Review
“String Theory” of c-kitpos Cardiac Cells
A New Paradigm Regarding the Nature of These Cells That May
Reconcile Apparently Discrepant Results
Matthew C.L. Keith, Roberto Bolli
Abstract: Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kitpos
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cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We
and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes,
suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis
of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct
“string theory” of c-kitpos cardiac cells because it reconciles multifarious and sometimes apparently discrepant
results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools
of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to
myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities,
or differentiation capacities of specific cardiac progenitors. C-kitpos cells derived from the first heart field
exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or
after birth. The residual c-kitpos cells found in the adult heart are probably of proepicardial origin, possess a
mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of
contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells).
If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic
potential of first heart field progenitors might be reconciled with recent results of c-kitpos cell lineage tracing
studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic
precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kitpos cell administration
to ischemically damaged hearts despite the observed paucity of cardiomyogenic differentiation of these cells. (Circ Res. 2015;116:1216-1230. DOI: 10.1161/CIRCRESAHA.116.305557.)
Key Words: muscle development
■
B
ecause of the encouraging results of both preclinical1–5 and
clinical6 studies, c-kitpos/cluster of differentiation (CD) 45neg/
hematopoietic lineage (lin)neg cardiac cells (herewith referred
to as c-kitpos cardiac cells) have emerged as one of the most attractive cell types for therapeutic application. At the preclinical
level, numerous investigations conducted by many independent
laboratories in a wide variety of animal models of ischemic cardiomyopathy have consistently documented salubrious effects of
exogenous c-kitpos cardiac cells on left ventricular function and
structure, including regeneration of dead myocardium.1–5 At the
clinical level, a small phase I study (the Cardiac Stem cells In
Patients with Ischaemic cardiOmyopathy [SCIPIO] trial) has
documented the safety of autologous c-kitpos cardiac cell administration in patients with ischemic heart failure.6 Although
SCIPIO was not designed to assess efficacy, its results suggest
that c-kitpos cardiac cells may impart beneficial effects on left ventricular function, quality of life, functional class, and infarct size,6
myocytes, cardiac
■
regeneration
thus providing a rationale for larger trials aimed at determining
efficacy.
Despite these promising results, however, there continues to be outspoken skepticism regarding the use of c-kitpos
cardiac cells as therapeutic agents.7–9 We believe that an
important factor fueling this skepticism is the inadequate
evidence that either endogenous or exogenous adult c-kitpos
cardiac cells differentiate into a relevant number of mature functional myocytes. Here we offer a new paradigm
aimed at reconciling discrepant results obtained by different laboratories with respect to the therapeutic utility and
differentiation potential of c-kitpos cardiac cells. Our conceptual construct is predicated on a comprehensive review
of a large amount of work published by many independent
groups over the past 2 decades. We believe that the theorem expounded herein provides a unifying theory that incorporates opposing, but perhaps not mutually exclusive,
Original received November 3, 2014; revision received January 20, 2015; accepted February 5, 2015. In January 2015, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 14.7 days.
From the Division of Cardiovascular Medicine, Department of Cardiology, University of Louisville, KY.
This article was sent to Joseph C. Wu, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to Roberto Bolli, MD, 550 S. Jackson St, ACB, Third Floor, Louisville, KY 40292. E-mail [email protected]
© 2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.116.305557
1216
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1217
Nonstandard Abbreviations and Acronyms
EMT
EPDC
ESC
FHF
GATA4
Isl-1
MACS
MSC
Nkx2.5
SHF
Tbx
WT1
epithelial to mesenchymal transition
epicardium-derived cell
embryonic stem cell
first heart field
GATA binding factor 4
islet-1 transcription factor
magnetic-activated cell sorting
mesenchymal stromal/stem cell
NK2 transcription factor related, locus 5
second heart field
T-box transcription factor
Wilm’s tumor protein
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positions regarding the direct contributions of c-kitpos cardiac cells to cardiomyogenesis.
The Controversy
In 2003, Beltrami et al10 reported the discovery, in a rodent
model, of resident c-kitpos/linneg cardiac cells that were able
to give rise to all cardiac lineages including cardiomyocytes.
Over the past decade, however, conflicting results have been
obtained with respect to the cardiomyogenic ability of c-kitpos
cardiac cells. Although some in vitro studies have suggested that these cells express stemness-associated markers and
early cardiac markers such as octamer-binding transcription
factor 4 (Oct4), NK2 transcription factor related, locus 5
(Nkx2.5), and GATA binding factor 4 (GATA4), among others, and some sarcomeric proteins,3,10,11 formation of mature
cardiomyocytes has not been observed2–4,11,12; furthermore,
the artificial in vitro conditions used in those studies may promote a pattern of protein expression that is not likely to occur in vivo.13,14 Indeed, in the in vivo setting, reports of adult
cardiomyocyte formation10,15,16 have not been reproduced by
several laboratories including our own.1–5,11,12,17–22 We1–5,21 and
others11,12,22 have found that c-kitpos cardiac cells transplanted
in infarcted hearts do not differentiate into mature myocytes
to a significant extent, implying that paracrine mechanisms
must be responsible for the functional improvement.1,3,5,17,22
Efforts to elucidate the multifaceted paracrine mechanisms
of c-kitpos cells, as well as other cells types, are currently
underway.23,24
Whether the aforementioned lack of maturation is due to
intrinsic inability of cells to differentiate into mature cardiomyocytes, extremely poor survival and engraftment, or compromised differentiation potential caused by suboptimal in
vitro expansion remains to be established. It is possible that
when they are removed from the heart and expanded in vitro,
these cells partially lose their differentiation potential because
of an impairment of complex in vivo cell signaling cascades
that are essential for signaling cells to start proliferating and
for eliciting targeted lineage commitment and differentiation.
However, consistent with our observations with exogenous
cells,1,2,4,5 recent work by the Molkentin group has also shed
doubt on the cardiomyogenic nature of endogenous c-kitpos
cardiac cells, suggesting instead a largely vasculogenic and
advential lineage predisposition.18 In part, the discrepant results regarding the in vivo cardiogenic ability of exogenous
c-kitpos cells1–5,10,15,17,19–21,25 might reflect differences in culture,
isolation, or expansion conditions; however, in the van Berlo
study,18 this was not an issue as the lineage-traced c-kitpos cells
were of endogenous origin. Regardless of its causes, the failure of transplanted postnatal c-kitpos cardiac cells to assume a
cardiac phenotype in most studies is a major limitation of cell
therapy, which mandates a reassessment of the nature of these
cells and commands a closer examination of their origins and
natural innate functions, in an effort to ascertain (and possibly
maximize) their potential for cardiogenic differentiation.
To this end, prior studies of fetal cardiac progenitors responsible for cardiomyogenesis and previous lineage tracing experiments in in vivo models may help evaluate the position of the
c-kitpos cardiac population(s) within the known hierarchy of
cardiac progenitors. This body of knowledge provides insights
into the lineage commitment capabilities of c-kitpos cardiac cells
and their likely predisposition toward mature phenotypes of the
contractile, vascular, or adventitial compartments.
Discovery and Ancestry of c-kitpos
Cardiac Cells
The initial discovery of c-kitpos cardiac cells was based on
the fact that the c-kit receptor is expressed in hematopoietic
progenitors10; it was postulated that the presence of c-kit may
identify an intramyocardial population of cardiac progenitors
similar to that of the hematopoietic compartment. In fact, this
is what Beltrami et al10 found. They observed colocalization
of c-kit with Nkx2.5, GATA-4, and Ki-67 but not with mature
sarcomeric proteins, suggesting a precursor cell, ie, a proliferating cell that is apparently committed to cardiac lineage but
lacks a mature phenotype. The absence of the hematopoietic
markers CD34 and CD45 indicated that the cells were not immediately from the bone marrow. Therefore, it was concluded
that the c-kitpos cardiac cells were derived from the embryonic
cardiac compartments that ultimately give rise to the adult
myocardium.10 Notably, this study did not address whether a
pool of intracardiac cells expressing a c-kitpos phenotype represents a population of progenitors persisting in a quiescent
state as remnants from embryonic development or whether
c-kitpos cells arise de novo from c-kitneg cells resident within
postnatal myocardium or even from c-kitneg cells in vitro.
Because the c-kit receptor (whose ligand is stem cell factor)
plays an important role in prosurvival and proproliferative signaling, it is possible that the c-kitpos phenotype may represent an
intermediate progenitor, derived from an upstream c-kitneg, more
undifferentiated cardiac progenitor in which c-kit expression increases in conjunction with cell cycle entry and differentiation.
Beltrami et al10 alluded to this possible hierarchy in their report
of c-kitpos cardiac cells, which were found to largely coexpress
Nkx2.5. This postulated upstream resident progenitor(s), however, has yet to be conclusively identified in the heart. Evidence
of a similar phenotypic progression, now widely accepted, was
observed in the bone marrow with the isolation in 2003 of ckitneg hematopoietic stem cells, which were found to give rise
to c-kitpos intermediate phenotypes that ultimately were able to
reconstitute all mature hematopoietic lineages.26
1218 Circulation Research March 27, 2015
So, what is the embryonic ancestry of c-kitpos cardiac cells?
Answering this question is important in order to ascertain their
regenerative capacity, ie, their ability to replace lost/damaged
cardiac cells of various lineages. Clues to the position of
c-kitpos cells within the hierarchy of established cardiovasculogenic phenotypes may be gleaned by examining their resident
locations within the myocardium, the coexpression of known
phenotypic, lineage-identifying transcription factors and cell
surface markers in vivo and in vitro, and the results of contradictory lineage tracing studies such as those conducted by the
Wu et al16 and Molkentin laboratories.18 Comparisons of these
data with the established characteristics of known cardiac
precursors should indicate a likely origin(s) of c-kitpos cardiac
cells, possible limitations of their differentiation capacity, and
their relative contribution(s) to the adult heart.
Mammalian Cardiac Developmental Biology
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The heart is the first functional organ formed during embryonic development, with cardiac progenitors specified in early
gastrulation. Three spatially and temporally distinct cardiac
precursors have been identified by lineage tracing experiments
in embryonic development: cardiac mesodermal cells, proepicardial cells, and cardiac neural crest cells. These individual
lineages have been established to give rise not only to specific cell types but also to regions of the mature heart.12,27,28
Understanding the specification of these lineages in forming
the mature heart is crucial if insights into the residual progenitors’ capacity to contribute to the contractile, vascular, and interstitial compartments, as well as response to injury, are to be
gained. A brief synopsis of embryonic cardiac development is
provided below (Figure 1).
Within the primitive streak, time-dependent differential
coexpression of vascular endothelial growth factor receptor
2 (VEGFR2) (also known as kinase insert domain receptor
[KDR] and fetal liver kinase 1 [Flk1]) allows the divergence
of hematopoietic and peripheral vasculature progenitors from
the cardiovascular progenitors that give rise to the heart and
central portions of the great vessels.12,27,29–32 The latter are
designated by upregulation of the T-box transcription factors
eomesodermin (Eomes) and mesoderm posterior 1 (Mesp1).
These Mesp1+/Eomes+/KDR+ progenitors give rise to cardiac mesodermal cells that create the first and second heart
fields (FHF and SHF, respectively) with thin endocardium and
the proepicardium.12,27,29–34 Cooperatively, these mesodermal
progenitors and their progeny form the near entirety of the
adult heart. The ectodermal originating cardiac neural crest
cells also contribute to fetal cardiomyogenesis, but their contributions to the contractile compartment are thought to be
minimal and, therefore, are not covered in this review.27,35,36
FHF progenitors in the cardiac crescent are exposed to
local cytokines and growth factors, which induce differentiation and upregulation of essential cardiac regulators such
as Nkx2.5, T-box transcription factor (Tbx)-5, and GATA4,
among others. These transcription factors induce commitment
to myocyte lineage and sarcomeric protein expression.12,27,29,30
Progenitor tracking and lineage tracing studies have shown
that the progeny of the FHF eventually gives rise to the myocytes and some smooth muscle cells that predominantly
make up the left ventricular and both atria.12,16,27,33–35,37 The
endocardium may also arise from FHF progenitors as early
simultaneous development is observed to form the primitive
heart tube, although efforts are ongoing to further delineate
early divergence of these 2 fields from one or more upstream
progenitors.16,27,29,38,39 Subsequent to FHF commitment and
formation of the primitive heart tube, the SHF progenitors,
identified by the expression of islet-1 transcription factor
(Isl1), Nkx2.5, and KDR, begin to proliferate and migrate, undergoing commitment and differentiation under the influence
of local fibroblast growth factors (FGFs), bone morphogenic
proteins (BMPs), and Wnt signaling.12,27,30,40,41 SHF progenitors have been shown to generate myocytes, some smooth
muscle, and some endothelial constituents of the right ventricle and ventricular outflow tract.12,27,29,32,35,37,42–44 Importantly,
Isl1+ progenitors have been found to lack c-kit and Sca-112,40,41
thus likely excluding this compartment as a source of residual
myogenic progenitors having a c-kitpos phenotype.
At this stage of cardiac development, the myocardium of
the FHF and SHF, possessing only a thin endocardial lining within the contorting primitive heart tube,38 is essentially
naked, lacking adventitia, perforating vasculature, or surrounding epicardium. These constituents have been traced to
arise from distinct proepicardial progenitor populations that
express the transcription factors Wilms’ tumor protein (Wt1)
and Tbx-18,12,27,28,35,43,45–48 largely giving rise to adventitial
and smooth muscle lineages, as well as Scleraxis (Scx) and
Semaphorin3D (Sema3D), giving rise to adventitia and some
vascular endothelium not of endocardial origin.49 Some of
these proepicardial progenitors have been found within endocardial cushions, areas well known to be formed by early
endocardial progenitors. This colocalization indicates that
these 2 fields undergo intermigration, essentially cooperating to form the mature structures of the AV valves and
cardiac septa through epithelial to mesenchymal transition
(EMT).39 It is currently unclear whether these proepicardial
populations stem from Isl1+/Nkx2.5+ precursors of the SHF
or are separately derived lineages. Tracing studies show that
these progenitors migrate over the surface of the exposed
myocardium, derived from the FHF and SHF, and form the
epicardium and epicardium-derived cells (EPDCs).12,45,47,50–53
Once formation of the epicardium is complete, epicardial cells proliferate in a direction parallel to the basement
membrane, resulting in thickening of the epicardial lining,
or perpendicular to the basement membrane, undergoing
EMT beginning around E12.5 to 13.5. Ultimately, penetrating mesenchymally transitioned EPDCs, which populate
the subepicardial region, migrate inward to form the coronary plexus (which later becomes the coronary vasculature, with contributions of endocardium-derived endothelial
cells54–56) and cardiac adventitial fibroblasts. Additionally,
the epicardium and EPDCs are involved in septation and
function to stimulate myocardial growth and myocyte division,12,27,28,51,53,57 specifically to aid formation of compact
myocardium. Endocardium-derived adventitia aids in forming the inner trabecular myocardium.56 A detailed hierarchy
of the aforementioned fetal cardiac progenitor phenotypes is
illustrated in Figure 1.
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1219
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Figure 1. Proposed position of c-kitpos intermediates in the hierarchy of cardiac progenitors in the fetal heart. A and B, Arising
first during cardiac development from a common pericardia mesodermal progenitor (brachyury T [Bry] +/ mesoderm posterior 1 [Mesp1]
+/ eomesodermin [Eomes] +/kinase insert domain receptor [KDR] +), endocardial (Bry+/KDR+/NK2 transcription factor related, locus 5
[Nkx2.5] +/islet-1 transcription factor [Isl-1] +) and first heart field (FHF; Bry+/Nkx2.5+/KDR−/c-kitpos) progenitors diverge early; the latter
have been shown to have dedicated cardiomyocyte and smooth muscle cell bipotential differentiation capacity and to include c-kitpos
intermediates. The expression of c-kit is postulated to be low because although these progenitors have been found to be c-kitpos, the
van Berlo study failed to demonstrate their lineage contribution to the cardiomyocyte compartment in the adult heart, and it has been
proposed that recombination in the van Berlo model is less effective in the presence of low levels of c-kit. Endocardial progenitors have
not been shown to express c-kit. C, Subsequently arising SHF progenitors (Isl-1+/Nkx2.5+/c-kitneg) are c-kit negative and contribute
to cardiomyocytes, smooth muscle cells, and vascular endothelium. D, Proepicardial progenitors (with diverging Wt1+/Tbx18+ and
Sema3D/Scx+ populations) arise later from a mesodermal or SHF progenitor, giving rise to vasculogenic lineages and nearly all of cardiac
adventitia including fibroblasts (lineages identified as having partially come from c-kitpos cells in the van Berlo study). Proepicardial
progenitors expressing WT1 and Tbx18 undergo epithelial-to-mesenchymal transition (EMT) characterized by an upregulation of c-kit,
resulting in c-kitpos intermediate phenotypes. The same is likely true for Sema3D/Scx expressing progenitors. The expression of c-kit
in these proepicardial progenitors is postulated to be high because, in the van Berlo study, it was sufficient to induce recombination
in adventitial lineages (which do not arise for FHF or SHF progenitors) and vascular lineages, in contrast to that of known c-kitpos
FHF progenitors, which remained unlabeled. The hierarchy illustrated herein shows that c-kit expression is not limited to one cardiac
progenitor and does not in itself define one specific cardiac precursor population. Shown at the bottom of the figure are the relative
contributions of epicardium-derived cells to each cardiac lineage in fetal development according to the evidence outlined above. MSCs
indicate mesenchymal stromal/stem cells.
1220 Circulation Research March 27, 2015
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It has recently been suggested that EPDCs may generate
cardiomyocytes in fetal development, but this is currently unresolved. Questions have been raised regarding the specificity
of the initial model that used Tbx-18 for in vivo tracing48,58 of
EPDCs. However, similar subsequent evaluation of EPDCs by
Zhou et al using Wilm’s tumor protein (WT1) also suggested
that EPDCs can in fact contribute to mature cardiomyocytes
during fetal cardiogenesis45 although this was rare. The same
group also performed tracing studies of WT1+ epicardial
cells in adult mice but did not find that these cells contribute
to cardiomyocytes or endothelium after infarction46; lineage
commitment after ischemic injury-induced epicardial activation was primarily limited to smooth muscle and adventitial
cells.46 Importantly, the study did observe that epicardial activation did occur as a result of ischemic injury, leading to
proliferation and migration of EPDCs into the damaged myocardium in a reparative role. However, the aforementioned
findings would support the concept that the differentiation
capacity of WT1+ epicardial cells that persists into adulthood
is less than that present in fetal development, because a more
limited lineage commitment, restricted almost entirely to nonmyocytes, was seen in adult mice.46 Scx/Sema3D+ cells were
found to be a distinct population of proepicardial cells having
only 33% overlapping coexpression of either WT1 or Tbx-18.
Scx/Sema3D+ cells were found to give rise predominantly
to coronary endothelial cells and adventitial cells with some
additional contributions to smooth muscle, and rarely cardiomyocytes in the embryonic heart.49 This disproportionally low
magnitude of cardiomyogenic potential mirrors that observed
by the Zhou et al45 tracing study of WT1+ cells. Although initial studies in zebrafish suggested that activation of epicardial
progenitors was responsible for cardiomyocyte replacement
after injury, more recent work has shown that they act by inducing division of existing cardiomyocytes; epicardial cells
were traced to give rise only to nonmyocyte lineages in that
model.28,49,59–62 The current consensus is that the direct contribution of EPDCs to the myocardium is minimal and that
cardiomyocyte differentiation is a rarity among EPDCs, at
least in the postnatal heart.28 A progenitor hierarchy of adult
EPDCs, with proposed phenotypic intermediates, is illustrated
in Figure 2.
Recent studies of the origin of the endocardium, its formation, and its eventual contribution to mature cardiac lineages
have found that its proportional contributions to mature lineages is similar to that attributed to proepicardium-derived cells.
The endocardium arises very early in cardiac embryogenesis,
simultaneously with the FHF, likely stemming from a common progenitor. Endocardial cells have been shown to arise
from brachyury T (Bry) +/fetal liver kinase 1 (Flk1/KDR) +/
Nkx2.5+ progenitors forming the primitive heart tube.38 These
progenitors are distinct from hemangioblast precursors and
are identified by a distinct expression profile (an E-cadherinlow,
Flk1low, nuclear factor of activated T-cells, cytoplasmic 1 [NFATc1] + phenotype).54 NF-ATc1 was found to be expressed exclusively in endocardium, providing a lineage specific marker
that enables differentiation of the endocardium from other endothelial cell types.63 Tracing and knockout studies performed
by de la Pompa et al63 demonstrated that endocardial cells not
only contribute to a subset of cardiac endothelial cells but
also are integral to cardiac cushion formation, valvulogenesis,
septation of the atria, ventricles, and aortopulmonary trunks,
as well as to guiding myocardial trabeculation.38 These processes are governed by EMT of endocardial cells (similar with
respect to mechanism and signaling pathways to that widely
recognized to occur in EPDCs39) that precipitates differential
commitment to various mature cardiac lineages. The complex
regulatory pathways underlying EMT of endocardial cells (as
well as that of EPDCs) involve Notch, transforming growth
factor-β superfamilies, small mothers against decapentaplegic (SMAD) proteins, Wnt/β-catenin, and bone morphogenic
proteins (BMP) signaling among others.39 Comprehensive
reviews of these signaling cascades have recently been published.39 NF-ATc1 null mice, which lacked endocardium and
therefore endocardial contributions to cardiac morphogenesis,
showed marked abnormalities in trunkal, valvular, and septal formation, which were ultimately embryonically lethal.
Interestingly, myocardial, adventitial, and most vascular endothelial compartments were found to be unaffected,38 indicating that the endocardium does not contribute significantly
to these compartments. Similarly, studies in Tie-1/TEK(Tie2)
null mice showed early embryonic lethality with impairment
not only of endocardium formation but also of valvular and
septal derivatives, and a lack of myocardial trabeculation.56
Interestingly, there was no impairment of early cardiomyocyte
formation.56 It remains unclear, however, whether there are
subpopulations of endocardial cells not defined by NF-ATc1
or Tie1/TEK expression that may contribute to these lineages.
Placing c-kitpos Cells Within the Developmental
Hierarchy of Cardiac Progenitor Phenotypes
As supposed residual progenitors remaining from embryonic development, c-kitpos cardiac cells should be able to be
attributed to derivation from one of these aforementioned
precursors; if so, this would provide insights into their predisposition to form the various mature cardiac phenotypes.
Clues to this assignment can be gained from available data on
the location and phenotype of c-kitpos cells and from lineage
tracing studies. In the aggregate, these data, detailed below,
support the concept that c-kitpos cardiac cells likely represent intermediate phenotypes from more than one progenitor compartment within embryonic cardiomyogenesis, and
that c-kit expression, in itself, does not define one specific
cardiac precursor. Indeed, c-kit expression has been found in
intermediate phenotypes in very early bipotential myogenic
FHF progenitors16 as well as in epicardium-derived cells that
undergo EMT to largely make vascular and advential lineages.35,37,38,49,51,53,55,64–68 The same may be true of c-kitpos cells
isolated from endocardial biopsies25,39 (this will be discussed
later). C-kit expression in these various progenitor lineages
in the developing heart may vary not only temporally and
spatially but also in the absolute levels of protein expressed.
We suggest that these factors may account for discrepant results obtained by many groups in characterizing c-kitpos cells.
We provide below a critical appraisal of the literature in an
attempt to reconcile these differences.
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1221
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Figure 2. Proposed origin of c-kitpos cells in the adult heart. The figure illustrates the concept that, in the adult heart, c-kitpos cells are
intermediates derived from c-kitneg epicardial progenitors that undergo epithelial-to-mesenchymal transition (EMT). (EMT is known to be
associated with expression of c-kit.) These c-kitpos intermediates have a mesenchymal phenotype and give rise to c-kitneg epicardiumderived cells (EPDCs) with differentiation potential limited to noncardiomyocytic lineages. A, Wilm’s tumor protein (WT)-1+/T-box
transcription factor (Tbx) 18+ epicardial progenitors contribute predominantly to smooth muscle and cardiac adventitial fibroblasts, with
minimal endothelial cell formation. B, Sema3D+/Scx+ epicardial progenitors (distinct from WT-1+/Tbx18+ cells) form vascular endothelium
and cardiac fibroblasts and contribute minimally to smooth muscle cells. Both of these epicardium-derived c-kitpos intermediates express
CD105 and possess a mesenchymal phenotype with canonical mesenchymal stromal/stem cell (MSC) markers; neither of these two
populations has shown any significant ability to form myocytes. The expression of c-kit in these epicardium-derived intermediates is
postulated to be high relative to that of c-kitpos intermediates from the FHF (shown in Figure 1), which have bipotent cardiomyocyte and
smooth muscle differentiation capacity. This differential c-kit expression among cardiac progenitors is currently a conjecture and has not
been demonstrated experimentally; nevertheless, it is inferred from the results of the van Berlo study, which did not detect significant
myogenesis from first heart field c-kitpos progenitors but detected robust evidence of a c-kitpos progenitor of fibroblasts, smooth muscle
cells, and endothelial cells (all of which are of epicardial origin), and from the proposed insensitivity of the van Berlo model to low levels
of c-kit expression, which could theoretically result in underestimation of low expressers of c-kit. Shown below the figure is the relative
contribution of mesenchymally transitioned EPDCs to cardiac lineages of the adult heart; this pattern is consistent with studies of adult
c-kitpos cells, which have shown preferential adventitial and vasculogenic differentiation and a paucity of direct cardiomyogenic potential.
Evidence for c-kit Expression in
Early FHF Progenitors
As mentioned above, the FHF progenitors give rise exclusively to cardiomyocytes and smooth muscle cells.12,33–35,37 It has
been shown that the simultaneously developing FHF progenitors and endocardium, although possibly originating from a
common upstream mesodermal precursor cell, diverge very
early with discrete specification to respective nonoverlapping
lineages.16,35,37–39,54
Direct evidence supporting a c-kitpos intermediate phenotype of FHF progenitor cells was provided in a seminal paper by Wu et al in 200616. In this work, the authors utilized
both in vitro studies of embryonic stem cells and in vivo
Nkx2.5-enhanced green fluorescent protein (eGFP) transgenic mice to examine the lineage specification of Nkx2.5+
cardiac progenitors throughout embryonic cardiomyogenesis.
They found that, in vitro, cardiac differentiation of embryonic
stem cells produced a subpopulation of Nkx2.5+/c-kitpos progenitors, lacking Flk1/Tie2(TEK) expression, which exhibited
specific bipotential differentiation capacity toward cardiomyocytes and smooth muscle cells.16 However, Nkx2.5+/ckitneg cells showed higher ability to directly differentiate into
cardiomyocytes and smooth muscle cells in vitro than did
Nkx2.5+/c-kitpos cells; therefore, c-kit positivity was viewed
to be dispensable for cardiomyogenesis. Once isolated from
E9.5 mouse hearts, Nkx2.5+/c-kitpos cells were able to form
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mature smooth muscle cells and cardiomyocytes.16 Thus,
Nkx2.5+/c-kitpos cells at E9.5 showed similar dedicated bipotential commitment to cardiomyocyte and smooth muscle
lineages as did those from in vitro studies of embryonic stem
cells and adoptive transfer studies in chick embryos. Evidence
of c-kit expression in FHF progenitors is also provided by a
study by Ferreira-Martins et al,15 in which c-kitpos cells were
directly visualized in murine embryonic hearts at embryonic
gestational day 6.5, a period of development currently thought
to be confined solely to FHF progenitors during primitive
heart tube formation, before the appearance of the SHF or the
proepicardium.27,35,69
In summary, the study by Wu et al16 demonstrates that a
subset of Nkx2.5+/eGFP+ cells coexpress c-kit in both in
vitro and in vivo and that the Nkx2.5+/eGFP+/c-kitpos cells
were able to generate smooth muscle cells as well as cardiomyocytes in single cell cloning. Interestingly, these cells were
dedicated solely to these 2 lineages, specifically showing only
bipotential differentiation capacity.16 Nkx2.5+/c-kitpos cells
showed no overlapping expression of Flk1 or Tie2(TEK), indicating a lack of endothelial commitment, and no endothelial
cells were observed to be generated from differentiation of
these early Nkx2.5+/eGFP+/c-kitpos progenitors in vitro. This
myogenic lineage restriction is consistent with that of FHF
progenitors. These results would appear to be in conflict with
the differentiation potential of c-kitpos cardiac cells observed
by Ferreira-Martins et al,15 who found formation of not only
cardiomyocytes and smooth muscle cells but also endothelial
cells. However, Ferreira-Martins et al15 isolated c-kitpos cells
much later in cardiac development (E16-18), a time when
FHF, SHF, and proepicardial development are all simultaneously taking place. Accordingly, the c-kitpos cardiac cell population utilized in that study may have been heterogeneous, with
c-kitpos cells originating from multiple compartments, which
would have resulted in a broader differentiation potential
compared with that observed by Wu et al.16 Further analyses
by Wu et al16 comparing c-kitpos and c-kitneg Nkx2.5+ progenitors supported the concept that the c-kitpos/Nkx2.5+ state is
an upstream intermediate progenitor phenotype, which, upon
commitment to smooth muscle and/or cardiomyocyte lineages, loses c-kit positivity, retaining only Nkx2.5. Importantly,
c-kit expression was observed to be down regulated, with very
few c-kitpos cells detected in the fetal murine heart by E15.5
despite ongoing cardiac development; thus, further myocyte
formation after E15.5 may be ascribable to c-kitneg progenitors
such as those described by Wu et al16 (Nkx2.5+/c-kitneg cells)
and/or to proliferation of cardiomyocytes themselves.62,70 In
this connection, division of existing cardiomyocytes, rather
than formation of new myocytes from pools of undifferentiated residual progenitors, appears to be the predominant mechanism for cardiomyogenesis in the neonatal heart, although this
ability is lost within weeks of birth.62
Evidence That Cells Expressing c-kit Are of
Proepicardial Origin and Mesenchymal in
Nature
Numerous independent laboratories have provided evidence
supporting the concept that c-kitpos cardiac cells, especially in
the postnatal heart, are derived from the proepicardium and
are mesenchymal in nature (Table). This body of evidence can
be summarized as follows.
Location of Adult c-kitpos Cells
C-kitpos cardiac cells in adult human and murine hearts inhabit predominantly the subepicardium and adjacent myocardial interstitium, regions derived from proepicardial
progenitors.64–67,71,72 Immunohistochemical labeling of c-kitpos
cells shows an epicardial to endocardial gradient.65,66
Expression of Proepicardial Markers in Some
c-kitpos Cells
Additional evidence for the proepicardial origin (and EMT)
of these cells is provided by recent studies showing that many
murine epicardial WT1 and Tbx18 expressing cells also coexpress c-kit and that this expression increases with epicardial
activation.67,71
In Vitro Generation of c-kitpos Cells by EMT of
Epicardial Cells
Human c-kitpos cells can be generated in vitro by inducing
EMT of human epicardial cells with transforming growth
factor-β.66 In vitro–generated c-kitpos cells exhibit expression
of mesenchymal markers at the mRNA level similar to that
of c-kitpos cardiac cells analyzed directly after isolation from
human cardiac tissue. This is in contrast to the expression
profile of directly isolated epicardial mesothelial cells.66 An
important implication of these observations is that a c-kitpos
phenotype can arise in vitro from c-kitneg cells, raising the
possibility that c-kitpos cells isolated and expanded in vitro
for therapeutic purposes may not represent, as commonly
thought, a resident c-kitpos embryonic remnant within the
myocardium.
Expression of Mesenchymal Markers in c-kitpos
Cells
Many studies by independent groups have consistently shown
that adult human c-kitpos cardiac cells express CD105, CD29,
and other mesenchymal-associated markers both in vivo and
in vitro.11,51,65–68,72–79 The in vivo expression, assessed by immunohistochemical staining, indicates that this mesenchymal
phenotype is inherent to c-kitpos cardiac cells from adult humans and mice and is not the result of in vitro artifacts or
culture drift.72 In the van Berlo study,18 small numbers of cardiomyocytes were found to originate from c-kitpos progenitors;
at least some of these were ascribed to cellular fusion, a phenomenon that is known to occur in mesenchymal stromal/stem
cells (MSCs).88–91
Differentiation Potential of c-kitpos Cells
When placed in directed differentiation conditions, adult ckitpos cells have shown a capacity to express markers of osteocytes, chondrocytes, and adipocytes typical of MSCs in
addition to some mature cardiac proteins.11,72,77,80
C-kit Expression in MSCs
MSC populations from various tissues (oral, adipose, bone
marrow, and cardiac tissue) express c-kit,72,81–86 indicating that
this protein is associated with mesenchymal lineages and that
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1223
Table. Evidence That c-kit Is Expressed in More Than One Progenitor Compartment and That c-kit
Expression in Itself Does Not Define a Capacity for Myogenic Differentiation Potential
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CD indicates cluster of differentiation; EPDC, epicardium-derived cell; ESC, embryonic stem cell; FHF, first heart field; Flk-1, fetal liver kinase
1; Nkx2.5, NK2 transcription factor related, locus 5; SHF, second heart field; Tbx, T-box transcription factor; and WT, Wilm’s tumor protein.
1224 Circulation Research March 27, 2015
those progenitor populations within various compartments
share a similar biology.
Lineage Tracing Studies
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Recently, van Berlo et al18 conducted a c-kitpos lineage tracing
study in mice utilizing permanent recombination to track all
progeny of c-kit expressing cells throughout cardiac organogenesis as well as after injury. Mature phenotypes arising from c-kitpos
progenitors were found to be mostly endothelial cells (which
also may have arisen from c-kitpos hemangioblast precursors
and not exclusively from cardiac progenitors) and few smooth
muscle and stromal interstitial cells including fibroblasts, but
rarely cardiomyocytes.18 Concerns have been raised regarding
the efficiency of recombination and the effect of the loss of a
c-kit allele in this study.87 However, even if one assumes that
there was suboptimal recombination in low expressers of c-kit
(which would result in underestimation of the contribution of ckitpos cells to adult cardiac lineages), this would not invalidate the
findings of positive recombination events in higher c-kit expressers and the mature cardiac lineage contributions thereof. Indeed,
no presumption of inaccurate recombination has been raised, nor
was such off target recombination observed by the authors in
the validation of their murine model.18 The lineage distribution
reported by van Berlo et al18 would imply that these supposed
high expressers of c-kit (c-kithigh cells) are likely derived from the
proepicardium because the FHF and SHF have not been shown
to contribute to fibroblasts or interstitial cells12,27,28 and smooth
muscle cells from the FHF share a common precursor with cardiomyocytes generated from that compartment.16 Lineage tracing studies of WT1+ and Tbx-18+ proepicardial progenitors in
fetal cardiomyogenesis have shown similar degrees of distribution toward noncardiomyocyte phenotypes as well as only
a small contribution to mature cardiomyocytes, mirroring the
observations of van Berlo et al.18,45,46,48 Further implications of
a possible insensitivity to lower expressers of c-kit in the heart
(c-kitlow cardiac cells) are discussed later.
Paracrine Mechanism of Action of Adult c-kitpos
Cells
Although bone marrow–derived MSCs have beneficial effects in
the setting of ischemic cardiomyopathy, differentiation of these
cells into cardiomyocytes seems unlikely23,88,90,91; rather, MSCs
are thought to work via paracrine actions.23,24 Similarly, we have
found that c-kitpos cardiac cells also appear to work via paracrine
actions.1–5,17 Although c-kitpos cells administered in animal models
of ischemic cardiomyopathy have been reported to differentiate
into phenotypically mature cardiomyocytes on tissue histopathologic examination,10,15,92 we1,3–5,17 and others11,19,20,22,72 have not
observed this phenomenon. Tracing studies of eGFP-labeled ckitpos cells have shown very limited engraftment, with isolated,
small eGFP+ cells displaying a disorganized pattern of staining
for sarcomeric proteins or smooth muscle actin1–5,17,19,20; rarely, if
ever, are mature cardiomyocytes observed that are derived from
transplanted cells. Despite this, administration of in vitro expanded c-kitpos cardiac cells has been reproducibly beneficial in preclinical and clinical studies of heart failure, implying a paracrine
mechanism, eg, antifibrotic or antiapoptotic actions, or activation
of endogenous precursors triggered by factors released from the
transplanted cells.3 This postulated paracrine mechanism would
be consistent with a proepicardial origin because throughout
development proepicardium-derived cells are known to support
the myocardium by secreting a variety of beneficial growth factors.12,27,30,35,37,46,71 The specific paracrine mediators responsible for
these beneficial effects are the focus of active investigation, and
likely involve a host of pathways including microparticles and
microRNA-mediated effects as well as release of growth factors
and cytokines such as stromal cell-derived factor 1, vascular endothelial growth factor, and many others. Regardless of the precise
mechanism(s) involved, the limited ability of adult transplanted
c-kitpos cells to acquire a mature cardiomyocytic phenotype is also
consistent with the limited ability of proepicardium-derived cells
to differentiate into myocytes.12,27,28,35,45,46
Some may point to results of in vitro differentiation of
adult c-kitpos cells, along with coexpression of factors such
as GATA4 in vitro and in vivo, as evidence to the contrary.
However, the expression of GATA4, like that of Nkx2.5, is
not restricted to cardiomyocyte precursors nor is it indicative
of specific cardiomyocyte commitment. GATA4 knockout
studies in murine embryos have concluded that this factor is
expressed in, and necessary for, formation of the proepicardium and its derivatives,93,94 which is again consistent with a
proepicardial origin of c-kitpos cardiac cells. The finding that
cardiac troponin T is expressed after in vitro differentiation or
in in vivo transplantation of c-kitpos cells has been construed as
evidence of cardiomyocyte differentiation; however, smooth
muscle cells may also express cardiac troponin T.16,95 These
facts highlight the fundamental importance of using multiple
markers and methodologies to document differentiation into
a specific lineage and to define an undifferentiated starting
population. In vitro differentiation conditions are highly artificial because they utilize nonphysiological stimuli that may
cause cellular drift potentially not indicative of what occurs
in vivo.13,14,77 Direct evidence supporting this concept is the
observation by Miyamoto et al96 that in vitro expanded c-kitpos
cardiac cells cultured in cardiac differentiation medium expressed not only some native cardiac markers but also markers typical of adipose and skeletal muscle lineages. Because
cells expressing these markers are not present within normal
myocardium, it may be concluded that this in vitro behavior
deviates from any normal function or derivation of c-kitpos cardiac cells in vivo, irrespective from which compartment (FHF,
proepicardial, or other) they originate, and can be considered a
culture artifact or drift. Such observations bring into question
the validity of relying on cardiomyogenic differentiation in
vitro as a true representation of in vivo capability (vide infra).
Although the evidence summarized above supports the notion
that adult c-kitpos cells may be of proepicardial origin and share a
mesenchymal-like phenotype, expressing canonical MSC markers, these cells appear to differ in a tissue-specific manner from
conventional MSCs; for example, they differ from MSCs isolated from the bone marrow both functionally and in their ability
to express multilineage markers of differentiation in vitro.19,72,97,98
C-kitpos Cells From Human Endomyocardial
Biopsies
One potential objection to the concept that c-kitpos cells originate
entirely from the FHF or are of proepicardial origin is that these
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1225
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cells have been isolated from endomyocardial biopsies obtained
from the right ventricular septum.25 Such observations are not
necessarily in conflict with the postulated origin of c-kitpos cardiac cells from the FHF or the proepicardium because it is possible that c-kit expression is not limited only to EMT of epicardial
cells but occurs more broadly as a part of EMTs. EMT is well
recognized to occur in endocardial epithelial cells that contribute
to various cardiac structures such as atrioventricular cushions,
valves, and septa as well as to vascular endothelium and cardiac adventitia,38,39 a pattern similar to the lineage capabilities of
EPDCs. In-depth reviews of these phenomena have been recently
published.39 Thus, endocardial cells obtained from endomyocardial biopsies (EMBs) may undergo EMT in vitro with resultant
upregulation of c-kit expression. This would parallel that which
has been observed in vitro in epicardial mesothelial cells.66
Besides the observations of increased c-kit expression in
epicardial EMT-induced in vivo and in vitro by transforming
growth factor-β, there is mounting evidence that similar c-kit
expression occurs in extracardiac tissues undergoing EMT as
well as in EMT leading to tumorigenesis.99,100 Studies of in vitro transforming growth factor-β–induced EMT in noncardiac
epithelial cell lines have shown an increase in expression of ckit and mesenchymal markers, essentially mirroring the results
obtained with induction of EMT in human epicardial mesothelium.66 These observations would indicate that c-kit upregulation
is biologically integral to the process of EMT itself, independent from the cell type of origin. If this hypothesis is correct, the
expansion of c-kitpos cells from endomyocardial biopsies could
be explained by EMT of endocardial cells in vitro.
Another potential explanation for the isolation of c-kitpos
cells from endocardial septal biopsies relates to the intermigration and cooperative function of EPDCs and endocardial
cells within the outflow tracts and adjacent AV cushions during cardiogenesis and/or as a part of septation. Cells from both
the epicardial and the endocardial fields work in tandem to
perform complex structural rearrangements to complete the
formation of a mature 4-chambered heart. It is possible that
the subendocardium and adjacent interstitial adventitia consist of cells with embryonic ancestral heterogeneity, being of
endocardial and proepicardial origin.
A Unifying Theory of c-kit Expression
in the Heart
Taken together, the evidence reviewed above supports the
concepts that (1) c-kit expression in the myocardium is not
limited to one progenitor but is a property of cells that originate from multiple pools of progenitors in the developing and
postnatal heart (eg, FHF, proepicardium), and (2) c-kit expression in itself does not define the embryonic origins, lineage
capabilities, or differentiation capacities of the various progenitors. C-kitpos cardiac cells from the FHF show marked
cardiomyogenic and smooth muscle differentiation capacity
early in fetal development.16 However, there is inconclusive
evidence that c-kitpos cells from this FHF compartment persist
in the postnatal heart into adulthood. More likely, any residual
progenitors from this field would exhibit only an Nkx2.5+
state because Wu et al16 observed a drastic downregulation of
c-kit expression in Nkx2.5+ cells, with c-kit becoming nearly
undetectable in E15.5 murine hearts. This may indicate depletion of the Nkx2.5+/c-kitpos early intermediate phenotypes
within the FHF progenitor pool. Any subsequent progenitor
proliferation and contributions to the contractile compartment
past E15.5 might be attributed to the more mature Nkx2.5+/ckitneg progenitors observed and characterized by Wu et al16 as
well as to cardiomyocytes62,70 and smooth muscle cells themselves, as mounting evidence suggests.62
Because no markers specific to the FHF have yet been identified that would allow segregation of c-kitpos cardiac populations, it is difficult to know what proportion of these cells
in the postnatal myocardium, if any, is a remnant from the
FHF with primary cardiomyogenic potential versus c-kitpos
cells stemming from other compartments such as the proepicardium whose contributions during cardiomyogenesis are
overwhelmingly to noncardiomyocyte lineages. It may reasonably be postulated that the number of c-kitpos cardiac cells
is proportional to the proliferative activity of their progenitors
and that the largest fraction of c-kitpos cardiac cells remaining
in the adult myocardium represents the compartments with
the largest proliferative and regenerative reserve. According
to this hypothesis, the lack of appreciable myocyte replacement in the contractile compartment, in contrast to the overwhelming plasticity and reserve of the vascular and adventitial
compartments (which encompass the progeny of non-FHF
progenitors), would indicate that the adult c-kitpos cardiac cells
represent intermediate phenotypes of these residual nonmyocyte contributing progenitor pools or even intermediates of
recently described transdifferentiating cell types undergoing
EMT such as vascular endothelial cells.101
So, then, how can studies such as those conducted by Wu
et al16 and van Berlo et al,18 with opposite conclusions regarding the cardiomyogenic capacity of c-kitpos cardiac cells, be
reconciled assuming that the findings of both may in fact be
valid? As discussed above, one possibility is that, as some have
proposed,87 the van Berlo model was not sensitive to recombination in cases of very low c-kit expression (c-kitlow cells) and
therefore only traced the lineage contributions of higher c-kit
expressers (c-kithigh cells). The van Berlo study clearly shows
that a large portion of cardiac endothelial cells (which may in
part be progeny of c-kitpos hemangioblasts and not dedicated
cardiac progenitors), as well as some smooth muscle and adventitial cells, arise from a progenitor with a c-kitpos intermediate phenotype. Again, this mature lineage distribution is not
inconsistent with a proepicardial and/or endocardial origin.
Additionally, this c-kithigh progenitor, which has a sufficiently
robust c-kit expression to induce recombination in the van
Berlo model, does not give rise to an appreciable number of
cardiomyocytes, thus leaving the contractile compartment as
the progeny of other progenitors. Assuming the validity of the
findings of Wu et al,16 who clearly demonstrated the bipotential
differentiation capacity (cardiomyocytes and smooth muscle
cells) of an Nkx2.5+/c-kitpos progenitor very early in embryonic cardiomyogenesis, and those of Ferreira-Martins et al,15 who
observed c-kitpos cardiac cells at embryonic gestational day 6.5,
both consistent with FHF progenitors, the differences between
the studies could be explained if these FHF c-kitpos cells possess lower levels of c-kit compared with cells of proepicardial/
1226 Circulation Research March 27, 2015
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endocardial origin (c-kithigh cells) and if the expression of ckit in these c-kitlow cells was insufficient to induce recombination and visualization in the van Berlo model. According to
this hypothesis, the contributions of FHF c-kitlow progenitors to
the adult myocardium would be underestimated, as some have
proposed.87 By segregating c-kitpos cardiac progenitors into ckithigh and c-kitlow expressers, this conceptual construct would
reconcile the Wu et al16 and van Berlo et al18 studies and allow
for both to be included under one unifying paradigm.
Whether these postulated FHF c-kitlow cardiac cells persist
into adulthood or are depleted early in embryonic development, as would be suggested by Wu et al16 and by studies of
neonatal cardiac regeneration,62 remains to be conclusively
elucidated. The evidence examined in this review regarding
the characteristics of adult c-kitpos cardiac cells that have been
isolated and expanded from adult human myocardial samples
would indicate that these c-kitlow cardiac progenitors are no
longer present in adult hearts. It is much more likely that cells
isolated from adult human cardiac specimens are c-kithigh cells,
not only for the reasons outlined above but also because of
the methodology of magnetic-activated cell sorting that is utilized to isolate cells for clinical or preclinical uses. Magnetic
immunoselection preferentially selects the highest expressers
and highest retainers of the immunomagnetic ferrous beads;
accordingly, low expressers of an antigen of interest are very
likely to pass through the selection column together with
negatively selected cells. In view of this, and considering the
entire body of evidence discussed in this article, we believe
that the cells expanded in vitro from adult cardiac tissue are
c-kithigh expressers of proepicardial origin.
The likely proepicardial origin and mesenchymal nature of
adult c-kitpos cells may explain their predisposition to form endothelium and some smooth muscle and adventitial cells, and
their lack of robust cardiomyocyte differentiation, which is
consistent with the recently published lineage tracing analysis.18
Additionally, the ability to form cardiomyocytes appears to differ significantly between neonatal and adult c-kitpos cells11,102–104;
the former can form cardiomyocytes, albeit to a limited extent,
whereas the latter either have lost this ability or do so at a minuscule rate. This difference mirrors the aforementioned differential cardiomyogenic capacity of EPDCs in fetal/neonatal and
adult mouse hearts,45,46 again suggesting a proepicardial origin.
Endogenous Versus Exogenous c-kitpos Cells
The evidence reviewed above pertains to c-kitpos cells residing in the heart (endogenous cells). An important question is
whether their properties can be extrapolated to c-kitpos cells
isolated, cultured, and expanded in vitro (exogenous cells).
What effect do in vitro conditions and expansion have on the
inherent differentiation capacity of these cells?
As previously mentioned, it is theoretically possible that in
vitro conditions increase or shift the differentiation capacity of
c-kitpos cells from certain lineages to others, possibly by disinhibition, resulting in increased cardiomyocyte formation, whereas
in the in vivo setting environmental signals, especially in the
adult heart, may limit this phenomenon, even in response to injury. However, evidence exists that this may not be the case.11 As
indicated above, data regarding exogenous (expanded) c-kitpos
cells are conflicting: while some studies have concluded that
these cells undergo full cardiomyogenic differentiation in the
recipient heart,10,15,92 we1–5,17,21 and others11,12,19,20,22 have found
that these cells do not assume a cardiomyocytic phenotype
when transplanted in vivo. The reason(s) for these discrepancies is unknown. Cells generated in one laboratory cannot be assumed to be identical to those generated in another laboratory,
as even subtle differences in culture conditions may bring about
phenotypic changes in cultured cells. In any case, the important
concept here is that the cardiomyogenic potential (as well as
other properties) of exogenous c-kitpos cells is likely different
from that of endogenous c-kitpos cells. The former have been expanded and cultured extensively in highly artificial conditions
that almost certainly affect cellular functions and may favor a
selection of the fastest replicating subsets of cells.
Indeed, considering the dramatic differences between culture and in vivo conditions, it would be surprising if many
cell properties were not affected. An obvious example is the
population doubling time of cultured c-kitpos cells (typically,
<30 hours) which is much shorter than that of endogenous
cells in vivo. Another example, described above, is the aberrant expression of noncardiac proteins that has been reported
in c-kitpos cells cultured in differentiation media.72,96 There are
likely many other differences, which are not unexpected when
one considers the very artificial (and often arbitrary) culture
conditions and the enormous differences between the environment to which c-kitpos cells are exposed in vitro and in vivo.
In our opinion, extrapolation from artificial (and largely arbitrary) culture conditions to the very complex environment in
the intact organism, with its myriad of signaling stimuli and
other modulating influences (most of which remain poorly
understood or unknown), is not warranted. Conclusions predicated on studies of exogenous c-kitpos cells should not be extrapolated to endogenous cells and vice versa.
Conclusions
In this essay, we have proposed a unifying theory that reconciles ostensibly discrepant results obtained in studies of c-kitpos
cardiac cells over the past 2 decades. We have (facetiously)
dubbed this construct the “string theory” of c-kitpos cardiac cells
(in analogy to the theory that has been proposed to explain the
physical universe105) because it reconciles multifarious and
sometimes apparently discrepant results. We have also cautioned against extrapolating studies of endogenous c-kitpos cells
to those of exogenous (expanded) c-kitpos cells and vice versa.
To recapitulate, multiple lines of evidence support the
concept that c-kit is expressed in more than one fetal cardiac
progenitor pool (ie, both FHF and mesenchymally transitioning proepicardium and EPDCs), and that its expression does
not define one specific myogenic precursor. C-kit expression
within these pools may vary not only temporally and spatially
throughout cardiac development but also in terms of absolute
protein levels. The apparently conflicting results of studies of
endogenous c-kitpos cells could be explained by the existence
of two populations of intermediate cardiac precursors, low
and high c-kit expressers (c-kitlow and c-kithigh). The former
would be derived from the FHF, give rise to cardiomyocytes
and smooth muscle cells, and are likely depleted during fetal
Keith and Bolli c-kitpos CSCs: Reconciling Discrepant Results 1227
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cardiomyogenesis, thus not persisting within the adult heart; if
they persist, they would likely escape isolation by conventional magnetic-activated cell sorting. The latter would be derived
from the proepicardium, display a mesenchymal phenotype,
give rise to some adventitial cells (including fibroblasts),
smooth muscle cells, and endothelial cells, and persist in the
adult heart, with a continuous cycle of epicardial cells undergoing EMT and migrating inward into the myocardium, especially in response to injury.65–67,106 These are likely the c-kitpos
cells that are isolated with magnetic-activated cell sorting from
adult myocardium. Because of their postulated lower levels of
c-kit expression, the former may not recombine efficiently in
a Cre knockin model such as the van Berlo study,87 thus yielding an underestimation of the contributions of FHF c-kitlow
progenitors to the contractile compartment (myocytes and
smooth muscle) during fetal development.
This paradigm accounts both for the robust cardiomyocytic differentiation of c-kitpos intermediates reported by
Wu et al16 during development and for the recently observed proclivity of endogenous c-kitpos cells to differentiate more toward interstitial and vascular lineages and less
toward contracting myocytes reported by van Berlo et al.18
Furthermore, it illuminates the apparent paradox regarding
the mechanism of action of exogenous c-kitpos cells isolated
from adult hearts. Because MSCs are known to work primarily via paracrine mechanisms,23,24 the recognition that
exogenous postnatal c-kitpos cardiac cells resemble the phenotype of traditional MSCs provides insights into the consistent functional benefits afforded by these cells despite the
paucity of their cardiomyocytic differentiation, and helps
to reconcile the recent report that endogenous c-kitpos cells
contribute minimally to restoring the cardiomyocyte compartment in the adult heart18 with the remarkable therapeutic
actions of exogenous c-kitpos cells.3
This paradigm does not exclude the possibility that an early
c-kitpos intermediate phenotype of FHF progenitors may give
rise to large numbers of cardiomyocytes, as was observed by
Wu et al.16 Although the data reviewed above indirectly support our theorem, the presence of two (or more) populations
of cardiac cells expressing different levels of c-kit (c-kitlow
and c-kithigh cardiac cells) is presently a conjecture and needs
to be verified experimentally. Clearly, more work is needed
to differentiate subsets of c-kit expressing cells on the basis
of multiple markers and to define residual pools of preferentially cardiomyogenic c-kitpos cells in the adult myocardium
if they are in fact still present. Currently, it appears that the
c-kitpos cardiac cells able to be isolated and expanded from
postnatal myocardium for therapeutic purposes are limited to
those without any significant cardiomyogenic capability and
represent intermediates from compartments other than the
FHF (ie, proepicardium). If the goal is to maximize formation
of new myocytes, new therapeutic approaches utilizing these
proepicardial/endocardial c-kitpos cardiac cells, such as reprogramming techniques, rather than simple in vitro expansion
and administration, may be useful to increase cardiomyocyte
differentiation, especially in cells harvested from adult hearts
that may show even more restricted lineage capabilities than
those in fetal or neonatal development.11
Sources of Funding
This work was supported by NIH grants P01 HL-78825 and 1 UM1
HL-113530 (CCTRN).
Disclosures
None.
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''String Theory'' of c-kitpos Cardiac Cells: A New Paradigm Regarding the Nature of
These Cells That May Reconcile Apparently Discrepant Results
Matthew C.L. Keith and Roberto Bolli
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Circ Res. 2015;116:1216-1230
doi: 10.1161/CIRCRESAHA.116.305557
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Correction
The Circulation Research article by Keith and Bolli (“String Theory” of c-kitpos Cardiac Cells: A New
Paradigm Regarding the Nature of These Cells That May Reconcile Apparently Discrepant Results. Circ
Res. 2015:116:1216-1230. doi: 10.1161/CIRCRESAHA.116.305557) states that van Berlo et al (2014)
observed that large numbers of fibroblasts and adventitial cells, some smooth muscle and endothelial
cells, and rare cardiomyocytes originated from c-kit positive progenitors. However, van Berlo et al reported that only occasional fibroblasts and adventitial cells derived from c-kit positive progenitors in
their studies. Accordingly, the review has been corrected to indicate that van Berlo et al (2014) observed
that large numbers of endothelial cells, with some smooth muscle cells and fibroblasts, and more rarely
cardiomyocytes, originated from c-kit positive progenitors in their murine model.
The authors apologize for this error, and the error has been noted and corrected in the online version
of the article, which is available at http://circres.ahajournals.org/content/116/7/1216.full
(Circ Res. 2015;116:e133. DOI: 10.1161/RES.0000000000000057.)
© 2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/RES.0000000000000057
e133