Timing of bone marrow cell therapy is more important than repeated injections after myocardial infarction.
ABSTRACT Bone marrow cell treatment has been proposed as a therapy for myocardial infarction, but the optimal timing and number of injections remain unknown.
Myocardial infarction was induced in mice followed by ultrasound-guided injection of mouse bone marrow cells at different time points post myocardial infarction (Days 3, 7, and 14) as monotherapy and at Days 3+7 as "double" therapy and at Days 3+7+14 as "triple" therapy. Controls received saline injections at Day 3 and Days 3+7+14. Left ventricular ejection fraction was evaluated post myocardial infarction prior to any therapy and at Day 28. Hearts were analyzed at Day 28 for infarct size and survival of donor cells.
Left ventricular ejection fraction decreased from 55.3±0.9% to 37.6±0.6% (P<.001) 2 days post myocardial infarction in all groups. Injection of bone marrow cells at Day 3 post myocardial infarction resulted in smaller infarct size (17.8±3.6% vs. 36.6±7.1%; P=.05) and improved LV function (left ventricular ejection fraction 40.3±2.0% vs. 31.1±8.3%; P<.05) compared to control. However, delayed therapy at Day 7 or 14 did not. Multiple injections of bone marrow cells, either double therapy or triple therapy, did not result in reduction in infarct size, but led to improvements in left ventricular ejection fraction at Day 28 compared to control (39.9±3.6% and 38.8±5.5% vs. 34.8±5.3%; all P<.05). The number of donor cells surviving at Day 28 did not correlate with improvement in left ventricular ejection fraction.
Injection of bone marrow cells at Day 3 reduced infarct size and improved left ventricular function. Multiple injections of bone marrow cells had no additive effect. Delaying cell therapy post myocardial infarction resulted in no functional benefit at all. These results will help inform future clinical trials.
- [Show abstract] [Hide abstract]
ABSTRACT: The ability of human pluripotent stem cells (hPSC) to differentiate into any cell type of the three germ layers makes them a very promising cell source for multiple purposes, including regenerative medicine, drug discovery, and as a model to study disease mechanisms and progression. One of the first specialized cell types to be generated from hPSC was cardiomyocytes (CM), and differentiation protocols have evolved over the years and now allow for robust and large-scale production of hPSC-CM. Still, scientists are struggling to achieve the same, mainly ventricular, phenotype of the hPSC-CM in vitro as their adult counterpart in vivo. In vitro generated cardiomyocytes are generally described as fetal-like rather than adult. In this review, we compare the in vivo development of cardiomyocytes to the in vitro differentiation of hPSC into CM with focus on electrophysiology, structure and contractility. Furthermore, known epigenetic changes underlying the differences between adult human CM and CM differentiated from pluripotent stem cells are described. This should provide the reader with an extensive overview of the current status of human stem cell-derived cardiomyocyte phenotype and function. Additionally, the reader will gain insight into the underlying signaling pathways and mechanisms responsible for cardiomyocyte development.Journal of Molecular and Cellular Cardiology 12/2013; · 5.15 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We investigate the extended (2+1)-dimensional shallow water wave equation. The binary Bell polynomials are used to construct bilinear equation, bilinear Bäcklund transformation, Lax pair, and Darboux covariant Lax pair for this equation. Moreover, the infinite conservation laws of this equation are found by using its Lax pair. All conserved densities and fluxes are given with explicit recursion formulas. The N-soliton solutions are also presented by means of the Hirota bilinear method.Chinese Physics B 05/2013; 22(5):050509. · 1.39 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Aging is associated with higher incidence of heart failure and death following myocardial infarction (MI). The molecular and cellular changes that lead to these worse outcomes are not known. Young and aging mice underwent induction of MI by LAD ligation. There was a significant increase in mortality in the aging mice. Neither the young nor aging hearts after MI had inducible ventricular tachycardia. Cardiomyocyte apoptosis increases early after MI in young and aging mice, but to a much greater degree in the aging mice. Caspase inhibition with Ac-DEVD-CHO resulted in a 61% reduction in activated caspase-3 and an 84% reduction in apoptosis in cardiomyocytes in young mice (P < 0.05), but not in aging mice. Gene pathway profiling demonstrated activation of both the caspase and Map3k1/Mapk10 pathways in aging mice following MI, which may contribute to their resistance to caspase inhibition. Aging hearts activate distinct apoptotic pathways have more cardiomyocyte apoptosis and are resistant to antiapoptotic therapies following MI. Novel or combination approaches may be required to improve outcomes in aging patients following MI.Cardiovascular Therapeutics 12/2013; 31(6):e102-10. · 2.85 Impact Factor
Timing of bone marrow cell therapy is more important than repeated
injections after myocardial infarction
Yan Zhanga, Richard E. Sieversa, Megha Prasada, Rachel Mirskya, Henry Shiha,
Maelene L. Wonga, Franca S. Angelia, Jianqin Yea, Junya Takagawaa, Juha W. Koskenvuoa,
Matthew L. Springera,b,c, William Grossmana, Andrew J. Boylea,c, Yerem Yeghiazariansa,c,⁎
aDivision of Cardiology, Department of Medicine, University of California, San Francisco, CA 94143-0103, USA
bCardiovascular Research Institute, University of California, San Francisco, CA 94143-0103, USA
cEli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, CA 94143-0103, USA
Received 7 December 2009; received in revised form 1 June 2010; accepted 21 June 2010
Background: Bone marrow cell treatment has been proposed as a therapy for myocardial infarction, but the optimal timing and number of
injections remain unknown. Methods: Myocardial infarction was induced in mice followed by ultrasound-guided injection of mouse bone
marrow cells at different time points post myocardial infarction (Days 3, 7, and 14) as monotherapy and at Days 3+7 as “double” therapy and
at Days 3+7+14 as “triple” therapy. Controls received saline injections at Day 3 and Days 3+7+14. Left ventricular ejection fraction was
evaluated post myocardial infarction prior to any therapy and at Day 28. Hearts were analyzed at Day 28 for infarct size and survival of donor
cells. Results: Left ventricular ejection fraction decreased from 55.3±0.9% to 37.6±0.6% (Pb.001) 2 days post myocardial infarction in all
groups. Injection of bone marrow cells at Day 3 post myocardial infarction resulted in smaller infarct size (17.8±3.6% vs. 36.6±7.1%; P=.05)
and improved LV function (left ventricular ejection fraction 40.3±2.0% vs. 31.1±8.3%; Pb.05) compared to control. However, delayed
therapy at Day 7 or 14 did not. Multiple injections of bone marrow cells, either double therapy or triple therapy, did not result in reduction in
infarct size, but led to improvements in left ventricular ejection fraction at Day 28 compared to control (39.9±3.6% and 38.8±5.5% vs.
34.8±5.3%; all Pb.05). The number of donor cells surviving at Day 28 did not correlate with improvement in left ventricular ejection
fraction. Conclusions: Injection of bone marrow cells at Day 3 reduced infarct size and improved left ventricular function. Multiple
injections of bone marrow cells had no additive effect. Delaying cell therapy post myocardial infarction resulted in no functional benefit at
all. These results will help inform future clinical trials. Published by Elsevier Inc.
Keywords: Mouse bone marrow cell; Myocardial infarction; Remodeling; Repeated injection; Intramyocardial injection
Bone marrow-derived cell treatment has been proposed as
a novel therapy for ischemic heart disease [1–4]. However,
the recent literature contains numerous contradictory results,
ranging from no improvement of cardiac function to
significant benefit in the contractile function after myocar-
dial infarction (MI) [3,5–14]. The exact mechanism(s) of the
possible benefits of cell-based therapies remains unclear
[3,5–7,10,15]. Additionally, it has been proposed that the
timing of stem cell therapy post-MI may influence whether
the cardiac function will improve after cell delivery. In the
largest randomized, multicenter, placebo-controlled, clinical
trial using bone marrow cells (BMCs) to date, the REPAIR-
AMI Trial, a significant improvement in left ventricular
ejection fraction (LVEF) was reported post-MI in those
patients with larger infarcts (e.g., LVEF prior to cell therapy
of b49%) or in those who received the cell therapy at least
Cardiovascular Pathology xx (2010) xxx–xxx
This work was supported in part by the UCSF Cardiac Stem Cell
Foundation to Y.Y. and a grant from the Wayne and Gladys Valley
Foundation to W.G. and Y.Y.
⁎Corresponding author. Division of Cardiology, Department of
Medicine, Box 0103, Eli and Edythe Broad Center of Regeneration
Medicine and Stem Cell Research, University of California, San Francisco,
CA 94143-0103, USA. Tel.: +1 415 353 3817; fax: +1 415 353 3090.
E-mail address: firstname.lastname@example.org (Y. Yeghiazarians).
1054-8807/10/$ – see front matter. Published by Elsevier Inc.
5 days after MI . No benefit was seen if therapy was
given within 5 days of the MI. More recently, Yao et al. 
reported in a small single-center study that therapy of
patients post-MI with repeated intracoronary administration
of BMC Days 3–7 post infarct and again at 3 months was
safe and more effective than single therapy at an early time
point. Whether the timing of therapy is indeed critical or
whether multiple injections of cell therapy post-MI result in
even more improvement of cardiac function is unknown and
these questions form the basis for this current research.
Small animal rodent models are frequently used to
assess the functional benefits of stem cell therapies
[5,10,15,17–20]; however, given the high mortality of
repeated open-chest surgery in these animals for cell
delivery, it has not been possible to reliably assess how
delayed cell therapy and multiple injections of stem cells
would affect the cardiac function post-MI. We have recently
developed a closed-chest ultrasound-guided intramyocardial
injection technique for use in the mouse MI model and this
approach has allowed us to perform studies at a clinically
more relevant time points by delivering the cells to the heart
several days post-MI, which mimics the conditions of
ongoing clinical trials [5,12,13,21,22]. Notably, the animal
mortality rate with this technique is almost zero, allowing
delayed or multiple injections to be undertaken successfully.
Using this approach, we have shown that injection of BMCs
3 days post-MI significantly improved cardiac function,
resulted in smaller left ventricular systolic and diastolic
volumes, and limited the extent of the infarct scar .
Despite these benefits, under our experimental conditions,
there was no evidence that donor cells (identified as GFP+
cells) differentiated into cardiomyocytes, and relatively few
implanted cells were detected several weeks later .
It has been suggested that there is a time window in early
post-MI in which delivery of cells will result in cardiac
functional improvement and that delaying this therapy longer
post-MI will have less or no benefit . In addition, the
effects of multiple injections of BMCs at different time
points post-MI on the cardiac function are unknown. This
study was designed to answer the following questions: (1)
Does the timing of BMC delivery post-MI affect the
outcome? (2) Do multiple cell injections post-MI have
additive benefits in improving cardiac function? These issues
have direct clinical implications.
to the guidelines of the Institutional Animal Care and Use
Committee of the University of California, San Francisco.
2.1. Myocardial infarction
MIs were induced surgically by a permanent ligation of
the left anterior descending coronary artery (LAD) as
previously described [21,24]. Briefly, permanent ligation
of the LAD is made by a 7-0 suture in the anterior
myocardium at 50% of the length of the heart from the
anterior–inferior edge of the left atrium to the apex. Each
group was allotted 10–12 animals for MI induction and eight
to nine survived to study completion without any differences
among the groups.
2.2. Collection of bone marrow cells
BMCs were harvested from 12- to 16-week-old C57BL/6
mice that constitutively express GFP [5,25]. The BMCs were
flushed from tibias and femurs with buffer (HBSS with 0.5%
BSA). The cell suspension was strained through a 70-μm
nylon filter and washed twice with buffer. The concentration
of the cells was adjusted to 105viable cells/μl, and the cell
viability was checked using Trypan blue with an average of
95% viability before injection.
Echocardiograms (Vevo 660, VisualSonics, Toronto,
ON, Canada) were obtained at baseline, 2 days post-MI
(before injection), and at Day 28 post-MI, and LVEF and LV
volumes [end-diastolic (EDV) and end-systolic (ESV)
volumes] were measured as previously described [5,26].
The analyses of the echocardiography images were
performed by a blinded investigator.
2.4. Ultrasound-guided cell delivery
Ultrasound-guided injection of cells into the myocar-
dium (Vevo 660) was performed as previously described in
detail [5,21]. Briefly, eight to nine hearts per group were
injected with 10 μl solution of either HBSS or BMCs (total
of 1×106cells divided into two adjacent 5 μl injections) into
the LV myocardium at different times post-MI as either
monotherapy (Day 3, 7, or 14) or at multiple time points as
follows: Days 3+7 as “double” therapy vs. Days 3+7+14 as
“triple” therapy (Fig. 1). Injections were targeted to the peri-
infarct border zone. Cardiac function was evaluated at Day
28 in all groups. Animals in the control group were
administered HBSS injections at Day 3 and also at Days 3+
7+14. We developed an echocardiographic scoring system
to assess successful injections: 1=no echocardiographic
evidence during or after injection; 2=contrast signal in the
LV cavity and no wall thickness changes notable during or
after injection; 3=either injection site wall thickening during
or after injection, or echo contrast enhancement at the
injection site during or after injection; 4=evidence of
injection site myocardial thickening and echo contrast
enhancement in the injected myocardium during or after
injection. Injections warranting a score of 3 or above were
considered to be successful. All animals were judged to be
optimally injected and none of the animals needed to be
removed from the study due to poor injections.
2Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
At Day 28, the hearts were injected with a saturated
solution of KCl (0.1 ml) to arrest in diastole. The hearts
were removed, and the left and right atria and large vessels
were resected. After a saline wash, the hearts were
immersed in 0.5% paraformaldehyde (PFA) solution for
1–2 h, then in 20% sucrose overnight. Hearts were then
embedded in OCT compound (Sakura Finetechnical USA,
Torrance, CA, USA), frozen in a bath of 2-methylbutane
with dry ice and stored at −80°C. The histologic analyses
were performed by investigators who were blinded to the
identity of the sections.
2.6. GFP Retention
Sections were first fixed in 1.5% PFA solution. The
fixed frozen sections were incubated in a Peroxidazed
solution (Biocare Medical, Concord, CA, USA) to quench
endogenous peroxidases followed by a 30-min incubation
in Rodent Block M (Biocare Medical) and rabbit
polyclonal antibody against GFP (Invitrogen/Molecular
Probes, 1:1000 dilution) for 1 h, washed with PBS, and
then incubated in an HRP-conjugated goat anti-rabbit IgG
(Biocare Medical) for 30 min. A negative staining control
lacking primary antibody was performed. Stain was
developed using DAB. GFP+ cells were then counted
manually in each of three zones: infarct zone (IZ), border
zone (BZ), and remote zone (RZ).
2.7. Infarct size
All histological sections were stained with Masson's
trichrome and quantified with a Nikon Eclipse E800
microscope using a 1× objective lens with the use of Openlab
software (Improvision, Lexington, MA, USA). ImagePro
Plus 6.0 software was used to measure lengths  and
infarcted LV areas. Scar size was analyzed at the mid-
papillary level by an investigator who was blinded to the
identity of the sections. The percent scar within the IZ was
determined by manually tracing the areas of interest and
measured automatically by the computer. IZ was defined as
an area continuously enclosed by scar spanning N50% of the
wall thickness. The area of the tissue staining positive with
analine blue was then traced manually and the percentage of
assess the circumferential extent of the infarct, the epicardial
and endocardial infarct lengths and epicardial and endocar-
dial circumferences were traced manually using ImagePro
Plus. Epicardial infarct ratio was obtained by dividing the
epicardial infarct length by the epicardial circumference.
Endocardial infarct ratio was calculated similarly. The
circumferential extent of the infarct was calculated as
[(epicardial infarct ratio+endocardial infarct ratio)/2]×100.
2.8. Measurement of vascularity
In order to assess vascularity, sections were stained for
the vascular markers CD31 and alpha smooth muscle actin
Fig. 1. Study scheme of the experiments. Myocardial infarction (MI) was induced in male mice by permanent mid-LAD ligation at Day 0. Ultrasound-
guided injection of mouse bone marrow cells (BMCs) (1×106/10 μl) to the peri-IZ was performed (n=8–9/group) at different time points post-MI (Days 3,
7, and 14) as monotherapy, and at Days 3+7 as double therapy and at Days 3+7+14 as triple therapy. Control group was injected with HBSS at Day 3 and
at Days 3+7+14.
3Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
(SMA). Sections were first fixed in 1.5% PFA solution.
After rinsing in distilled water, the sections were treated
with a peroxidase solution (Biocare). The sections were
rinsed with Tris buffered solution (TBS) and then treated
with a universal block (Rodent Block M, Biocare) for
30 min. A primary rat antibody against CD31 (Biocare,
1:50 dilution in Da Vinci Green Diluent) was applied to the
sections for 2 h at room temperature. A Rat Detection Kit
(Biocare) was used to detect the rat anti-CD31 antibody.
The stain was developed using DAB (Biocare). Sections
were again blocked using Rodent Block M and then stained
using a mouse primary antibody against alpha SMA
(Sigma-Aldrich, St. Louis, MO, USA; 1:200 dilution in
TBS) for 1 h, and a Mouse on Mouse Polymer (Biocare)
conjugated to alkaline phosphatase was applied to the
sections for 20 min at room temperature. The sections were
washed in TBS again, and the stain was developed using a
Ferangi Blue Chromogen Kit (Biocare). The negative
controls were treated using the same methodology, omitting
the primary antibody and replacing it with the Da Vinci
Green Diluent. Low power (4×) photomicrographs were
taken of three regions: IZ, BZ, and RZ. ImagePro Plus 6.0
software was used to select brown CD31+ staining after
manually selecting the region of interest, and the percentage
vessel area was obtained for each of the three regions.
Additionally, the number of vessels staining positive for
alpha SMA per high-power field (HPF) was counted within
each region. The number of vessels in the IZ and BZ
regions was adjusted by the number of vessels in the RZ
region for both small and large vessels.
Fig.2. Therapeuticeffect of BMCs at differenttime points,andmultiple vs.monotherapyon left ventricular function measuredby echocardiography.Injection of
BMCs at Day 3, Days 3+7, and Days 3+7+14 led to similar and significant improvements in LVEF at Day 28 compared to the control group (Pb.05). There were
no significant differences between these three groups at Day 28. Delaying the injection time to Day 7 or Day 14 post-MI did not improve LV function compared
to the controls. (A and D) LVEF: each line represents the mean of one experimental group. (B and E) ESV and (C and F) EDV at Day 28. Data are shown as
4 Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
2.9. Statistical analyses
Data were presented as mean±S.E.M. For the comparison
of echocardiographic parameters and histological data
between groups, ANOVA with LSD post hoc tests was
used. For the comparison of LVEFchanges at Day 2 and Day
28 within each group, paired t test was used. A P value of
b.05 was considered statistically significant.
3.1. Timing of therapy
The results of the monotherapy groups are described
first. In all groups, LVEF was uniformly reduced from an
average of 55.1±2.8% before MI to 37.9±1.3% (Pb.001) at
2 days post-MI (Fig. 2). At 28 days post-MI, LVEF
improved significantly in the Day 3 group compared to the
control group (40.3±2.0% vs. 31.1±8.3%, Pb.05). In
contrast, groups injected with BMC at Day 7 or Day 14
showed no improvement in LVEF compared to control
(35.5±6.1% and 34.5±3.9% vs. 31.1±8.3% respectively;
P=ns). LVESV and LVEDV were significantly smaller in
the Day 3 BMC injection group compared to the control
group (Pb.05; Fig. 2A–C), demonstrating less LV dilation
in this group. BMC injection at Day 7 reduced LVESV, but
not LVEDV, suggesting a modest beneficial effect when
cell therapy was delayed to Day 7, but no effect when
delayed to Day 14 post-MI. Of note, none of the animals in
any group suffered from sudden death using the echo-
guided delayed injections.
The infarct size was reduced when BMC therapy was
administered at Day 3 compared with control (17.8±3.6%
vs. 36.6±7.1%, P=.05) (Fig. 3A). BMC therapy at Day 7
or at Day 14 was not significantly different from control.
There appears to be a trend towards decreased benefit
with BMC therapy with delaying the injections when
comparing treatments at Days 3, 7, and 14 (Fig. 3A–B).
Early BMC therapy at Day 3 resulted in significantly
more viable tissue within the IZ (44.7±4.8%) compared to
monotherapies at Day 7 (29.2±4.2%; P=.03) and Day 14
(20.3±5.7%; P=.002) and control (27.1±9.1%; P=.01)
differences were observed between any of the treatment
groups for CD31+ vessel density in the IZ (Days 3, 7, and 14,
respectively; 0.66±0.26, 0.66±0.14; 0.58±0.22; P=NS) and
the BZ (0.72±0.10, 1.03±0.19; 1.1±0.2; P=NS) or the
number of SMA+ vessels per HPF in the IZ (Days 3, 7, and
14, respectively; 2.33±1.44, 1.88±0.6; 1.69±0.72; P=NS)
and the BZ (1.77±0.34, 4.10±1.66; 3.61±1.32; P=NS).
Fig. 3. Early therapy at Day 3 was associated with reduction in scar size. The infarct size was reduced when BMC therapy was administered at Day 3 compared
with Day 14 and control (A). Early therapy (Day 3) was associated with more viable myocardium in the IZ compared to Day 7 and Day 14 therapy, and control
(B). On the other hand, monotherapy at Day 3 was associated with reduction of infarct size (C) and an increase in viable tissue within the IZ at Day 28 (D)
compared with BMC triple therapy. Data are shown as mean±S.E.M.; n=8–9/group.
5Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
The numbers of GFP+ donor cells were quantified at
Day 28. No GFP+ cells were detected in the HBSS group.
The total number of GFP+ cells per section increased with
delayed injections comparing Days 3, 7, and 14, respectively
(2.33±1.28 vs. 5.57±3.61 vs. 29.17±11.88 cells per section)
(Fig. 4), and it was significantly higher in the Day 14 group
vs. the Day 3 group (Pb.05). The persistent presence of
donor BMCs does not correlate with the functional benefit of
cell therapy. The majority of GFP+ cells appear to localize
at the IZ and the least are present in the remote myocardium.
3.2. Multiple injections
In all groups, LVEF was uniformly reduced from an
average of 56.1±0.9% before MI to 37.5±1.1% (Pb.001) at
2 days post-MI (Fig. 2). At Day 28, LVEF improved
significantly with Day 3 monotherapy, Days 3+7 double
therapy, and Days 3+7+14 triple therapy groups compared to
HBSS control triple therapy group (40.3±2.0%, 39.9±3.6%
and 38.8±5.5%, vs. 34.8±5.3%, Pb.05) (Fig. 2D). However,
there was no significant difference between the triple and
double therapy groups vs. the Day 3 monotherapy group.
EDV and ESV were significantly preserved in the Day 3
monotherapy and double therapy groups vs. the HBSS triple
therapy group (all Pb.05) (Fig. 2E–F). However, the trend of
preservation of the left ventricular volumes in the triple
therapy group was not significant (P=.07). Of note, none of
the animals in any group suffered from sudden death using
the echo-guided repeated injections.
The infarct size was significantly reduced with BMC
monotherapy at Day 3 compared with triple therapy and
HBSS triple therapy control [17.8±3.6% vs. 37.8±8.1%
(P=.03) and 43.9±6.1% (P=.01)] (Fig. 3C). As shown in
Fig. 3D, in terms of the percent of viable tissue within the IZ,
monotherapy at Day 3 showed significantly more viable
myocardium (44.7±4.8%) compared to double therapy
(30.8±4.6%; P=.04), triple therapy (22.3±4.0%; P=.002),
and HBSS triple therapy (29.0±6.3%; P=.03).
In regard to blood vessel numbers at Day 28, capillary
density within the BZ was increased with triple therapy
(1.3±0.4 %) compared to monotherapy (Day 3) (0.72±0.1%,
P=.03). However, there were no significant differences in the
number of arterioles between groups (triple, double,
monotherapy Day 3, and control, respectively: 3.1±1.7,
2.1±0.9, 1.8±0.3, 1.8±1.2 per HPF).
No GFP+ cells were detected in the HBSS group. The
number of GFP+ cells was greater in triple therapy vs.
double therapy, and Day 3 monotherapy (34.0±21.5 vs.
5.33±2.47 and 2.33±1.28, respectively, both Pb.05). Most
donor BMCs were located in the IZ and least in the RZ. The
results show that the persistent presence of BMCs does not
correlate with the functional benefit of cell therapy.
In this study, we report the following: (1) delivery of cells
into the mouse myocardium many days post-MI can be
Fig. 4. Retention of GFP+ cells at the IZ at Day 28. The total number of GFP+ cells per section was greater in triple therapy vs. double therapy, Day 3 and Day 7
monotherapies (Pb.05). However, there was no significant difference between triple therapy and Day 14 monotherapy. Arrows demonstrate the presence of
GFP+ cells by brown DAB staining to avoid the artifacts of autofluorescence.
6Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
safely and successfully accomplished using the ultrasound-
guided technique without a need of resternotomy; (2)
multiple intramyocardial injections can be successfully
made with minimal mortality; (3) there appears to be a
critical time window in post-MI (around 3 days) during
which cell therapy is most successful in improving cardiac
function; and (4) multiple injections do not appear to have an
additive beneficial effect on cardiac function post-MI. In this
infarct model, double therapy at Days 3+7 or triple therapy at
Days 3+7+14 did not improve the cardiac function any more
than monotherapy at Day 3. The results also show that
despite persistence of BMCs, there is no additional
functional benefit with multiple injections.
The closed-chest ultrasound-guided approach has allowed
us to successfully perform repeated injections with almost
zero mortality and enabled us to address a number of
important and clinically relevant issues. Based on the
echocardiographic scoring system outlined in the Methods
section, all animals were judged to be optimally injected and
none of the animals needed to be removed from the study due
to poor injections. We found that injection at Day 3 defined
the best time window for therapy in the mouse infarct model
and that delaying the injection did not have any effect on
improving the heart function. This is of interest because Day
3 post-MI in the mouse model may correspond to
approximately Days 6–7 post-MI in humans when consid-
ering inflammatory changes [22,27], although direct extrap-
olation of the timing in the mouse model to the human
situation is not known.
As pointed out earlier, the REPAIR-AMI Trial 
reported on the benefit of cell therapy if the cells were
delivered 5 days or more after MI. In that study, most
patients with benefit of cell therapy were treated at Days 6–7
post-MI and it is unclear whether patients who received the
therapy much later post-MI had any benefit. Our results
would suggest that delaying the cell therapy too long post-
MI might no longer result in any clinical benefit. It is likely
that once the scar tissue begins to form several days after an
MI, the tissue is less receptive to therapies and thus has no
benefit. In fact, we have recently shown that early therapy
post-MI with BM-derived cells results in decreased cardio-
myocyte apoptosis and more blood vessel formation which
results in a smaller scar early after MI . It is notable that
too early a therapy post-MI may also be of no benefit as
shown in the REPAIR-AMI trial and recently reported in a
meta-analysis by Zhang et al. . The reason(s) for this is
not known, but it is possible that the inflammatory milieu
that is present immediately post-MI creates an environment
that may not benefit from the delivery of even more cells into
the myocardium. In our study, we did not test cell delivery at
the time of the infarct as this is not clinically very relevant
since bone marrow-derived cells are not available clinically
for use in patients at the time of their acute MI.
In addition to the timing of therapy, our current study is
the first to comprehensively assess the safety and efficacy of
repeated intramyocardial cell injections post-MI. We found
that multiple injections did not have an additive effect on
improving the heart function. This is consistent with the
Danish Stem Cell study, which was a prospective non-
randomized study assessing repeated intracoronary BMC
infusion in patients with ischemic heart disease . Even
though this study reported an association with symptomatic
relief and transiently improved exercise capacity, the authors
were unable to detect any improvement in LVEF or in the
wall motion score index. In fact, in our study, multiple
injections were associated with worsening trends in the left
ventricular volumes. One possibility for this is that repetitive
needle puncture may have resulted in significant local
damage at the peri-infarct myocardium in these small hearts,
and repeated injections could have disturbed the healing of
Our research also differs from the findings of Yao et al.
 that therapy of patients post-MI with repeated
intracoronary administration of BMC Days 3–7 post-MI
and again at 3 months was more effective than single therapy
at the early time point. The disparities may have resulted
from numerous differences in the study designs. Our study
involved a direct intramyocardial injection in the rodent
model in contrast to the intracoronary infusion in patients
used by Yao et al. . The studies also used different cell
fractions (whole BMCs vs. component mononuclear cells).
Notably, our study compared single to multiple deliveries
within the first several weeks, while their patients came back
3 months later after a first-week infusion.
We were able to detect the presence of the injected GFP+
cells in the treated groups. Interestingly, increased presence
of cells, either via delayed injection or via repeated
injections, did not show a direct correlation in terms of the
therapeutic functional benefit. It seems conceptually enticing
that more cells would result in greater benefit, but we could
not demonstrate this in our model . Notably, cell retention
in the heart even after direct intramyocardial delivery is poor.
We have previously shown a significant disappearance of
GFP transcripts over time despite the use of closed-chest
ultrasound-guided approach . Consistently, others have
also shown poor retention of stem cells in the heart after
direct cell delivery [29,30]. Yasuda et al.  have reported
that only 50% of cells remained in the myocardium after only
1 h and that cell survival progressively decreased further
over the 4-week study. In our study, while Day 3 therapy
leads to fewer surviving cells at Day 28, the cells (or their
extract as we have reported) exert their beneficial effects at a
critical time point post-MI. Thus, we propose that additional
injections are not as important as figuring out the best timing
of therapy post-MI.
It should be noted that in order to restrict analyses to a
constant time during the remodeling process post-MI
(28 days), the Day of functional analysis must necessarily
be at different times after BMC implantation; that is, 28 days
post-MI analysis of a Day 3 injection is 25 days post-
injection, whereas 28 days post-MI analysis of a Day 14
injection is 14 days postinjection. However, we feel that this
7Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
is the most valid approach because it is the only way to
compare different treatments at comparable stages of cardiac
remodeling. Moreover, we have previously reported that the
number of remaining BMCs declines rapidly  and it is
unlikely that additional therapeutic effect will be exerted
during the last 14 days.
The mechanisms underlying the beneficial effects of
BMC implantation into post-MI hearts are complex and
controversial. We and others have reported that BMCs can
aid in tissue preservation post-MI via a paracrine mecha-
nism. In our previous report , we demonstrated an early
increase in vascularity at Day 6 but not at Day 28, and that
injection of BMCs increased vessel area density and had a
higher number of arterioles in the infarct BZ. At Day 28, this
increase in vascularity equilibrated back to the expected level
in normal tissue, while the overall cardiac functional benefits
appear to have lasted for a longer time period. We
hypothesize that increased vascularity early after MI rescues
ischemic cardiomyocytes from apoptosis, thereby salvaging
myocardium. After the first few weeks post-MI, the viable
myocardial tissue and scar remodel to have their appropriate
vascular supply. What we see as the infarct BZ, then, in the
treated animals would have become scar tissue in the
untreated animals. Enhancing neovascularization post-MI
“moves” the BZ. These results are consistent with prior
studies by Schuleri et al.  who demonstrated that an early
increase in myocardial perfusion resulted in longer term
functional improvement. Indeed, these authors also show no
increase in capillary number after 8 weeks.
Our study has several limitations. First, the duration of the
study is only 28 days. Longer duration and survival studies
should be performed in the future, especially in the delayed
and multiple therapeutic arms. Secondly, earlier time points
before Day 3 or other time points post-MI were not tested to
allow successful completion of an already challenging
experimental design. Our experimental protocol was
designed before the results of the REPAIR-AMI Trial were
available and Day 3 post-MI was selected to mimic a
reasonable time period that a human subject may be treated
with bone marrow-derived cell therapies clinically post-MI.
Third, histology at early time points after cell delivery are not
available as the size of the study would have been too large
to include more animals for early sacrifice time points.
Fourth, it is unknown which stem cells hold the best
therapeutic promise in treating patients post-MI. Whether
other stem cells would have additive benefits with multiple
injections post-MI is unknown and this is outside the scope
of our current research. Lastly, this is a descriptive report and
our previous publication has addressed the detailed mecha-
nistic questions in regard to the cells which were used in this
manuscript and because of space limitations these data have
not been presented here .
In summary, we have shown that in the mouse MI
model, multiple injections of BMC did not have an additive
effect on improving LVEF post-MI compared to a single
therapy at Day 3. Moreover, further delaying the time of
cell therapy post-MI resulted in no functional benefit and, in
fact, led to a worse outcome. These results have direct
clinical implications and further clinical research is
warranted to delineate the optimal timing of cell therapy
in patients post-MI.
The authors acknowledge Mr. Petros Minasi for his
administrative support in conducting the experiments.
 Lee N, Thorne T, Losordo DW, Yoon YS. Repair of ischemic heart
disease with novel bone marrow-derived multipotent stem cells. Cell
 Reffelmann T, Konemann S, Kloner RA. Promise of blood- and bone
marrow-derived stem cell transplantation for functional cardiac repair:
putting it in perspective with existing therapy. J Am Coll Cardiol 2009;
 Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T,
Endresen K, Ilebekk A, Mangschau A, Fjeld JG, et al. Intracoronary
injection of mononuclear bone marrow cells in acute myocardial
infarction. N Engl J Med 2006;355(12):1199–209.
 Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV,
Kogler G, Wernet P. Repair of infarcted myocardium by autologous
intracoronary mononuclear bone marrow cell transplantation in
humans. Circulation 2002;106(15):1913–8.
 Yeghiazarians Y, Zhang Y, Prasad M, Shih H, Saini SA, Takagawa J,
Sievers RE, Wong ML, Kapasi NK, Mirsky R, et al. Injection of
bone marrow cell extract into infarcted hearts results in functional
improvement comparable to intact cell therapy. Mol Ther 2009;17(7):
 JanssensS, Dubois C, BogaertJ, Theunissen K,Deroose C, DesmetW,
Kalantzi M, Herbots L, Sinnaeve P, Dens J, et al. Autologous bone
marrow-derived stem-cell transfer in patients with ST-segment
elevation myocardial infarction: double-blind, randomised controlled
trial. Lancet 2006;367(9505):113–21.
 Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D,
Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of
ischemic myocardium by human bone-marrow-derived angioblasts
prevents cardiomyocyte apoptosis, reduces remodeling and improves
cardiac function. Nat Med 2001;7(4):430–6.
 Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R, Sheiban I,
Bartunek J, Vanderheyden M, Kim HS, Kang HJ, Strauer BE, et al.
Impact of intracoronary cell therapy on left ventricular function in the
setting of acute myocardial infarction: a collaborative systematic
review and meta-analysis of controlled clinical trials. J Am Coll
 Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S,
Hecker H, Schaefer A, Arseniev L, Hertenstein B, et al. Intracoronary
bone marrow cell transfer after myocardial infarction: eighteen
months' follow-up data from the randomized, controlled BOOST
(BOne marrOw transfer to enhance ST-elevation infarct regeneration)
trial. Circulation 2006;113(10):1287–94.
 Murry CE, Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO,
Rubart M, Pasumarthi KB, Virag JI, Bartelmez SH, Poppa V, et al.
Haematopoietic stem cells do not transdifferentiate into cardiac
myocytes in myocardial infarcts. Nature 2004;428(6983):664–8.
 Orlic D, Kajstura J, Chimenti S, JakoniukI, AndersonSM, Li B, Pickel
J, McKay R, Nadal-Ginard B, Bodine DM, et al. Bone marrow cells
regenerate infarcted myocardium. Nature 2001;410(6829):701–5.
 Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R,
Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, et al.
8Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx
Intracoronary bone marrow-derived progenitor cells in acute myocar-
dial infarction. N Engl J Med 2006;355(12):1210–21.
 Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R,
Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, et al.
Improved clinical outcome after intracoronary administration of bone-
marrow-derived progenitor cells in acute myocardial infarction: final
1-year results of the REPAIR-AMI trial. Eur Heart J 2006;27(23):
 Roncalli J, Leobon B, Massabuau P, Galinier M, Parini A, Pathak A,
Bourin P, Hagege AA, Menasche P, Fournial G, et al. Cardiac cellular
therapy: from cells to the first clinical uses. Arch Mal Coeur Vaiss
 Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F,
Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Mobilized bone
marrow cells repair the infarcted heart, improving function and
survival. Proc Natl Acad Sci U S A 2001;98(18):10344–9.
 Yao K, Huang R, Sun A, Qian J, Liu X, Ge L, Zhang Y, Zhang S, Niu
Y, Wang Q, et al. Repeated autologous bone marrow mononuclear cell
therapy in patients with large myocardial infarction. Eur J Heart Fail
 Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R, Torella
D, Tang XL, Rezazadeh A, Kajstura J, et al. Cardiac stem cells
delivered intravascularly traverse the vessel barrier, regenerate
infarcted myocardium, and improve cardiac function. Proc Natl Acad
Sci U S A 2005;102(10):3766–71.
 Kudo M, Wang Y, Wani MA, Xu M, Ayub A, Ashraf M. Implantation
of bone marrow stem cells reduces the infarction and fibrosis in
ischemic mouse heart. J Mol Cell Cardiol 2003;35(9):1113–9.
 Uemura R, Xu M, Ahmad N, Ashraf M. Bone marrow stem cells
prevent left ventricular remodeling of ischemic heart through paracrine
signaling. Circ Res 2006;98(11):1414–21.
 Urbanek K, Rota M, Cascapera S, Bearzi C, Nascimbene A, De
Angelis A, Hosoda T, Chimenti S, Baker M, Limana F, et al. Cardiac
stem cells possess growth factor-receptor systems that after activation
regenerate the infarcted myocardium, improving ventricular function
and long-term survival. Circ Res 2005;97(7):663–73.
 Springer ML, Sievers RE, Viswanathan MN, Yee MS, Foster E,
Grossman W, Yeghiazarians Y. Closed-chest cell injections into
mouse myocardium guided by high-resolution echocardiography. Am
J Physiol Heart Circ Physiol 2005;289(3):H1307–1314.
 Yang F, Liu YH, Yang XP, Xu J, Kapke A, Carretero OA. Myocardial
infarction and cardiac remodelling in mice. Exp Physiol 2002;87(5):
 Zhang S, Sun A, Xu D, Yao K, Huang Z, Jin H, Wang K, Zou Y, Ge J.
Impact of timing on efficacy and safety of intracoronary autologous
bone marrow stem cells transplantation in acute myocardial infarction:
a pooled subgroup analysis of randomized controlled trials. Clin
 Takagawa J, Zhang Y, Wong ML, Sievers RE, Kapasi NK, Wang Y,
Yeghiazarians Y, Lee RJ, Grossman W, Springer ML. Myocardial
infarct size measurement in the mouse chronic infarction model:
comparison of area- and length-based approaches. J Appl Physiol
 Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green
mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407(3):
 Zhang Y, Takagawa J, Sievers RE, Khan MF, Viswanathan MN,
Springer ML, Foster E, Yeghiazarians Y. Validation of the wall motion
score and myocardial performance indexes as novel techniques to
assess cardiac function in mice after myocardial infarction. Am J
Physiol Heart Circ Physiol 2007;292(2):H1187–1192.
 Fishbein MC, Maclean D, Maroko PR. The histopathologic evolution
of myocardial infarction. Chest 1978;73(6):843–9.
 Diederichsen AC, Moller JE, Thayssen P, Junker AB, Videbaek L,
Saekmose SG, Barington T, Kristiansen M, Kassem M. Effect of
repeated intracoronary injection of bone marrow cells in patients with
ischaemic heart failure the Danish stem cell study—congestive heart
failure trial (DanCell-CHF). Eur J Heart Fail 2008;10(7):661–7.
 Beeres SL, BengelFM, Bartunek J, Atsma DE, Hill JM, Vanderheyden
M, Penicka M, Schalij MJ, Wijns W, Bax JJ. Role of imaging in
cardiac stem cell therapy. J Am Coll Cardiol 2007;49(11):1137–48.
 Yasuda T, Weisel RD, Kiani C, Mickle DA, Maganti M, Li RK.
Quantitative analysis of survival of transplanted smooth muscle cells
with real-time polymerase chain reaction. J Thorac Cardiovasc Surg
 Schuleri KH, Amado LC, Boyle AJ, Centola M, Saliaris AP, Gutman
MR, Hatzistergos KE, Oskouei BN, Zimmet JM, Young RG, et al.
Early improvement in cardiac tissue perfusion due to mesenchymal
stem cells. Am J Physiol Heart Circ Physiol 2008;294(5):
9Y. Zhang et al. / Cardiovascular Pathology xx (2010) xxx–xxx