Stem Cells and the
Cardiovascular System
20 October 2011
Robert Siggins, Ph.D.
stemcellschool.com
Outline and Objectives
• What are stem cells (SCs)?
• Which SCs contribute to the adult CV
system?
• What are the physiological and
pathophysiological roles for SCs in the
CV system?
Stem Cell “Theory”
• 1855  First eluded to by (Remak) Virchow 
Omnis cellula e cellula
– RediOmne vivum ex ovo
• 1875  ”Embryonic rest” proposed by Cohnheim
(cancer stem cells)
• 1917  HSC proposed by Pappenheim
• 1961  McCulloch and Till describe CFU-s
Definition of SCs
• Totipotent, pluripotent, or multipotent
• Self-renewal
• Clonogenic
• Niche
http://icanhascheezburger.com
Totipotent = ability to give rise to all
cells of the body and all cells that
constitute the extraembryonic
tissues (i.e. placenta)
Self-renewal
Pluripotent = ability to make all cells
of the body (all three germ layers)
Multipotent = ability to
differentiate into more than
one cell type (usually germ
layer restricted)
autismpedia.org
Embryonic SCs (ESCs) are pluripotent
Tissue-restricted SCs are Multipotent!
stemcellresources.org
stemcellresources.org
toonpool.com
UEA-1 (ulex europaeus agglutinin-1) or SSEA-4 (stage-specific
embryonic antigen-4) eliminates teratoma-initiating cells!
Physiol Rev 85:1373-1416, 2005
Definition of SCs
• Totipotent, pluripotent, or multipotent
• Self-renewal
• Clonogenic
• Niche
Symmetric and Asymmetric Divisions
http://xarquon.jcu.cz/edu/hematologie/03kmenove_bunky/03stem_lineages.htm
Cell Extrinsic and Intrinsic Models
Regulated by niche
attachment
Regulated by niche
cell polarity
Regulated by
intrinsic polarity
Asymmetric Divvision
Definition of SCs
• Totipotent, pluripotent, or multipotent
• Self-renewal
• Clonogenic
• Niche
Cardiac
Stem Cells
(CSCs)
Large clone
of c-kit+ cells
(green)
generated by
a single rat
c-kit+ CSC.
Physiol Rev
85:1373-1416, 2005
Cardiac
Stem Cells
(CSCs)
Clonogenic
cells in
differentiating
medium
acquire the
myocyte
phenotype (αsarcomeric
actin, red).
Physiol Rev
85:1373-1416, 2005
Cardiac
Stem Cells
(CSCs)
Clonogenic
cells acquire
the SMC
phenotype
(α-smooth
muscle actin,
magenta).
Physiol Rev
85:1373-1416, 2005
Cardiac
Stem Cells
(CSCs)
Clonogenic
cells acquire
the EC
phenotype
(vWF, yellow).
Physiol Rev
85:1373-1416, 2005
Definition of SCs
• Totipotent, pluripotent, or multipotent
• Self-renewal
• Clonogenic
• Niche
Niches are
specialized
microenvironmnets
comprising support
cells, soluble
factors, ECM,
SCs, and
progenitor cells
Every niche is
unique!
http://stemcells.nih.gov/info/
Cardiac niches and putative
supporting cells.
Apical (A, B, and F) and atrial (C–
E) niches contain CSCs and LCCs.
(A) c-kit+ (green) Ets1+ (white; EC
progenitors). (B) MDR1+ cells
(white) GATA-4+ (magenta dots);
α-sarcomeric actin+GATA-4+ (red;
myocyte precursors). GATA-4+
cardiac progenitors (arrowheads).
(C) Sca-1+MEF2C+ (yellow &
white dots; myocyte progenitors);
Sca-1+von Willebrand factor+ (green; EC precursor). (D–F)
Connexin 43 and E- and N-cadherin in niches containing c-kit+ (D
& F, green) and Sca-1+ (E, yellow) CSCs and LCCs. GATA-4+ (D,
magenta dots) and MEF2C+ (E & F, yellow dots). Connexin 43
(yellow dots), E-cadherin (green dots), and N-cadherin (white
PNAS 103:9226-9231, 2006
dots) Nuclei are stained by propidium iodide (blue).
Cardiac niches and putative supporting cells.
(G–J) Connexin 43 and 45 and N- and E-cadherin in atrial niches
containing c-kit CSCs–LCCs (green) are represented by yellow
dots located between two CSCs–LCCs (arrowheads), a CSC–
LCC and a fibroblast (arrows), and a CSC–LCC and a myocyte
(double arrowheads); see Insets. Nuclei are stained by propidium
iodide (blue).
PNAS 103:9226-9231, 2006
Formation of Gap Junctions
PNAS 103:9226-9231, 2006
PNAS 103:9226-9231, 2006
Myocyte turnover and life span
(A and B) Apical section illustrating bright
(arrows), intermediate (open arrowheads), and
dim (arrowheads) BrdU+ myocyte nuclei (yellow)
after 10 weeks of chasing. Myocytes are stained
by cardiac myosin (B, red).
Myocyte Turnover
(C) % of BrdU-bright, -int, and -dim myocyte nuclei.
(Right) Bars document the total number of BrdU+
myocytes and total number of myocytes in the atria,
base–mid region, and apex.
PNAS 103:9226-9231, 2006
Myocyte Turnover
PNAS 103:9226-9231, 2006
Myocyte Turnover
PNAS 103:9226-9231, 2006
Myocyte Lifespan
PNAS 103:9226-9231, 2006
ESCs versus Adult SCs (ASCs)
• ESCs
– Embryolically
derived
– Not tissue-specific
– Pluripotent
– Requires in vitro
fertilized egg for
derivation (ethical
concerns)
• ASCs
– Tissue resident
– Tissue-specific
– Multipotent
– No ethical
problems with their
clinical application
nature.com
americanprogress.org
http://stemcells.nih.gov/info/media/
Outline and Objectives
• What are stem cells (SCs)?
• Which SCs contribute to the adult CV
system?
• What are the physiological and
pathophysiological roles for SCs in the
CV system?
A
B
C
D
Identification of Mitotic Spindles in Dividing Myocytes from
Infarcted Hearts
(A) blue = organization of tubulin in the mitotic spindle (arrows)
(B) green = metaphase (arrowheads)
(C) green and blue = combination of tubulin and metaphase
chromosomes (arrows and arrowheads)
(D) red = sarcomeric α-actin, blue = tubulin, and green = N Engl J Med
344:1750-1757,
chromosomes in metaphase (arrows and arrowheads)
2001
A Myocyte in the Process of Cytokinesis
Arrows = actin
Red = staining of myocyte sarcomeric α-actin
green = PI labeling of chromosomes
N Engl J Med 344:1750-1757, 2001
Effects of MI on the Number of Mitotic Myocytes
N Engl J Med 344:1750-1757, 2001
The Y Chromosome in
Transplanted Hearts.
•Myocytes (A and B)
•Smooth-muscle cells (C
and D), Endothelial cells in
both coronary arterioles (E
and F), and capillary
endothelial cells (G and H)
•Blue areas = PI staining in
nuclei, Green areas
indicate Y chromosome
•The red areas indicate
sarcomeric α-actin in B, of
smooth-muscle α-actin in
D, and of factor VIII in F
and H.
N Engl J Med 346:5-15, 2002
The Y Chromosome in
Transplanted Hearts.
In I, J, and K, the bright
blue, fluorescent areas and
the arrows indicate the
presence of Ki-67, and the
yellow areas show the Y
chromosomes.
N Engl J Med 346:5-15, 2002
ASCs of the CV system
• Isolated based on functional assessments
or surface phenotypes
• Cardiac Stem Cells (CSCs) and
Endothelial Progenitor Cells (EPCs)
• Possible Vascular Progenitor Cell (VPC);
resident in vessel walls
Surface Markers for EPCs
• Mouse
– VEGFR2, LinNeg, c-kit, Sca-1, VE-cadherin
• Human
– CD133, VEGFR2, VE-cadherin, CD34
Cell 127:1137-1150, 2006
Vasculogenesis
• Aggregation of de-novo-forming angioblasts
into a primitive vascular plexus
• Hemangioblast Flk1+ cells produce ECs
and hematopoietic cells
• Earliest marker of angioblast precursors 
Flk1/VEGFR-2
• Complex remodeling processgrowth,
migration, sprouting and pruning lead to
development of functional circulatory system
Outline and Objectives
• What are stem cells (SCs)?
• Which SCs contribute to the adult CV
system?
• What are the physiological and
pathophysiological roles for SCs in the
CV system?
N Engl J Med 363:1638-1647, 2010
N Engl J Med 363:1638-1647, 2010
N Engl J Med 363:1638-1647, 2010
N Engl J Med 363:1638-1647, 2010
Evidence for Cardiomyocyte
Renewal in Humans
Olaf Bergmann, Ratan D. Bhardwaj, Samuel
Bernard, Sofia Zdunek, Fanie Barnabe-Heider, Stuart
Walsh, Joel Zupicich, Kanar Alkass, Bruce A.
Buckholz, Henrik Druid, Stefan Jovinge, Jonas Frisen
Science 324:98-102, 2009
Science 324:98-102, 2009
Fig. 1. Cell turnover in the
heart. (A) Schematic figure
demonstrating the strategy
to establish cell age by 14C
dating. The black curve in
all graphs shows the
atmospheric concentrations
of 14C over the decades
since 1930 [data from (14)].
The vertical bar indicates
the date of birth of the
individual. The measured
14C concentration (1) is
related to the atmospheric
14C concentration by use of
the established atmospheric
14C bomb curve (2). The
average birth date of the
population can be inferred
by determining where the
data point intersects the x
axis (3).
Science 324:98-102, 2009
Fig. 1. Cell turnover in
the heart. 14C
concentrations in DNA
of cells from the left
ventricle myocardium in
individuals born after (B)
or before (C) the nuclear
bomb tests correspond
to time points
substantially after the
time of birth, indicating
postnatal cell turnover.
The vertical bar
indicates the date of
birth of each individual,
and the similarly colored
dots represent the 14C
data for the same
individual.
Science 324:98-102, 2009
Fig. 1. Cell turnover in the heart. For individuals born before the increase in
14C concentrations, it is not possible to directly infer an age because the
measured concentration can be a result of 14C incorporation during the rising
and/or falling part of the atmospheric curve, and thus the concentration is
indicated by a dotted horizontal line.
Science 324:98-102, 2009
Fig. 2. Isolation of
cardiomyocyte nuclei. (A to
C) Flow cytometric analysis
of cardiomyocyte nuclei
from the left ventricle of the
human heart with an
isotype control antibody or
antibodies to the
cardiomyocyte-specific
antigens cTroponin I or T.
Boxes denote the
boundaries for the positive
and negative sorted
populations. (D) cTroponin I
and T are present in the
same subpopulation of
heart cell nuclei.
Science 324:98-102, 2009
Fig. 2. (E) Western blot analysis of flow cytometry–isolated nuclei
demonstrates nearly all detectable cTroponin T (analyzed with two different
antibodies) and I protein in the cTroponin T–positive fraction. Brain and heart
tissue were used as negative and positive controls, respectively. (F) The
cardiac troponin T–positive population is enriched for the cardiomyocytespecific transcription factors Nkx2.5 and GATA4. Both fractions contain
similar amounts of the nuclear protein histone 3 (loading control).
Science 324:98-102, 2009
Fig. 2. (G) Gene expression analysis of flow cytometry–isolated nuclei shows
high expression of cardiomyocytespecific genes in the cTroponin T–positive
fraction (cTroponin I and T, Nkx2.5), whereas marker genes for endothelial
cells (vWF), fibroblasts (vimentin), smooth muscle (ACTA2), and leukocytes
(CD45) are highly expressed in the cTroponin T–negative fraction (H). Bars in
(G) and (H) show the average from three independent experiments (TSD).
Science 324:98-102, 2009
Fig. 3. Cardiomyocyte turnover in adulthood. (A) The 14C concentrations in
cardiomyocyte DNA from individuals born before the time of the atmospheric
radiocarbon increase correspond to time points after the birth of all
individuals. The vertical bar indicates year of birth, with the correspondingly
colored data point indicating the Δ14C value. (B) 14C concentrations in
cardiomyocyte DNA from individuals born after the time of the nuclear bomb
test.
Science 324:98-102, 2009
Fig. 3. (C) Average DNA content (2n = 100%) per cardiomyocyte nucleus
from individuals of different ages. Ploidy was measured by flow cytometry.
(D) 14C values corrected for the physiologically occurring polyploidization of
cardiomyocytes during childhood for individuals born before and after the
bomb-induced spike in 14C concentrations, calculated on the basis of the
individual average DNA content per cardiomyocyte nucleus. The 14C content
is not affected in individuals where the polyploidization occurred before the
increase in atmospheric 14C concentrations.
Science 324:98-102, 2009
Fig. 4. Dynamics of cardiomyocyte turnover. (A) Individual data fitting
assuming a constant turnover (see supporting online text) reveals an almost
linear decline of cardiomyocyte turnover with age (R = −0.84; P = 0.001). A
constant-turnover hypothesis might therefore not represent the turnover
dynamics accurately. (B) Global fitting of all data points (see supporting
online text, error sum of squares = 1.2 × 104) shows an age-dependent
decline of cardiomyocyte turnover.
Science 324:98-102, 2009
Fig. 4. (C) The gray area depicts the fraction of cardiomyocytes remaining
from birth, and the white area is the contribution of new cells. Estimate is
from the best global fitting. (D) Cardiomyocyte age estimates from the best
global fitting. The dotted line represents the no-cell-turnover scenario, where
the average age of cardiomyocytes equals the age of the individual. The
black line shows the best global fitting. Colored diamonds indicate computed
data points from 14C -dated subjects. Error bars in (A) are calculated from the
errors on 14C measurements. Error bars in all other graphs are calculated for
each subject individually and show the interval of possible values fitted with
the respective mathematical scenario.
http://stemcells.nih.gov/info/
Postnatal
neovascularization in
physiological or
pathophysiological
events is through the
processes of
angiogenesis and
vasculogenesis.
Angiogenesis = EC
activation.
Vasculogenesis =
BM EPC
mobilization and
activation
Isolation of Putative
Progenitor Endothelial Cells
for Angiogenesis
Takayuki Asahara, Toyoaki Murohara, Alison Sullivan,
Marcy Silver, Rien van der Zee, Tong Li, Bernhard
Witzenbichler, Gina Schatteman, Jeffrey M. Isner
Science 275:964-967, 1997
Fig. 1. Attachment, cluster formation, and capillary network
development by progenitor ECs in vitro
(A) Spindle shaped attaching cells (ATCD34+) 7 days after
platingMBCD34+ (50 cells/mm2) on fibronectin in standard medium (14).
(B) Number of ATCD34+ cells 12 hours and 3 days after culture of
MBCD34+ on plastic alone (CD34+/non), collagen coating (CD34+/Col),
or fibronectin (CD34+/Fn), and MBCD34- on fibronectin (CD34-/Fn).
Fig. 1. Attachment, cluster formation, and capillary network
development by progenitor ECs in vitro
Network formation (C) and cord-like structures (D) were observed 48
hours after plating coculture of MBCD34+, labeled with DiI, with
unlabeled MBCD34- cells (ratio of 1:100) on fibronectin.
At 12 hours after coculture, MBCD34+-derived cells had
formed multiple clusters (E and F). After 5 days, uptake of
acLDL-DiI was detected in ATCD34+ cells at the periphery but
not the center of the cluster (G and H).
Flow cytometric evaluation of EPC progression to EC-like
phenotype, analyzing leukocyte and EC markers.
Fig. 3. Progenitor ECs express
ecNOS, Flk-1/KDR, and CD31
mRNA and release NO
(A) Complementary DNA (from
106 cells) was amplified by PCR
(40 cycles) with paired primers
(23) (B) NO release from ATCD34+
and ATCD34- cells cultured in sixwell plates was measured as
described (24). NO production
was measured in a well with
incremental doses of VEGF and
Ach. HUVECs and bovine aortic
ECs were used as positive
controls, and human coronary
smooth muscle cells (HCSMCs)
as negative control. The values
are means ± SEM of 10
measurements for each group.
Fig. 4. Heterologous
(panels A to L),
homologous (M), or
autologous (panels
N and O) EC
progenitors
incorporate into sites
of angiogenesis in
vivo. (A and B) DiIlabeled MBCD34+
(red, arrows)
between skeletal
myocytes (M),
including necrotic
(N) myocytes 1
week after injection; most are colabeled with CD31 (green, arrows). Note a
preexisting artery (A), identified as CD31-positive, but DiI-negative. (C and D)
Evidence of proliferative activity among several DiI-labeled MBCD34+-derived cells
(red, arrows), indicated by coimmunostaining for antibody to Ki67 ( Vector Lab,
Burlingame, California) (green). Proliferative activity is also seen among DiInegative, Ki67-positive capillary ECs (arrowheads); both cell types contribute to
neovasculature.
Fig. 4. (E) DiI (red)
and CD31 (green) in
capillary ECs (arrows
in E and F) between
skeletal myocytes,
photographed
through a double
filter 1 week after DiIlabeled MBCD34+
injection. (F) A single
green filter shows
CD31 (green)
expression in DiIlabeled capillary ECs
integrated into the capillary with native (DiI-negative, CD31-positive) ECs
(arrowheads in E and F). (G) Immunostaining 1 week afterMBCD34+ injection
showing capillaries comprising DiI-labeled MBCD34+-derived cells expressing Tie-2
receptor (green). Several MBCD34+-derived cells (arrows) Tie-2 positive and
integrated with some Tie-2–positive host capillary cells (arrowheads) identified by
the absence of red fluorescence. (H) Phase-contrast photomicrograph of the
same section shown in (G) indicates the corresponding DiI-labeled (arrows) and unlabeled (arrowheads) capillary ECs.
(I and J) Six weeks after
administration, MBCD34+-derived cells
(red, arrows) colabel for CD31 in
capillaries between preserved
skeletal myocytes (M). (K and L) One
week after injection of MBCD34-,
isolated MBCD34--derived cells (red,
arrows) are observed between
myocytes but do not express CD31.
(M) Immunostaining of β-Gal in a
tissue section harvested from
ischemic muscle of C57BL/6J,129/SV
mice 4 weeks after the administration
of MBFlk-1+ isolated from transgenic
mice constitutively expressing β-Gal.
(Flk-1 cell isolation was used for
selection of EC progenitors because
of the lack of a suitable antibody to
mouse CD34.) Cells overexpressing
β-Gal (arrows) were incorporated into
capillaries and small arteries; these
cells were identified as ECs by antiCD31 and BS-1 lectin (16).
(N and O) Section of muscle harvested from rabbit ischemic
hindlimb 4 weeks after administration of autologous MBCD34+
cells. Red fluorescence in (N) indicates localization of MBCD34+derived cells in capillaries seen (arrows) in the phase contrast
photomicrograph in (O). Each scale bar is 50 μm.
Questions?
Comments?
Ideas?
rsiggi@lsuhsc.edu