Concise review: stem cells, myocardial regeneration, and methodological artifacts.

Abstract

This review discusses the current controversy about the role that endogenous and exogenous progenitor cells have in cardiac homeostasis and myocardial regeneration following injury. Although great enthusiasm was created by the possibility of reconstituting the damaged heart, the opponents of this new concept of cardiac biology have interpreted most of the findings supporting this possibility as the product of technical artifacts. This article challenges this established, static view of cardiac growth and favors the notion that the mammalian heart has the inherent ability to replace its cardiomyocytes through the activation of a pool of resident primitive cells or the administration of hematopoietic stem cells.

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DOI: 10.1634/stemcells.2006-0623
2007;25;589-601; originally published online Nov 22, 2006;
Stem Cells
Urbanek, Jan Kajstura and Roberto Bolli
Piero Anversa, Annarosa Leri, Marcello Rota, Toru Hosoda, Claudia Bearzi, Konrad

Artifacts
Concise Review: Stem Cells, Myocardial Regeneration, and Methodological
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Concise Review: Stem Cells, Myocardial Regeneration, and
Methodological Artifacts
PIERO ANVERSA,aANNAROSA LERI,aMARCELLO ROTA,aTORU HOSODA,aCLAUDIA BEARZI,a
KONRAD URBANEK,aJAN KAJSTURA,aROBERTO BOLLIb
aCardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla, New York,
USA;bInstitute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, USA
Key Words. Heart • Confocal and light microscopy • Mitosis • Chimerism • Bone marrow cell transdifferentiation
Enhanced green fluorescent protein autofluorescence
ABSTRACT
This review discusses the current controversy about the
role that endogenous and exogenous progenitor cells have
in cardiac homeostasis and myocardial regeneration fol-
lowing injury. Although great enthusiasm was created by
the possibility of reconstituting the damaged heart, the
opponents of this new concept of cardiac biology have
interpreted most of the findings supporting this possibil-
ity as the product of technical artifacts. This article chal-
lenges this established, static view of cardiac growth and
favors the notion that the mammalian heart has the in-
herent ability to replace its cardiomyocytes through the
activation of a pool of resident primitive cells or the
administration of hematopoietic stem cells. STEM CELLS
2007;25:589–601
INTRODUCTION
This article discusses the fundamental results that have contrib-
uted to create the view that the postnatal and adult heart cannot
renew its parenchymal cells and that the number of myocytes
that are present at birth dictates the destiny of this organ
throughout life [1, 2]. According to this premise, myocyte
turnover does not occur, and the cells originally formed during
embryonic and fetal cardiac development are responsible for the
preservation of myocardial performance in the young, adult, old,
and senescent heart. Coronary vascular endothelial cells (ECs)
and smooth muscle cells (SMCs) divide rarely, have a very long
cell cycle, and are replaced slowly by division of resident ECs
and SMCs within the wall, without changing the interaction
between the established, irreplaceable myocytes and the mod-
estly more dynamic coronary vessels. Importantly, circulating
progenitor cells or primitive cells mobilized from the bone
marrow do not transdifferentiate and therefore are incapable of
acquiring the myocyte, EC, and SMC lineages [3]. By necessity,
myocyte apoptosis has to be an occasional event of no or, at
most, trivial consequence; if not, the heart would rapidly lose a
significant number of parenchymal cells, and the small group of
myocytes left would be unable to sustain ventricular perfor-
mance. Together, these concepts project a rather static biologi-
cal view of cardiac homeostasis and pathology. In addition, this
perception of the heart imposes severe limitations on the devel-
opment of therapeutic strategies that can be implemented for the
management of human disease [4].
We will review the work that has promoted a shift in
paradigm of the heart from a terminally differentiated postmi-
totic organ to a self-renewing organ. This dramatic change in
understanding of cardiac behavior and function was originated
by observations that were made in the 1940s and that have
continued to accumulate over the years (for reviews, see [5, 6]).
However, the disagreement that persists among the members of
the scientific community today was present then and has per-
sisted for nearly 60 years. The reason for the controversy is
unclear, but it seems to reflect unshakable positions based on
preconceived beliefs more than on careful analysis and proper
consideration of published results [7]. In this article, we focus
on methodological issues because, in the course of the contro-
versy, recurrent statements have been made to undermine the
technical protocols used in studies supporting the existence of
cardiac regeneration. Consistently, the data in favor of myocar-
dial regeneration are claimed to be the product of methodolog-
ical artifacts [1–4, 8–11]. To properly evaluate the validity of
these criticisms or lack thereof, methodologies need to be com-
pared to provide readers with a better understanding of the
substrate that governs the debate.
The old dogma has profoundly conditioned basic and clin-
ical research in cardiology for the last three decades [7, 12].
Based on this paradigm, cardiomyocytes undergo cellular hy-
pertrophy but cannot be replaced either by the entry into the cell
cycle of a subpopulation of nonterminally differentiated myo-
cytes or by the activation of a pool of primitive cells that
become committed to the myocyte lineage. However, the efforts
made to introduce a highly dynamic perspective of the heart
have led to the identification and characterization of a resident
pool of stem cells that can generate myocyte, and ECs and
SMCs organized in coronary vessels [13]. This discovery has
created a new, heated debate concerning the implementation of
adult cardiac stem cells in the treatment of heart failure of
ischemic and nonischemic origin.
Correspondence: Piero Anversa, M.D., Cardiovascular Research Institute, Vosburgh Pavilion, New York Medical College, Valhalla, New
York 10595, USA. Telephone: 914-594-4168; Fax: 914-594-4406, e-mail: piero_anversa@nymc.edu Received October 3, 2006; accepted
for publication November 7, 2006; first published online in STEM CELLS EXPRESS November 22, 2006; available online without subscription
through the open access option. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2006-0623
STEMCELLS2007;25:589–601 www.StemCells.com
TISSUE-SPECIFIC STEM CELLS
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POSSIBILITY 1: THE HEART IS A
TERMINALLY DIFFERENTIATED
POSTMITOTIC ORGAN
The majority of organs contain dividing and nondividing cells.
However, nondividing cells can rest in the G0phase and reenter
the cell cycle following growth activation or become terminally
differentiated and die without undergoing further division [14].
The latter category of cells is not dormant in G0and cannot
reenter the cell cycle. This is typically seen at the tip of the villi
of the small intestine [15], in the auditory hair cells of the ear
[16], and in the upper layer of the epidermis [17]. Hepatocytes
in vivo are in a G0state, and after partial hepatectomy or severe
injury, liver regeneration is accomplished by proliferation of
hepatocytes, biliary epithelial cells, and fenestrated endothelial
cells [18]. Because the liver contains a population of primitive
cells, the oval cells, the growth and maturation of this cell
category may contribute to the reconstitution of liver mass [19].
Probably both cell mechanisms are involved in the repair of the
organ, although controversy exists in the field [20]. Multipoten-
tial cells have also been identified in the central nervous system,
but questions have been raised concerning their ability to grow
and generate Purkinje neurons in the adult brain [21–24]. Con-
versely, the regeneration of the skin [25], bone marrow [26], and
skeletal muscle [27] is regulated by activation of skin stem cells,
hematopoietic stem cells (HSCs), and satellite cells, respec-
tively. These mechanisms of cellular growth have not been
considered possible in the myocardium, and the notion has been
proposed that ventricular myocytes cannot be replaced once cell
division ceases immediately after birth in the mammalian heart
[1, 2, 9, 10].
Thus, the dogma was established that the postnatal heart is
composed of a fixed number of myocytes and that, if myocytes
die, they are permanently lost and the myocardium must main-
tain its vital role with a reduced number of cells. The remaining
myocytes are not in G0and cannot be triggered into the repli-
cating phase [28]; they continue to perform their physiological
function, undergo cellular hypertrophy, and ultimately die [29].
Based on this paradigm, the age of myocytes, organ, and organ-
ism was assumed to coincide, implying that myocytes in humans
may have a lifespan that exceeds 100 years [30]. For several
decades, no effort was made to reexamine this rather unusual
view of the biology of the heart and cardiac homeostasis. Re-
markably, there is not a single piece of evidence that demon-
strates the inability of the heart to replace its dying myocytes. It
seems rather extravagant that cardiomyocytes can contract 70
times per minute over 100 years and continue to be functional.
During this period, they would have contracted 3.7 billion times
and still be operative. If this were to be the case, adult myocytes
would be essentially immortal cells.
POSSIBILITY 2: THE HEART IS NOT A
TERMINALLY DIFFERENTIATED
POSTMITOTIC ORGAN
Historically, the foundations for the view of the heart as a
terminally differentiated postmitotic organ incapable of regen-
eration were established in the mid-1920s [31]. This conclusion
was dictated by the difficulty in identifying mitotic figures in
myocyte nuclei of the human heart with the unsophisticated
histologic preparations available 80–90 years ago. This notion
gained support from autoradiographic studies of tritiated thymi-
dine incorporation in the myocardium during postnatal devel-
opment and pathological overloads [32–35], conducted in the
mid- and late 1960s. DNA synthesis in myocyte nuclei was
negligible, and this observation, together with the inability to
recognize myocyte mitotic figures, led to the belief that the adult
heart is composed of parenchymal cells that are permanently
withdrawn from the cell cycle. Myocytes can increase in volume
but not in number [36]. Again, this work used standard histo-
logic preparations in combination with photographic emulsions,
which imposed severe limitations in terms of microscopic res-
olution [37]. These factors made it highly problematic to dis-
tinguish whether DNA replication or mitosis was present exclu-
sively in interstitial cells or, at times, involved cardiomyocytes
as well. In fact, the possibility of myocyte division was ac-
knowledged in two of the early reports [34, 35].
The qualitative results discussed so far were in sharp con-
trast with quantitative measurements of myocyte volume and
number performed in human hearts obtained from patients who
died because of decompensated cardiac hypertrophy and con-
gestive heart failure [5]. In the late 1940s and early 1950s,
Linzbach documented that, in the presence of a cardiac weight
equal to or greater than 500 g, myocyte proliferation represented
the predominant mechanism of increased muscle mass [38, 39].
An interesting aspect of this work is that these determinations
were all based on two very simple but critical parameters:
myocyte length, assessed by the distance between two nuclei of
largely mononucleated myocytes, and myocyte diameter across
the nucleus. These variables are easily recognizable in standard
histologic sections and do not require high resolution and an
impeccable preparation. Based on a cylindrical shape configu-
ration, myocyte cell volume was computed. Moreover, the ag-
gregate volume of the myocyte compartment of the myocardium
was obtained, and the quotient of this quantity and myocyte cell
volume yielded the total number of ventricular myocytes. Be-
cause of the simplicity of the approach, the original results were
confirmed several years later in other laboratories in which more
refined techniques were applied [40, 41]. In all cases, hearts
weighing 500 g or more were characterized by a striking in-
crease in myocyte number that was more prominent than cellu-
lar hypertrophy; this adaptation involved both the left and right
ventricles [38–43].
In the mid- and late 1990s, new studies of the human heart
examined the distribution of mononucleated and binucleated
myocytes in 72 normal and 176 diseased hearts [43]. Aging,
cardiac hypertrophy, and ischemic cardiomyopathy were char-
acterized by the lack of changes in the relative proportion of
mononucleated and multinucleated myocytes in the ventricular
myocardium, confirming that the early and more recent mea-
surements of myocyte proliferation were valid and did not
represent and erroneous interpretation dictated by nuclear hy-
perplasia in the absence of myocyte division [6, 44]. Mono-
nucleated cells constitute ?75% of myocytes in the human
heart, differing significantly from mice [45], rats [46], dogs [47,
48], and pigs [49].
Mononucleated cells are smaller than binucleated cells, and
this cellular property may influence the ability of myocyte to
divide [50]. Human myocytes larger than 30,000 ?m3cannot
reenter the cell cycle and proliferate [50]. In fact, cycling and
mitotic myocytes are predominantly small and mononucleated,
and this feature may account for the massive cardiac hypertro-
phy that can be achieved in humans (Fig. 1). It would be
inefficient for large myocytes to divide once or at most twice to
expand the cardiac mass. Heart weight in humans can increase
nearly threefold, reaching values of 1,000 g or larger [5, 38, 41,
43]. Heart failure typically shows increases in myocyte number
that vary from 20%–100% or more [5, 38, 41, 43, 51–53]. This
phenomenon is not affected by age; in an analysis of 7,112
human hearts, from birth to 110 years of age, Linzbach and
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Akuamoa-Boateng have shown that extreme forms of organ
hypertrophy are detectable up to the ninth decade of life, and
heart weights of 500 and 600 g are present in patients at 100
years of age and older [54].
Importantly, scar formation, foci of replacement fibrosis
scattered throughout the ventricular wall, diffuse interstitial
fibrosis, and ongoing myocyte death by apoptosis and necrosis
are typically present in the hypertrophied failing heart [44, 55,
56]. These factors markedly affect the number of cardiomyo-
cytes and indicate that the extent of myocyte formation deter-
mined by the morphometric methodologies summarized above
constitutes an underestimation of the actual magnitude of myo-
cyte regeneration in the pathological heart. In addition, there is
no alternative methodology able to demonstrate unequivocally
myocyte proliferation. By definition, myocyte replication cor-
responds to an increase in the number of myocytes; in the
absence of this documentation, biochemical, molecular, physi-
ological, autoradiographic, and structural results can only be
suggestive of myocyte regeneration but in themselves cannot
provide the critical information, that is, an increased number of
cells. Quantitative morphology is the only technique that can
identify myocyte formation. Contrary to expectations, the ob-
servations in favor of myocyte regeneration were not received
with enthusiasm by the scientific community [57–63]. With
some small variations, the morphometric results in the human
heart were questioned, and the possibility of myocyte formation
in the adult myocardium was first dismissed and subsequently
attacked. This new idea challenged the comfortable paradigm of
myocyte hypertrophy that had dominated the field of molecular
cardiology and cell signaling for the previous 25 years. Heart
failure was viewed as the consequence of a defective myocyte
hypertrophy in which alterations of effector pathways regu-
lating myocyte growth and contractility were considered re-
sponsible for the depression in organ, tissue, and cell function
[64, 65].
However, the quantitative data collected in the human heart
suggested that a reexamination of the mechanisms of myocyte
growth needed to be considered and that additional studies
needed to be performed to clarify the apparently contradictory
results. For this purpose, experiments were conducted in animal
models of cardiac failure and in human diseased hearts to
provide relevant information in support of or against the notion
of the regeneration potential of the adult myocardium. New
methodologies were introduced to determine whether a subset of
cardiomyocytes had the ability to reenter the cell cycle and
divide and whether this process involved the activation of cell
cycle-related genes, cyclins, and cyclin-dependent kinases and
the expression of proteins modulating karyokinesis and cytoki-
nesis [45, 50, 55, 66–80]. Rounds of cell division result in
progressive telomere attrition, and critical telomeric shortening
triggers irreversible growth arrest [81–83]. Thus, the telomere-
telomerase axis, together with the telomere-binding proteins, is
a fundamental component of the multiple mechanisms that
regulate the regeneration of non-postmitotic organs; because of
this function, this system was evaluated and found to be oper-
ative in the adult heart of animals and humans [50, 74–77, 79,
84]. Unfortunately, these multiple studies left the controversy
unresolved.
MYOCYTE DIVISION
In 1925, a highly significant paper was published in which the
claim was made that there are no detectable mitotic figures in
myocytes of the human heart, and therefore myocyte regenera-
tion does not occur in mammals [31]. This work challenged the
view proposed in numerous studies published from 1850 to
1911 in which the general belief was that cardiac hypertrophy is
the consequence of hyperplasia and hypertrophy of existing
myocytes [6]. The 1925 study can be considered the one that
introduced the concept that the heart is a terminally differenti-
ated postmitotic organ. The impact of this research was enor-
mous. To the best of our knowledge, for several decades, many
generations of pathologists and cardiovascular scientists have
accepted the view that mitotic myocytes are not to be found in
the adult myocardium, and only a few rare exceptions have been
published, mostly restricted to the fetal-neonatal heart [85–88].
In the last 10–12 years, however, mitotic images in human
cardiomyocytes have been documented in acute and chronic
ischemic cardiomyopathy [72, 89, 90], idiopathic dilated car-
diomyopathy [72], chronic aortic stenosis with moderate ven-
tricular dysfunction [50], myocardial aging [84], acromegaly
(Fig. 2A), and diabetes (Fig. 2B). Using light microscopic
examination of routine histologic sections, an extensive search
was required for the recognition of occasional mitotic figures in
myocytes. Because in the early successful observations, divid-
ing myocytes were seen only in the fetal and diseased failing
heart [89], the assumption was made that replicating myocytes
are essentially undetectable in normal adult myocardium. This
hypothesis, in fact, suggested a rather minor role of myocyte
regeneration in cardiac homeostasis and pathology in humans.
However, in spite of the caution exercised in the interpretation
of these results, this work was considered to reflect the incorrect
interpretation of proliferating interstitial fibroblasts as dividing
myocytes [1, 2]. Surprisingly, this conclusion was reached based
on electron microscopic images that illustrated nondividing fi-
broblasts in proximity to differentiated nonreplicating cardio-
myocytes [1]. This represents the normal organization of the
myocardium and makes it difficult to understand how this
well-established morphological characteristic of cardiac struc-
ture can be used as an argument against myocyte division and a
case in favor of its erroneous identification. So Artifact 1 was
introduced in the literature as the only logical explanation for
these unexpected results.
An important point that necessitates discussion is the
belief by some that electron microscopy is the only tool that
can unequivocally prove or disprove the presence of myocyte
mitotic images in the myocardium. In invited reviews ques-
tioning the existence of myocyte regeneration, interstitial
fibroblasts have been described as extremely mobile cells
capable of penetrating by several microns the thickness of
myocytes, positioning themselves in the central portion of
Figure 1. Myocyte proliferation and isch-
emic cardiomyopathy in humans. Small di-
viding left ventricular myocytes (?-sarco-
meric actin; red) are identified by Ki67
labeling (A) (white, arrow), MCM5 expres-
sion (B) (yellow, arrow), and metaphase
chromosomes (C) (arrow). The boundaries
of myocytes and interstitial cells are defined
by laminin (green). Nuclei are stained by
4,6-diamidino-2-phenylindole (blue).
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these cells [1, 2]. By this mechanism, the myocyte nucleus
was apparently displaced from its original location, which
was occupied by the “penetrating fibroblast.” Schemes were
created to strengthen this unlikely possibility [1]. However,
this highly imaginative understanding of cardiac morphology
fell short on several accounts: the examples of “penetrating”
interstitial cells did not show dividing cells, the myocyte
nucleus was at its expected site in the middle of the myocyte
cytoplasm, and the electron microscopic preparation was of
poor quality. Myocytes were hypercontracted and had sarco-
meres of ?1.4–1.5 ?m in length. Normally, diastolic sarco-
mere length is 2.1–2.2 ?m, and during systolic contraction, it
becomes 1.8–1.9 ?m [91]. Hearts with sarcomeres ?1.4 ?m
in length are dead, since no contraction can be generated
under this condition. Contraction bands produced by the
inappropriate fixation of the myocardium were analyzed to
reinterpret myocardial ultrastructure. Surprisingly, these ar-
tifacts were accepted and introduced in the literature as facts.
The frequency of mitosis in myocytes of normal human,
mouse, or rat heart, as assessed by confocal microscopy, is low
and averages 14/106myocytes [72], 37/106myocytes [45], and
85/106myocytes (J.K., unpublished data), respectively. The
values for dividing interstitial fibroblasts are similar to those of
cardiomyocytes [89]. Based on these data, ?350, 140, and 60
mm2of tissue need to be examined to identify a dividing
myocyte in the human, mouse, and rat heart, respectively. This
is why the suggestion to implement electron microscopy for this
analysis is not realistic. The area of myocardium that is sampled
in each thin section by electron microscopy is ?0.2 mm2, and
the area of the microscopic field at a magnification of approx-
imately ?2,000 is 0.0054 mm2. Thus, the numbers of micro-
graphs that would have to be collected to identify a single
myocyte mitotic image are 66,000 pictures for humans, 25,000
for mice, and 11,000 for rats. If one wishes to use electron
microscopy to challenge the existence of myocyte division and
to demonstrate that proliferating fibroblasts were improperly
interpreted as mitotic myocytes, that is what needs to be in-
vested to obtain at least one example in support of or against this
thesis [92].
Major advances in understanding myocyte replication in
the adult human heart have been made by the use of confocal
microscopy and immunolabeling in the analysis of the normal
and diseased heart. With this approach, the identification of
mitotic images in myocytes became easier and more accurate.
These new results strengthened the notion that parenchymal
cells are formed continuously in the normal myocardium and
myocyte regeneration is markedly potentiated in the patho-
logical failing heart [6, 50, 72, 84, 90]. This technology was
also applied to the study of animal models of human diseases,
and myocyte replication was unequivocally demonstrated in
myocardial sections and isolated cell preparations [6, 45, 73,
93]. The latter observation conclusively refuted the claim that
dividing myocytes represent proliferating fibroblasts inserted
within or attached to terminally differentiated myocytes. The
formation of the mitotic spindle by the arrangement of tubu-
lin and the generation of the contractile ring by the accumu-
lation of actin in the region of cytoplasmic separation were
clearly documented, together with karyokinesis and cytoki-
nesis (Fig. 2C–2E). However, these results did not change the
position of certain groups, who continued to reject the regen-
erative ability of the adult mammalian myocardium [2, 4, 8,
9, 28]. These groups introduced the possibility of a new
source of artifacts, Artifact 2, related to confocal microscopy;
they stated that traditional light microscopy is greatly supe-
rior to confocal microscopy and, with this personal view-
point, tried to invalidate the results obtained with the latter
technique.
An important issue that requires discussion relates to the
notion that the analysis of histochemical preparations by
conventional light microscopy is superior to fluorescence
labeling and confocal microscopy [94, 95]. The resolution of
the micrographs provided by light microscopy is markedly
inferior to that obtained by confocal microscopy, ?0.62
versus 0.29 ?m [37, 96]. In addition, only the very superficial
layer of a tissue section can be examined by light microscopy
(Fig. 3). This inherent problem with light microscopy was
recognized immediately when confocal technology became
available [97]. Clear examples were published in the Journal
of Cell Biology in 1987 demonstrating the impossibility of
identifying microtubules, mitotic spindle, cytoplasmic pro-
teins, and chromosomal structures by light microscopy in
cultured cells. Conversely, these morphological details were
apparent when the same cells were examined by confocal
microscopy [98]. The difference between epifluorescence
light microscopy and confocal microscopy is even greater in
tissue sections. When confocal microscopy was not available
to us, we estimated the myocyte mitotic index in chronic
heart failure to be 11 myocytes per million [89]. When
comparable hearts were evaluated by confocal microscopy,
the mitotic index reached a value of 152 cells per million
[72]. Even more striking is the difference between the mitotic
indices obtained by light and confocal microscopy in the
border zone of acute infarcts in humans: light microscopy
yielded 3.3 mitotic myocytes per million [89] and confocal
microscopy 775 mitotic cells per million [90]. In all cases,
Figure 2.
eased human heart. Metaphase chromosomes
(blue, 4,6-diamidino-2-phenylindole [DAPI];
arrows) are apparent in two small dividing
cardiomyocytes (red, ?-sarcomeric actin) in
patientsaffectedbyacromegaly(A)ordiabetes
(B). The organization of tubulin in the mitotic
spindle (C) (white, arrows), the accumulation
ofactininthecontractilering(D)(red,arrows),
and cytokinesis (E) (arrows) in dividing cardi-
omyocytes are apparent. In all cases, chromo-
somes are labeled by DAPI (blue) and the
myocyte cytoplasm by ?-sarcomeric actin
(red). Green indicates laminin.
Myocyte generation in the dis-
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