Cardiovascular Research 2005 65(2):293-295; doi:10.1016/j.cardiores.2004.11.031
Copyright © 2004, European Society of Cardiology
Bone marrow cells for cardiac regeneration: the quest for the protagonist continues
Buddhadeb Dawn* and
Roberto Bolli
From the Institute of Molecular Cardiology, University of Louisville, ACB, 3rd Floor 550 South Jackson Street, Louisville, KY 40292, USA
* Corresponding author. Tel.: +1 502 852 7959; fax: +1 502 852 7147. Email address: buddha{at}louisville.edu
Received 22 November 2004; accepted 30 November 2004
See also article by Hattan et al. [11] (pages 334-344) in this issue.
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1. Introduction
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The past few years have witnessed an explosion of research on
methods to achieve myocardial repair via cellular transplantation
or mobilization of endogenous cells
[1]. Several different cell
types, including skeletal myoblasts
[2], c-kit
+/Sca-1
+/lin
– bone marrow (BM) cells
[3], BM mesenchymal stem cells (MSCs)
[4], BM ‘side-population’ (SP) cells
[5], endothelial
progenitor cells (EPCs)
[6], and cardiac stem cells (CSCs)
[7],
have been utilized in an effort to achieve cardiac reconstitution
and restoration of function following myocardial infarction
(MI). The evidence accumulated over the past few years indicates
that left ventricular (LV) function and adverse LV remodeling
can be ameliorated via cell-based therapies, and the preliminary
results from several small-scale, mostly non-randomized, clinical
trials have been encouraging
[8–10]. However, the mechanism(s)
underlying the observed functional improvement remains unclear
and the debate regarding the advantages of one candidate cell
versus another continues unabated.
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2. The ideal cell for therapeutic use
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So, what characteristics should a cell have in order to be selected
for therapeutic use in humans? The answer is simple: it has
to differentiate into cardiomyocytes that are connected both
mechanically and electrically with native myocytes (contracting
in synchrony with adjacent cells) and be able to form vascular
structures in order to get blood supply in the scar. Thus far,
very few cell types have been shown to do all of these things.
In this issue of the Journal, Hattan, Kawaguchi, and colleagues report the isolation and purification of a ‘cardiomyogenic’ subpopulation from BM MSCs [11]. MSCs were treated with 5-azacytidine followed by transfection with an EGFP cDNA driven by the myosin light chain (MLC)-2v promoter. EGFP-positive cells were isolated by flow cytometry. These cells expressed cardiomyocyte-specific genes in culture, incorporated BrdU, exhibited sarcomeric organization, and demonstrated spontaneous beating after 3 weeks. Electrophysiologic studies revealed a cardiomyocyte-like action potential. Following transplantation into non-injured mouse hearts, EGFP-expressing cardiomyocytes could be detected within the myocardium.
These results [11] clearly demonstrate that BM MSC-derived cardiomyocytes can survive following transplantation in vivo and integrate into the host myocardium. The findings are important because they raise the possibility of using large-scale isolation of BM-derived cells destined to differentiate into cardiomyocytes for therapeutic use in myocardial repair. However, before this type of preselection can be applied to humans, several issues need to be resolved. First, although the expression of MLC-2v and the concomitant expression of a marker identify a cell as ‘cardiomyogenic’, these cells have not been characterized with respect to antigen expression and lineage commitment. Previous studies of BM cells have yielded conflicting results. For example, evidence has been provided that c-kit+/Sca-1+/lin– cells differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells following intramyocardial injection [3] and that BM-derived SP cells also transdifferentiate into cardiomyocytes and endothelial cells [5]. Others, however, have reported that c-kit+/Sca-1+/Thy1.1lo/lin– long-term repopulating hematopoietic stem cells failed to differentiate into anything but adult hematopoietic lineages [12]. So, to which subpopulation do these cardiomyogenic cells belong? A thorough phenotypic characterization of these cells may help to resolve these issues. Second, it is doubtful that EGFP labeling can be used for selecting cells for human therapeutic use, since overexpression of EGFP or other cytoplasmic proteins can potentially cause adverse alterations in the cellular milieu [13]. An ideal marker would be one that is expressed on the cell surface and helps sorting of positive cells by flow cytometry. Third, the ultimate proof-of-principle in cardiac ‘regeneration’ lies in the demonstration that cardiac function improves following transplantation into the damaged myocardium. Although these cells express connexin 43 in culture and exhibit a cardiomyocyte-like action potential, it remains to be proven whether following transplantation into the infarcted myocardium they will also survive and improve LV function. Finally, an alternative to the hypomethylating agent 5-azacytidine should be explored to address any potential concern regarding tumorigenesis [14,15].
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3. Regeneration of the vasculature
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Another unanswered question revolves around the issue of blood
supply to the dead or regenerated myocardium. The current literature
does not specifically address the importance of cardiomyocyte
regeneration vis-à-vis vascular regeneration. Peripheral
blood-derived EPCs have been successfully employed to improve
myocardial function
[6,16], and cardiomyocyte regeneration with
scar repair has been reported to occur without vascular differentiation
[4]. So, do we necessarily need to select only those cells that
generate cardiomyocytes but not vessels? The answer is presently
unclear. However, an alternative has been offered by Anversa's
group that reported the existence of resident CSCs that differentiate
not only into cardiomyocytes but also into endothelial cells
and smooth muscle cells
[7]. Because of their ability to differentiate
into several different cell types, CSCs offer a more balanced
reparative approach that would not only restore ventricular
function, but also secure blood supply for continued growth
and sustained function amidst a bloodless scar. As the natural
progenitor of cardiac cells, the CSC would appear best suited
for the complex task of reconstituting tissue that is lost after
a myocardial infarction
[7].
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4. The existence of ‘tissue-committed stem cells’ (TCSCs) in the BM
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The selection process used by Hattan, Kawaguchi, and colleagues
was based on the ability of BM MSCs to express MLC-2v
[11].
These results raise a very fundamental question: is it possible
that have these cells were already expressing MLC-2v even before
the harvest and thereby were "committed" to differentiate into
cardiomyocytes anyway? Does the adult BM harbor cells that are
ready to repair other tissues in the body when necessary? Ratajczak
et al.
[17] have recently demonstrated the existence of cells
in the adult BM that express markers for various adult tissues,
including brain, skeletal muscle, and endothelial cells. These
TCSCs express CXCR4 and can be isolated by chemoattraction to
stromal-derived factor-1
[18]. Intriguingly, we
[19] have recently
identified a subpopulation of BM-derived CXCR4
+/Sca-1
+/lin
–/CD45
– cells that express not only GATA-4, Nkx2.5/Csx, and MEF2C but
also cardiac-specific myosin heavy chain and troponin I in culture,
and thus appear to be cardiac TCSCs. These cardiac TCSCs are
present in both mice and humans
[19]. Recently, cardiac, endothelial,
and skeletal muscle TCSCs have been shown to be mobilized following
MI in humans
[20]. The ability to select cardiomyogenic cells
on the basis of promoter activation, as shown by Hattan, Kawaguchi,
et al., supports the concept that within the adherent cell population
in the BM reside subpopulations of TCSCs that can be identified
by their ability to express tissue-specific proteins. These
TCSCs for multiple tissues can be potentially selected or isolated
from the BM, expanded in culture, and utilized for organ repair.
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5. Future directions
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Regenerative medicine is still in its infancy and many hurdles
need to be overcome before effective repair of the heart becomes
a reality. The study by Hattan, Kawaguchi, and colleagues
[11] is important because it provides solid evidence that the adult
BM contains cells that can be selected on the basis of cardiac-specific
gene expression and that these cells not only survive following
transplantation but also interdigitate with the native cardiomyocytes.
Further studies will be necessary to investigate the nature
of these cells vis-à-vis other BM cells, such as the
recently described cardiac TCSCs
[19], to determine whether
regeneration of cardiomyocytes alone is sufficient to improve
LV function following MI, and to establish whether therapy with
these cells offers any tangible advantage over CSCs with respect
to myocardial regeneration.
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Acknowledgments
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This publication was supported in part by the NIH grants R01
HL-72410 (B.D.), HL-55757, HL-68088, HL-70897, and HL-76794
(R.B.), and the American Heart Association Scientist Development
Grant 0130146N (B.D.).
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1. Introduction
2. The ideal cell...