Brief UltraRapid Communication
Asymmetric Chromatid Segregation in Cardiac Progenitor
Cells Is Enhanced by Pim-1 Kinase
Balaji Sundararaman, Daniele Avitabile, Mathias H. Konstandin, Christopher T. Cottage,
Natalie Gude, Mark A. Sussman
Rationale: Cardiac progenitor cells (CPCs) in the adult heart are used for cell-based treatment of myocardial
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damage, but factors determining stemness, self-renewal, and lineage commitment are poorly understood.
Immortal DNA strands inherited through asymmetric chromatid segregation correlate with self-renewal of adult
stem cells, but the capacity of CPCs for asymmetric segregation to retain immortal strands is unknown.
Cardioprotective kinase Pim-1 increases asymmetric cell division in vivo, but the ability of Pim-1 to enhance
asymmetric chromatid segregation is unknown.
Objective: We aimed to demonstrate immortal strand segregation in CPCs and the enhancement of asymmetric
chromatid distribution by Pim-1 kinase.
Methods and Results: Asymmetric segregation is tracked by incorporation of bromodeoxyuridine. The CPC DNA
was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of
immortal versus newly synthesized strands. Intensity ratios of binucleated CPCs with bromodeoxyuridine of >70:30
between daughter nuclei indicative of asymmetric chromatid segregation occur with a frequency of 4.57%, and
asymmetric chromatid segregation is demonstrated at late mitotic phases. Asymmetric chromatid segregation is
significantly enhanced by Pim-1 overexpression in CPCs (9.19% versus 4.79% in eGFP-expressing cells; Pⴝ0.006).
Conclusions: Asymmetric segregation of chromatids in CPCs is increased nearly two-fold with Pim-1 kinase
overexpression, indicating that Pim-1 promotes self-renewal of stem cells. (Circ Res. 2012;110:1169-1173.)
Key Words: asymmetric cell division 䡲 asymmetric chromatid segregation 䡲 bromodeoxyuridine 䡲 cytochalasin B
䡲 immortal DNA strand hypothesis
T
silent sister hypothesis also is supported by the correlation of
ACS with asymmetric cell division, self-renewal, and fate
determination.7–10 Discovery of resident adult stem cells specific
to the heart, termed cardiac progenitor cells (CPCs),12 have
opened new possibilities for cell-based treatment of heart
disease that could be mitigated by improved myocardial
regeneration.13 CPCs found in cardiac niches are c-kit–
positive, clonogenic, multipotent, self-renewing, and capable
of asymmetrical division.12,14 Results of a phase I clinical trial
with CPCs are promising,15 but clinical potential of CPCs is
limited by donor age and disease state.16,17 CPCs in aged
myocardium are senescent, with limited replication potential.17 Therefore, a subset of CPCs capable of ACS would be
desirable to preserve youthful self-renewal of the stem cell
he immortal strand hypothesis states that adult stem cells
selectively retain chromatids with old DNA strands (the
mother strand), whereas all differentiating progeny acquire
the newly synthesized daughter to protect stem cells from
DNA replication errors1 and aging.2 The silent sister hypothesis presumes template strand segregation of all or some
chromatids to regulate gene expression to determine cell
fate.3,4 The immortal strand and silent sister hypotheses are
supported by observations of absolute to partial asymmetric
chromatid segregation (ACS) in adult stem cells like mouse
intestinal and colon epithelial stem cells,5,6 neural stem cells,7
satellite cells8,9 and many cancer stem cells.10,11 Stem cells
also may undergo symmetric segregation on an intermittent
basis and switch back to asymmetric segregation.7,8,10,11 The
Original received February 22, 2012; revision received March 12, 2012; accepted March 13, 2012. In February 2012, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13.77 days.
Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive
observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, Brief UltraRapid
Communications present seminal findings that have the potential to open up new avenues of research. A decision on Brief UltraRapid Communications
is rendered within 7 days of submission.
From the SDSU Heart Institute and Biology Department, San Diego State University, 5500 Campanile Drive, San Diego, CA.
This manuscript was sent to Ali J. Marian, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.267716/-/DC1.
Correspondence to Mark A. Sussman, SDSU Heart Institute and Biology Department, San Diego State University, 5500 Campanile Drive, San Diego,
CA 92182. E-mail [email protected]
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.112.267716
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Circulation Research
April 27, 2012
Non-standard Abbreviations and Acronyms
ACS
BrdU
CPC
CPCe
CPCeP
asymmetric chromatid segregation
5-bromo-2⬘-deoxyuridine
cardiac progenitor cell
CPC expressing eGFP
CPC expressing eGFP and Pim-1
phenotype. ACS in CPCs is enhanced two-fold in our study
by overexpression of Pim-1, a kinase that enhances cardiac
repair,18 increases asymmetric cell division,19 and promotes
CPC self-renewal.
Editorial, see p 1154
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Methods
Cell culture, treatments, BrdU immunostaining, and image quantification are described in the detailed Methods provided in the Online
Supplement. Experimental design for label release and retention
assay are explained and depicted in Online Figure I.
Label Release Assay
“Immortal” strands are never labeled, as per the initial postulate of
Cairns.1 Short-time BrdU labeling for a single S-phase results in
CPCs with one labeled and one unlabeled DNA strand (Online
Figure IA). After completion of mitosis when cells are chased in
BrdU free media, chromatids with unlabeled stands segregate from
chromatids with newly made BrdU-labeled strands. Therefore, stem
cells release the label because of retention of the oldest immortal
strands not being labeled throughout the assay.7,11
Label Retention Assay
Long-term BrdU exposure of CPCs allows for labeling of both
DNA strands (Online Figure IB), assuming switching occurs
between symmetric and asymmetric segregation as previously
published.6 – 8,10,11 After completion of a single mitotic division,
chasing cells in BrdU free medium results in CPCs with one labeled
and one unlabeled strand in a chromatid. After a second round of
mitosis, labeled immortal strands will segregate from the unlabeled
new strands; therefore, label retention will occur for CPCs continuing to asymmetrically segregate their chromatids.6,7,10
Results
Cell-cycle distribution of CPCs after drug-induced mitotic
synchronization of the cell population is shown in Online
Figure II. Use of a label release assay (Online Figure IA) was
devised to demonstrate presence of unlabeled “immortal
stands.” Asymmetric segregation of unlabeled immortal
strands from BrdU-labeled newly synthesized strands is
revealed by rare binucleated CPCs with one daughter nucleus
having little or no BrdU staining, whereas the other nucleus is
positive for BrdU (Figure 1A). CPCs exhibiting daughter
nuclei with equal BrdU intensity indicate symmetrical segregation (Figure 1B).
Label retention assay (Online Figure IB) validates results
of the release assay that is limited to CPCs undergoing DNA
synthesis at the time of labeling. Both strands are BrdUlabeled in the label retention assay, in which CPCs are
continuously labeled and passaged at least four times to dilute
slowly cycling and exclusively asymmetrically segregating
CPCs.6 – 8,10 Daughter nuclei with approximately 50:50
BrdU intensity ratio (only symmetric segregation) are observed in CPCs blocked at first mitosis during chase (Figure
2A), consistent with model predictions (Online Figure IB).
Both symmetric and asymmetric BrdU staining is observed at
second mitosis (Figure 2B–D). Asymmetric segregation of
BrdU-labeled chromatids is demonstrated by binucleated
cells having daughter nuclei with disproportionate BrdU
intensity (Figure 2B, D). CPCs never exhibited a completely
negative paired with positive BrdU daughter nuclei pair,
consistent with previous reports of punctate BrdU staining.10,20 Therefore, ACS is defined as a daughter nuclei pair
having BrdU ratio of ⱖ70:30, similar to chromosome in situ
hybridization experiment,5 instead of absolute ratio (100:0),
confirming that not all chromatids are asymmetrically segregated.4,21,22 CPCs with asymmetric segregation of chromatids
with BrdU intensity ratio of ⱖ70:30 between the daughter
nuclei are found in 4.59% of the population (n⫽4; 53–194
binucleated cells per experiment). CPCs during late stage
mitosis collected through mitotic shake-off exhibit asymmetric (Figure 3A) and symmetric BrdU intensity (Figure 3B),
demonstrating that ACS occurs without requiring druginduced cell-cycle arrest. Intensity differences between
daughter nuclei are not attributable to differences in focal
plane, as confirmed by confocal optical sectioning (Figure
3C), validating asymmetrical segregation of BrdU-labeled
chromatids.
CPCs overexpressing Pim-1 and enhanced green fluorescent protein (CPCeP) or enhanced green fluorescent protein
alone (CPCe) were used to test the capacity of Pim-1 to
Figure 1. Cardiac progenitor cells
(CPCs) retain unlabeled immortal
strands in label release assay. A,
Binucleated cell with one daughter
nucleus exhibiting low BrdU immunostaining (red arrowhead) and another showing
intense BrdU signal. B, Binucleated cell
with daughter nuclei exhibiting symmetric
BrdU immunostaining. Binucleated CPCs
are confirmed by reflection scanning (cell).
Scale bar⫽10 ␮m.
Sundararaman et al
Pim-1 and Chromatid Segregation
1171
Figure 2. Cardiac progenitor cells
(CPCs) asymmetrically segregate BrdUlabeled chromatids. A and B, BrdU
intensity distribution among daughter
nuclei in binucleated cells at first (A) and
second (B) mitosis during chase in label
retention assay. The 70:30 distribution
threshold for BrdU intensity ratio is shown
as dotted box in (B), and events above
this threshold are considered asymmetrically segregating cells. C and D, Representative binucleated cells with symmetric
(C) and asymmetric (D) BrdU immunostain.
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enhance ACS by label retention assay. Morphological differences were not observed between CPCs, CPCe, CPCeP,
indicating that the genetic modification does not significantly
affect cell function. Binucleated CPCs with daughter nuclei
exhibiting marked differential BrdU intensity are observed in
both CPCe and CPCeP (Figure 4A). Asymmetric segregation
of chromatids is increased nearly two-fold in CPCeP (9.19%;
Figure 3. Asymmetric BrdU segregation at late mitotic stages.
A, A telophase cell with symmetric BrdU staining. B, An anaphase
cell with asymmetric BrdU segregation. C, Z stacks of BrdU immunostaining of (B). Mitotic phases are confirmed by differential interference contrast (DIC) images, labeled as cell.
n⫽5; 72–135 binucleated cells per experiment) compared to
CPCe (4.79%; P⫽0.006; Figure 4B–D) with no significant
difference between nontransduced control versus CPCe
(4.59% versus 4.79%; P⫽0.375).
Discussion
Autologous cell therapy with CPC is an efficacious treatment
for heart failure.15 Beneficial effects of naïve cells are
improved by modification and selection to promote reparative
potential in the harsh milieu of a postinfarct heart.16 Modification with Pim-1 facilitates CPC survival and proliferation
after infarction injury,18,23 in addition to numerous other
signaling molecules and pathways that facilitate stem cell–
mediated repair.16 However, CPC-mediated regeneration
could be further augmented by identification of a cell subset
that possesses enhanced potential for self-renewal as evidenced by ACS. Therefore, there is a need to identify signal
transduction cascades that enhance ACS to confer superior
self-renewal properties.
The presence of immortal/template strands and ACS in
adult stem cells remains controversial.2– 4 The immortal
strand hypothesis assumes ability of stem cells to retain old
strands to minimize transformational risk associated with
DNA replication errors.1 Our findings of predominant rather
than total ACS are consistent with direct observation of
chromatid segregation5 and punctate BrdU retention in nonmyocardial stem cell studies,10,20 indicating partial ACS
might be specific to stem cell type. The silent sister hypothesis presumes ACS to regulate differential gene expression
profile to determine cell fate and self-renewal,3,4,24 which is
strengthened by the observation of cosegregation of template
strands with self-renewal markers in stem cells7–10 and of
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April 27, 2012
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Figure 4. Pim-1 kinase
increases asymmetric DNA
segregation. A, Representative
binucleated cell at second
mitosis for cardiac progenitor
cells (CPCs) expressing eGFP
(CPCe; upper) and CPCs
expressing eGFP and Pim-1
(CPCeP; lower) with asymmetric BrdU staining indicated by
red arrowhead. B and C,
BrdU distribution at second
mitosis between daughter
nuclei of CPCe and CPCeP,
respectively. Dotted box represents 70:30 threshold of BrdU
intensity ratio. D, Percentage of
asymmetrically segregating
cells from five independent
experiments. *P⫽0.006 compared to CPCe.
conformational changes in histone variant H2A.Z on template
strands.20 Functional significance of ACS through study of
live cells is technically challenging because of protocols of
fixation and immunolabeling required for detection by fluorescent cell sorting to isolate asymmetrically segregating
cells, although utilization of directly labeled thymidine analogues recently has been used in cancer stem cells to
circumvent fixation requirements.25,26 Future studies will
capitalize on the functional characterization of asymmetrically segregating CPCs to determine their regenerative potential relative to comparable CPCs undergoing symmetric
segregation, with the expectation that asymmetric segregation
correlates with enhanced myocardial repair.
Previous research in our laboratory established multiple
beneficial roles for Pim-1, including enhancement of proliferation, survival, and commitment of CPCs without causing
tumor formation after transplantation.18,19 Pim-1–mediated
increase in ACS can be explained by the multifaceted
participation of Pim-1 in mitosis and transcriptional regulation. Pim-1 colocalizes at spindle poles during mitosis to
phosphorylate NuMA and promote complex formation with
HP1␤, dynein, and dynactin during chromosome segregation.27 The left–right dynein motor is necessary for selective
segregation of mouse chromosome 7.22 Pim-1 also binds and
phosphorylates HP1␥ on serine-rich clusters, modulating its
transcriptional repression activity.28 Pim-1–mediated phosphorylation of HP1 proteins, which are involved in chromatin
structure, indicates that Pim-1 likely modulates chromatin
dynamics. Chromatin dynamic regulation by Pim-1 could
explain the functional significance of ACS in conjunction
with the silent sister hypothesis and stem cell self-renewal.
Pim-1 increases asymmetrically dividing CPCs in the heart
after pathological injury to balance self-renewing versus
differentiated cell populations.19 Pim-1 decreases spontaneous differentiation of embryonic stem cells29 and is highly
expressed in long-term populating hematopoietic stem cells,30
supporting the premise that Pim-1 regulates stemness. Future
studies are warranted to address the direct role of Pim-1 in
ACS and stem cell self-renewal to further enhance the
regenerative potential of CPCs and other stem cell types.
Acknowledgments
The authors thank the Sussman laboratory members for their critical
review of the manuscript, and Brett Hilton of SDSU FACS Core
facility for his help in flow cytometry analysis.
Sources of Funding
M.A.S. was supported by National Heart, Lung, and Blood Institute
grants 1R21HL102714-01, 2 R01 HL067245, IR37 HL091102-01, 5
P01HL085577-05, RC1HL100891-02, 1 R21 HL102613-02, 1 R21
HL102714-02, 1 R21 HL104544-02, and R01 HL105759-01. C.T.C.
is supported by the Rees-Stealy Research Foundation, the Achievement Rewards for Colleges Scientists Foundation, American Heart
Association Predoctoral Fellowship 10PRE3060046, and an Inamori
Foundation Fellowship. M.H.K is supported by Deutsche Forschungsgemeinschaft (DFG) grant KO 3900/1-1.
Disclosures
None.
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Novelty and Significance
What Is Known?
●
●
Preferential partitioning of old versus newly synthesized DNA strands
known as asymmetric segregation is hypothesized to regulate
self-renewal and aging in several cell types.
The cardioprotective kinase Pim-1 increases proliferation, asymmetric
cell division, and regenerative potential of cardiac progenitor cells
(CPCs).
What New Information Does This Article Contribute?
●
●
Asymmetric partitioning of old versus newly synthesized DNA formed
during mitosis occurs in a small subpopulation of CPCs.
Asymmetric segregation of chromatids into daughter CPCs is enhanced by Pim-1 kinase overexpression.
CPCs are potentially effective as agents of cell-based regenerative treatment for myocardial damage. However, factors that
determine self-renewal and lineage commitment of CPCs are
poorly understood. The regenerative potential of aged CPCs may
be compromised in elderly patients with chronic heart disease.
Identification and characterization of subsets of CPCs that
exhibit preservation of proliferative potential and enhanced
self-renewal might be valuable for use in the clinical setting to
augment stem cell– based repair. Superior self-renewal properties and protection from phenotypic characteristics of aging are
associated with asymmetric chromatid segregation wherein old
DNA strands preferentially distribute to a single daughter cell
during mitosis. This study confirms that asymmetric DNA
segregation occurs in CPCs at a low frequency consistent with
rates observed for other adult stem cell types. Moreover, the
frequency of asymmetric DNA segregation in CPCs can be
significantly increased by overexpression of Pim-1 kinase. The
observation of increased asymmetric segregation with Pim-1
overexpression may help explain the enhanced regenerative
properties of CPCs engineered to overexpress Pim-1 and provide
another mechanistic basis for increasing self-renewal of CPCs
that might be essential for effective myocardial repair.
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Asymmetric Chromatid Segregation in Cardiac Progenitor Cells Is Enhanced by Pim-1
Kinase
Balaji Sundararaman, Daniele Avitabile, Mathias H. Konstandin, Christopher T. Cottage,
Natalie Gude and Mark A. Sussman
Circ Res. 2012;110:1169-1173; originally published online March 22, 2012;
doi: 10.1161/CIRCRESAHA.112.267716
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2012/03/22/CIRCRESAHA.112.267716.DC1
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1
Supplemental Material
Detailed Methods
Cell culture
Mouse CPCs are isolated, maintained and lenti virally engineered to stably over express
either eGFP alone (CPCe) or eGFP and human Pim-1 kinase (CPCeP) together as bicistronic
transcripts by previously established methods1. For label release and retention assays cells
were plated at 250 cells/mm2. Cells were labeled with 10 μmol/L BrdU for one generation or with
1 μmol/L BrdU for longer labeling period. BrdU concentration used to label DNA replication is
well below typical concentrations used to track chromosomal activities such as DNA repair (30100 μmol/L).
Cell cycle analysis and treatments
Cells were fixed in 70% ethanol and stored overnight at -20°C. Cell cycle distribution
was analyzed by staining with propidium idodie (PI) RNase staining buffer (BD) at 37°C for 3
hours using BD FACSCanto and data processed by FlowJo software. Cells were incubated with
2.5 mmol/L thymidine for indicated times in full medium to block in G1-S phase. Cells were
released by trypsinizing and washing twice with PBS and plated in full medium for synchronized
progression of cell cycle. 2 μmol/L cytochalasin B was used whenever necessary to block in
cytokinesis. For mitotic shake-off cells were grown at 70% confluence, blocked at S phase by
2.5 mmol/L thymidine blocking for 24 hours, released in thymidine free media for 18 hours.
Loosely adherent mitotic cells were collected by gently knocking the plate against a hard
surface once in every 1.30 hours for up to 5-6 times after thymidine release and cells were fixed
in 70% ethanol.
Cell cycle analysis of CPCs indicated most of the cells complete mitosis 12-18 hours
after thymidine removal. Thymidine block for 30 hours and releasing immediately in cytochalasin
B for up to 24 hours blocks cells in first cytokinesis. Release in thymidine free medium for first
24-30 hours followed by cytochalasin B for another 30 hours block cells in second cytokinesis.
These conditions are followed to block cells in first and second mitosis during chase period in
BrdU free media to see asymmetric DNA segregation.
2
BrdU staining
Cells were fixed in 70% ethanol, stored at 4°C until use. Cells were denatured with 2N
HCl/0.2% Triton-X100 for 1 hour at RT, then neutralized with 0.1 mol/L TrisCl pH 8.5 and
blocked with 10% horse serum. For detecting BrdU, mouse-anti-BrdU (clone B44, BD # 347580)
antibody was used at 1:100 dilution, 1 hour at RT followed by donkey-anti-mouse IgG-549
(1:150) as secondary antibody. Cells were mounted in Vectashield containing 1:1000 of Sytoxblue nuclear dye and scanned using Leica SP2 confocal microscope. Images were acquired
using 20X lens with 2X electronic zoom for BrdU measurement and 15X electronic zoom for
high resolution single cell panels. Differential interference contrast (DIC) images were acquired
to confirm binucleated cells.
Image processing and density measurements
All images and panels were identically processed. ImageJ software was used for BrdU
intensity measurements. Threshold levels of the single scan images were set using auto adjust
then the image color was inverted. Mean gray value (d) of two daughter nuclei of a binucleated
cell was measured separately and multiplied by their corresponding area (A). Fraction of BrdU
intensity of individual daughter nuclei to the total (T= A1*d1 + A2*d2) was plotted (D1= A1*d1/T
versus D2=1-D1).
Method of deriving tumor free Pim-1 CPCs
Recent results from our lab indicate that Pim-1-mediated proliferation of CPCeP is transient.
Increased proliferation of CPCeP observed at early passages diminishes to normal levels with
repeated cell passage without loss of Pim-1 expression (manuscript in preparation). We are also
exploring alternative non-lentiviral based methods for Pim-1 gene delivery including episomal
minicircle vectors and cell penetrant proteins.
3
Online Figure I
Online Figure I. Experimental design for label release and retention assay. A, In label
release assay, cells are labeled for 6hrs and then chased in BrdU free media for 24hrs followed
by a cytochalasin B treatment to induce binucleated cells. By retaining the unlabeled ‘immortal
strands’, the stem cell will lose the BrdU labeled chromosomes. B, In the label retention assay
cell are labeled for up to 15-25 days and chased for two consecutive cells divisions. Stem cells
will retain the BrdU labeled immortal strands for ever while the daughter cells dilute the label.
4
Online Figure II
Online Figure II. Cell cycle distribution of CPCs with drug treatments. Establishing CPCs’
cell cycle kinetics is necessary to successfully block at specific mitosis. Cell cycle distribution is
determined by propidium iodide staining and flow cytometry. Population distribution of DNA
content is plotted to examine different phases of cell cycle. CPCs grown without any drugs or
treatment showed that more than 70% are in G1 and 5% of cells in G2 phase of cell cycle (Ctrl).
Thymidine treatment for 30 hours blocked almost all cells in G1-S phase boundary (T30C0).
Cells grown with thymidine (T) are washed and plated immediately with medium containing 2μM
cytochalasin B (C) and fixed at six hour intervals. Cytochalasin blocks cells in cytokinesis
preventing from further G1 phase, creating binucleated cells (cells with 4n DNA content). Six
hours after thymidine removal, cells progressed to S phase (T30C6) and by 12 hours a small
percentage of cells entered G2 phase. Eighteen hours after thymidine removal the majority of
cells entered G2/M phase and by 24 hours more than 85% cells completed mitosis but are
binucleated (T30C24). Analysis at thirty hours after thymidine removal and cytochalasin
treatment confirmed that cells are permanently blocked in G2.
Supplementary references
1.
Fischer KM, Cottage CT, Wu W, Din S, Gude NA, Avitabile D, Quijada P, Collins BL, Fransioli J,
Sussman MA. Enhancement of myocardial regeneration through genetic engineering of cardiac
progenitor cells expressing pim-1 kinase. Circulation. 2009;120:2077-2087