment of stroke.
Acknowledgments
We acknowledge Yisheng Cui, MD, Hongqi Xin, PhD, and Qi Gao,
PharmD, employees at Henry Ford Hospital; and Michael Romanko,
PhD, Brian Kramer, PhD, and Anthony Kihm, PhD, employees at
ATRM, for technical, editorial, and logistical support during the
conduct of this study.
15.
16.
17.
18.
19.
20.
Sources of Funding
The research presented here was funded by Advanced Technologies
and Regenerative Medicine, LLC.
21.
22.
Disclosures
K.H., Stroke Team Leader, and A.G., Senior Director of R&D, are
both employees of ATRM.
23.
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Delayed Administration of Human Umbilical Tissue-Derived Cells Improved Neurological
Functional Recovery in a Rodent Model of Focal Ischemia
Li Zhang, Yi Li, Chunling Zhang, Michael Chopp, Anna Gosiewska and Klaudyne Hong
Stroke. 2011;42:1437-1444; originally published online April 14, 2011;
doi: 10.1161/STROKEAHA.110.593129
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34
Stroke 日本語版 Vol. 6, No. 2
Abstract
げっ歯類の局所脳虚血モデルにおいてヒト臍帯組織由来細
胞の遅延投与が神経学的機能回復を改善する
Delayed Administration of Human Umbilical Tissue-Derived Cells Improved Neurological
Functional Recovery in a Rodent Model of Focal Ischemia
Li Zhang, MD1; Yi Li, MD1; Chunling Zhang, MD1; Michael Chopp, PhD1,2; Anna Gosiewska, PhD3;
Klaudyne Hong, PhD3
1 Department of Neurology, Henry Ford Hospital, Detroit, MI; 2 the Department of Physics, Oakland University, Rochester, MI; and 3 Advanced
Technologies and Regenerative Medicine, LLC, c/o Ethicon, Inc, Somerville, NJ
背景および目的：虚血性脳卒中患者の治療を成功させるた
めの神経保護戦略は短い治療適応時間が要件となるため，
大部分の治療が妨げられる。しかし，神経再生に基づく臨
床治療によりこの治療時間枠が延長され，それによりこれ
まで対応できなかった重要な課題に取り組むことができる
可能性がある。ヒト臍帯組織由来細胞は脳卒中の神経再生
治療薬候補として大きな可能性を示している。
方法：ヒト臍帯組織由来細胞の静脈内投与の有効性を，
げっ歯類の中大脳動脈脳卒中モデルを用いて，用量漸増試
験（検討した用量：3 × 105，1 × 106，3 × 106，または 1 ×
107 cells/ 回）とそれに続く投与時期に関する試験（脳卒中
後の時間：第 1 日，第 7 日，第 30 日，第 90 日に用量 5 ×
106 cells/ 回）において検討した。対照はリン酸緩衝食塩
水注射およびヒト骨髄由来間葉系間質細胞の注射とした。
治療後の転帰測定には改変神経学的重症度スコアおよび接
着除去試験を用いた。全例について組織診断を実施し，シ
ナプス形成，神経発生，血管新生，および細胞アポトーシ
スを評価した。
結果：脳卒中後最長 30 日目に 3 × 106 cells 以上の用量を
投与した場合，ヒト臍帯組織由来細胞ではリン酸緩衝食塩
水と比較して改変神経学的重症度スコアおよび接着剤試験
結果に統計学的に有意な改善が認められた。3 × 106 cells
以上の用量では，組織学的評価により，リン酸緩衝食塩水
の投与を受けた対照動物と比較してヒト臍帯組織由来細胞
の投与を受けた動物の虚血性境界域でシナプス形成，血管
密度の増大，およびアポトーシスの低下が確認され，また
脳室下帯における前駆細胞の増殖の増加が確認された。
結論：これらの結果は，リン酸緩衝食塩水の対照群と比較
した場合のげっ歯類脳卒中モデルにおけるヒト臍帯組織由
来細胞の静脈内投与の有効性を示すものであり，ヒトに対
する使用の可能性について研究を進める根拠となる。
Stroke 2011; 42: 1437-1444
A
PBS
B
hUTC
1000
800
400
200
LV
PBS
LV
stroke6-2.indb 34
*p ＜ 0.05
（8 例 / 群）
600
0
C
対側
PBS
hUTC hUTC hUTC hUTC hMSC
0.3M
1M
3M
10M
3M
hUTC
LV
D
DCX（＋）領域の割合（％）
（平均値± SE）
LV
細胞数 /mm2（平均値± SE）
同側
20
10
0
PBS hUTC
（ A ）PBS（上のパネル）および 3 × 106
cells の用量の hUTC（下のパネル）の投与を
受けたラットの同側 SVZ に BrdU 免疫反応
が認められる典型的な顕微鏡写真。（ B ）同側
および対側の脳室壁の BrdU 陽性細胞密度（ 1
mm2 あたりの細胞数）の定量的解析。（ C ）
PBS（左のパネル）および 3 × 106 cells の
用量の hUTC（右のパネル）の投与を受けた
図2
ラットの同側脳半球における DCX 免疫反応
を示す典型的な顕微鏡写真。（ D ）SVZ 内の
DCX 発現の定量的解析。バー＝ 50 μ m。
*PBS 投与群との比較で p ＜ 0.05。BrdU：
ブロモデオキシウリジン，SVZ：脳室下帯，
PBS：リン酸緩衝食塩水，hUTC：ヒト臍帯
組織由来細胞，hMSC：ヒト骨髄由来間葉系
間質細胞，DCX：ダブルコルチン。
11.9.27 10:54:06 AM

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