stem cells in cardiopulmonary development: implications for novel approaches to therapy for...

logy 25 (2008) 37–49www.elsevier.com/locate/ppedcard

Progress in Pediatric Cardio

Stem cells in cardiopulmonary development: Implications for novelapproaches to therapy for pediatric cardiopulmonary disease

Karen Young a, Joshua M. Hare b,⁎

a Departments of Pediatrics/Neonatology, The Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, USAb Departments of Internal Medicine/Cardiology, The Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, USA

Available online 7 March 2008

Abstract

One of the most exciting discoveries of recent times is that of the stem cell. While hematopoieitic stem cells were identified in the 1960's,representing the prototypic “adult stem cell”, the pace of knowledge about embryonic stem cells has advanced substantially in the past decade. Newdata is emerging regarding the continuum between embryonic cardiac precursors and cardiac stem cells found postnatally and in adulthood. The newbiological insights offer new opportunities to understand cardiomyogenesis, physiologically and pathophysiologically. Importantly, new therapeuticopportunities are beginning to emerge as well. While the pace of the cell-based therapy field has already led to clinical trials for adult cardiac diseases,this new field has the potential to advance dramatically insights and therapies for congenital heart disease and other cardiopulmonary diseases ofneonates and children.© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Stem cells; Progenitor cells; Pediatric cardiomyopathies; Pulmonary hypertension; Cardiogenesis; Congenital heart disease

1. Introduction

Moderate to severe congenital heart defects occur in approxi-mately 6/1000 live births, and the incidence approaches 75/1000live births when tiny muscular ventricular septal defects and othertrivial lesions are included [1]. Additionally, given the improve-ments in the survival rate of extremely low birthweight infants inthe past decade, the incidence of bronchopulmonary dysplasia andpulmonary hypertension continues to increase [2]. Despite ad-vances in pharmacotherapeutic and surgical interventions, cardi-opulmonary disease in children remains a significant cause ofinfantmorbidity andmortality. Therefore, enhanced understandingof etiology and pathophysiology and the development of newtherapeutic strategies for heart disease in infants and children isincreasingly important.

⁎ Corresponding author. Leonard M Miller School of Medicine, Interdisci-plinary Stem Cell Institute and Division of Cardiology, 1124 Clinical ResearchBuilding, 1120 NW 14th Street, Miami, FL 33136, USA. Tel.: +305 243 1998;fax: +305 585 5710.

E-mail address: [email protected] (J.M. Hare).

1058-9813/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ppedcard.2007.11.005

One of the most exciting new areas of biology is that of stemcells. These cells, defined by their ability to self-replicate anddifferentiate, are found in embryos and importantly in postnatallife. There have been major recent advances in understanding ofthe molecular signals that govern the development of cardiacdifferentiation from the most primitive cells. These studies alsoinform the very exciting new field of adult cardiac stem cells.This biology has important implications for understanding anddesigning new treatments for congenital heart disease and othercardiopulmonary diseases of newborns. This review will sum-marize the role of cardiac progenitor cells in cardiogenesis anddiscuss stem cell therapeutic interventions for pediatric patientswith cardiomyopathy and pulmonary hypertension.

1.1. Cardiogenesis and stem cells

1.1.1. Overview of normal heart developmentDerived from the mesoderm, the heart is one of the first

functional embryonic organs to develop in humans. Arising fromthe primitive streak, progenitor cells which are committed to acardiac fate migrate anterior-laterally and then extend across themidline to form the cardiac crescent (E7.75). At approximately,

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38 K. Young, J.M. Hare / Progress in Pediatric Cardiology 25 (2008) 37–49

E8.0, these progenitors coalesce in the midline to form the linearheart tube. By E9.5, the heart undergoes rightward looping, ex-pansion followed by septation and the subsequent development ofthe four-chambered heart.

1.1.2. The primary and secondary heart fieldsWhile earlier studies suggested that the heart develops from a

single source of progenitor cells in the anterior splanchnic me-soderm or primary heart field, it is now known that the heart alsodevelops from a second population of cardiac progenitor cells(secondary heart field) which are derived from the pharyngealmesoderm [3–6]. These so-called “primary and secondary heartfields” are distinguished by their contribution to the left ventricleand outflow tract respectively, the timing of their contribution toheart development and their expression of molecular markers(Fig. 1) [6]. It is now known that cells in the secondary heart fieldmay be identified by their expression of the LIM homeodomaintranscription factor islet-1 (isl-1) and that null mutants of isl-1fail to develop the outflow tract, right ventricle, or much of theatria [7]. Lineage tracing of isl-1-expressing progenitorsdemonstrate that isl-1 is a marker for a distinct population ofundifferentiated cardiac progenitors that gave rise to the cardiacsegments missing in isl-1 mutants [7].

Adding further intrigue to the complex organogenesis of theheart, Christoffels et al. recently described a possible third heartfield which, unlike cells in the first and second heart lineages,does not express the transcription factor nkx2-5. In contrast,these cells express the T-box transcription factor gene tbx18 and

Fig. 1. Primary and secondary heart fields. Oblique views of whole embryos and fro(First panel) First heart field (FHF) cells form a crescent shape in the anterior embryo wSHF cells lie dorsal to the straight heart tube and begin to migrate (arrows) into the an(CT), and part of the atria (A). (Third panel) Following rightward looping of the heartfrom the neural folds to septate the outflow tract and pattern the bilaterally symmetriatria, and atrioventricular valves (AVV) results in the four-chambered heart. V, ventricPA, pulmonary artery; RSCA, right subclavian artery; LSCA, left subclavian arte(Reproduced with permission from Srivastava D., Cell 2006).

contribute exclusively to the formation of the myocardium sur-rounding the sinus horns [8].

This question of one or two or multiple heart fields remainsopen, and as suggested by Moorman et al., it is possible that theheart develops from a single heart field that transition in a time-dependent manner corresponding to the phenotype of the two heartfields.While themolecular markers of these so called fields may berelevant in our search for the etiology of congenital heart defects,another important question to be answered for regenerativemedicine is that of the progenitor cells within these heart fields.

1.1.3. Multipotent embryonic cardiac progenitor cellsSince the beginning of the 20th century, experiments in the

chick embryo have provided important clues into the progenitorcells which are committed to a cardiac fate [9]. By labelingmesodermal as well as endodermal cells in avian embryos withthymidine-3H or Dil (a fluorescent label), researchers haveutilized cardiac fate maps to accurately define the progenitor cellswhich develop into the anatomic heart [10, 11]. Since these studiesdid not determine whether the progenitor cells were solelycommitted to a cardiac fate, other investigators cultured theexplants of tissue obtained from the precardiac mesoderm anddemonstrated the formation of cardiac tissue [12]. Yet, thisinformation did not conclusively delineate cardiac commitment ofthe progenitor cells, as culture of the mesodermal cells obtainedfrom the precardiac regions under conditions which promotedhematopoiesis resulted in the production of red blood cells,monocytes, macrophages, granulocytes and thrombocytes [13].

ntal views of cardiac precursors during human cardiac development are shown.ith second heart field (SHF) cells medial and anterior to the FHF. (Second panel)terior and posterior ends of the tube to form the right ventricle (RV), conotruncustube, cardiac neural crest (CNC) cells also migrate (arrow) into the outflow tractc aortic arch arteries (III, IV, and VI). (Fourth panel) Septation of the ventricles,le; LV, left ventricle; LA, left atrium; RA, right atrium; AS, aortic sac; Ao, aorta;ry; RCA, right carotid artery; LCA, left carotid artery; DA, ductus arteriosus

39K. Young, J.M. Hare / Progress in Pediatric Cardiology 25 (2008) 37–49

These data therefore suggested that the precardiac mesodermmaycontain multipotential cells.

Within the last year three independent groups have isolatedmultipotent embryonic cardiac progenitor cells which have thepotential to develop into cardiomyocytes, smooth muscle andendothelial cells. Gordon Keller and his colleagues utilized anembryonic stem cell line with the green fluorescent protein (GFP)cDNA targeted to the early mesodermal gene brachyury (GFP–Bry) to define the developmental progression of cardiacmesoderm[14]. They followed the maturation of GFP–Bry+/Flk-1− cellsand demonstrated the sequential development of a hemangioblastFlk-1+ population which was distinct from a cardiogenic Flk-1+

population. The hemangioblast Flk-1+ cells gave rise to hema-topoeitic and endothelial cells while the cardiogenic populationgave rise to cardiomyocytes, endothelial cells and vascular smoothmuscle cells. Moreover the molecular analyses of the latter pop-ulation, so called “cardiovascular colony forming cells” (CV-CFCs), demonstrated that Flk-1 expression preceded the onset ofexpression of transcription factors associatedwith the earliest stageof cardiac development (Fig. 2). Additionally, these CV-CFC'sappeared to give rise to both the primary and secondary heartfields. These findings provided further credence to the earlierfindings of Meilhac and colleagues who utilized a retrospectiveclonal analysis in the mouse to demonstrate the existence of twoheart fields that segregate from a common precursor [15].

Utilizing knowledge that Nkx2.5 is one of the earliest cardiactranscription factors expressed in the developing heart field, Wuand colleagues sought to isolate cardiac progenitor cells from themouse embryo. They generated transgenic mice which expressedenhanced green fluorescent protein (eGFP) controlled by nkx2-5enhancer and demonstrated EGFP expression in E8.5–11.5 mouseembryos. In addition, these researchers identified a c-kit+Nkx2.5+

Fig. 2. Gene expression profiles of sorted GFP-Bry/Flk-1 populations anddeveloping cardiac cultures. Expression profiles representing endothelium(Flk-1, CD31), mesoderm (Mesp1), and cardiac markers (Isl1, Nkx2.5, GATA4,Mef2C, Tbx5, Tbx20, and Mlc2v) were analyzed. The star and bolding at D6indicate the onset of contraction. The endothelial markers Flk-1 and CD31 wereexpressed in all isolated and replated contracting populations, however thismodel positions the D4.25 Flk-1+cardiac progenitor population at a develop-mental stage preceding the upregulation of the expression of the transcriptionfactors Nkx2.5 and Mef2c, (reproduced with permission from Kattman et al.,Dev Cell 2006).

population in E9.5 embryos which differentiated spontaneouslyinto both myocardial and smoothmuscle cells, as observed in vitrodifferentiating embryonic stem cells and in embryonic transplanta-tion studies [16]. It is noteworthy that these cells did not differen-tiate into endothelial cells, suggesting that the CV-CFC's isolatedby Keller and colleagues may be an earlier progenitor cell [14].

Searching for other cardiac progenitor cells in the embryo,Moretti and colleagues sought to explore the possibility that islet-1(isl-1) may be a marker of multipotent cardiovascular progenitorcells which could give rise to distinct cell lineages in the secondaryheart field [17]. This group identified isl-1+/Nkx2-5+/flk-1+ cellsin vivo during the early stages of heart development (E8–8.5embryo) and demonstrated that these cells were capable of spon-taneously differentiating into cardiomyocytes, endothelial andsmooth muscle cells (Fig. 3). Similarly distributed isl+ cells werealso demonstrated in the postnatal hearts of 1–5 day old rats byLaugwitz and colleagues, suggesting that these cells may be de-velopmental remnants [118]. While these cells expressed the earlycardiac markers Nkx2-5 and GATA4, they did not express stemcell antigen-1 (Sca-1) or c-kit. Co-culture studies of these cells withneonatal myocytes indicated that the isl-1+ cells converted effi-ciently to a mature cardiac phenotype which expressed myocyticmarkers (25%), intact Ca2+-cycling, and generated action po-tentials [18]. It is however noteworthy that these cells were veryfew in number and could only be isolated from the young heart.

1.1.4. Postnatal cardiac progenitorsIn addition to the isl+ cells isolated by Laugwitz et al., several

other studies have documented the existence of cells in thepostnatal heart with stem cell-like characteristics [18].While it isunclear whether these cells are developmental remnants or cellswithin the bone marrow which may migrate in response to injury(Fig. 4), the previous dogma that the postnatal heart does nothave the capacity to regenerate requires revision.

In one of the earlier reports of native cardiac stem cells in theadult heart, Beltrami et al. reported the existence of approximately1/10,000 Lin (−) c-kit(+) cells in 20–23 month old rodent hearts.Lacking expression of CD34 or CD45, 7–10% of the cells isolatedalso exhibited the early cardiacmarkers Nkx2-5,MEF2C (myocyteenhancer factor 2C) and GATA-4 (Fig. 5). While the origins ofthese progenitors is not clear, the recent finding by Wu et al. ofmultipotent cardiac progenitors in the embryonic heart which areNkx2-5+c-kit+ suggests that these cells may be a developmentalremnant that persisted in the postnatal heart [16]. Indeed, these cellswere self-renewing, clonogenic, and multipotent, giving rise tocardiomyocytes, smooth muscle, and endothelial cells [19].

Similarly, Oh and colleagues demonstrated the existence ofadult heart-derived cardiac progenitor cells which expressed stemcell antigen-1(Sca-1+). Distinct from hematopoeitic cells or en-dothelial progenitor cells, these cells did not express CD34,CD45, flt-1 or flk-1. They did however express high levels oftelomerase reverse transcriptase (associated with self-renewalpotential) as well as the early cardiac markers GATA4 andMEF2C, and were able to differentiate in vitro in response to 5-azacytidine via a process which partly involved a BMP receptor.Intravenous injection of these cells to mice with ischemic–reperfusion injuries resulted in homing and engraftment of these

Fig. 4. Mesodermal progenitor cells may give rise to progenitors in the primary and secondary heart field. Remnants of these mesodermal progenitor cells or possiblybone marrow derived cells may be detected in the postnatal heart.

Fig. 3. Existence of Isl1+/Nkx2.5+/Flk1+ cardiac precursors in the developing embryo in vivo. Immunofluorescence analysis for transcription factors isl1(green) and Nkx2.5 (cyan) and the surface marker flk1 (red) in transverse cryosections of embryos at ED8.25. Sections correspond to the position indicated by the linesdrawn through the adjacent embryo view and are shown at 20x magnification in the small left panels and at 63X magnification of the area of interest in the big panels.A. Triple positive cells in splanchnic mesoderm are indicated by white arrows. B. Yellow arrows mark double isl/flk1-labeled cells and green arrows cells of the foregutendoderm and adjacent mesoderm, which costain positively for isl1 and Nkx2.5. C. Cells coexpressing Nkx2.5 and flk1 are found in differentiating myocardium andare indicated by pink arrow. (reproduced with permission from Moretti et al., Cell 2006).


Fig. 5. Clusters of primitive and early committed cells in the heart. Cluster of 11c-kitPOS cells (green) with three expressing c-kit only (arrows), seven ex-pressing Nkx2.5 (white dots; arrowheads) in nuclei (blue, propidium iodide, PI),and 1 Nkx2.5 and α-sarcomeric actin in the cytoplasm (red) (Reproduced withpermission from Beltrami et al., Cell 2003).


cells in the damaged heart. A similar proportion of the donor cellswere shown to differentiate or fuse with the native myocardium[20]. The concept of cell fusion remains highly controversial asother investigators have shown very low to non-existent rates offusion events [18,19].

Confirmation of the presence of Sca-1+ cells in the postnatalheart was also provided by Matsuura and colleagues. Theyisolated Sca-1+ cells from adult murine hearts and demonstratedthat adult cardiac Sca-1+ cells can differentiate into beatingcardiomyocytes in vitro by treatment with oxytocin. Followingtreatment with oxytocin, the Sca-1+ cells expressed cardiacgenes including Nkx-2.5, GATA4 and MEF-2C, and cardiacproteins including cardiac troponin T, tropomyosin, sarcomericmyosin heavy chain, and connexin 43. Furthermore, some of theSca-1+ cells showed well organized sarcomeres and sponta-neous beating [21].

Several other investigators have isolated other cardiac pro-genitors. Hierlihy et al. demonstrated the presence of cells in theadult myocardium which exude the Hoechst dye, so called SPpopulation. These SP cells represented 1% of the total cellnumber in the adult heart [22]. Similarly, given that the ability ofSP cells to efflux the Hoechst dye is dependent on the expressionof the ATP-binding cassette transporter (Abcg2), Martin et al.also identified an Abcg2 expressing SP cell population anddemonstrated that these progenitor cells were capable of pro-liferation and differentiation [23].

In addition to the identification of cardiac stem cells byantigen panning and their capacity to exude the Hoechst dye,Messina and colleagues obtained cells which formed self-adherent clonal clusters (cardiospheres) from subcultures ofpostnatal atrial or ventricular human biopsy specimens and frommurine hearts. These cells expressed c-kit as well as Sca-1 andwere shown to be clonogenic and capable of long-term self-renewal. They differentiated in vitro and after transplantation inSCID mice to yield cells which had contractile activity as wellas cardiomyocyte markers [24].

Taken together, while the results of these studies do not clearlydemonstrate the relationship of these isolated multipotentembryonic cardiac progenitors to each other or to the postnatalcardiac progenitors, the identification of the committed progenitorcells in the embryo may provide a target for embryonic stem cell-derived therapies for cardiac regeneration.

1.1.5. Signaling pathways in cardiogenesisIn order to utilize any progenitor cell with cardiogenic potential

for regenerative therapy, the molecular pathways which drive astem cell to differentiate into cardiac tissuemust be clarified.Whilethere are still many gaps in our understanding of the process ofcardiac specification of mesodermal cells, there are several sig-nalingmolecules and transcription factorswhich are known to playessential roles. It is now apparent that mesodermal cells becomecommitted to a cardiac fate after exposure to signals derived fromthe endoderm [25]. Positive signals from fibroblast growth factors,the transforming growth factor-β family (TGF-β/activin andBMP), and possibly negative effects from members of wingless(wnts) family are known key steps in establishing cardiac musclelineage [26–28]. These proteins may regulate the expression ofseveral lineage-restricted transcription factors including neuren-berg kim homeobox (Nkx2.5) and GATA-4, early markers ofcardiac fate commitment [28,29]. The subsequent expression ofcardiacmuscle genes and their productsmay be directed by severaltranscription factors including MEF-2C, transcription enhancerfactor (TEF-1), and members of the GATA family [26,29].Utilization of this knowledge may be a potent strategy to isolateembryonic cardiac progenitor cells and direct them to differentiateinto functional cardiomyocytes.

2. Applications in pediatric cardiopulmonary disease

2.1. Stem cell sources

2.1.1. Adult bone marrow derived stem cellsThe ability of the bone marrow to regenerate the heart was

perhaps first demonstrated in adult female dystrophic mdx micewho received bonemarrow transplantation from normal congenicmale donor mice. Seventy days after transplantation, histologicalsections of cardiac muscle from mdx transplanted female micerevealed single cardiomyocytes with donor derived nuclei sug-gesting the possibility that bone marrow could be capable ofregenerating cardiac muscle cells [30].

The bone marrow is known to be a mesodermal derived tissueconsisting of a hematopoeitic cellular component supported by amicroenvironment composed of stromal cells embedded in anextracellular matrix [31].Within the bone marrow, there are severalstem cell populations. The main ones are hematopoeitic (HSCs),mesenchymal (MSCs), and endothelial progenitor cells (EPCs).These BM-derived cells have been shown to have the capacity todifferentiate into heart, brain, liver, skeletal muscle, lung,gastrointestinal tract, pancreatic cells and bone [30,32–51].

HSCs are defined as cells which are capable of self-renewal anddifferentiate into all mature hematopoeitic cells in the body [52].These cells express several markers including CD34 andCD45 andare also positive for c-kit. The ability of bonemarrowderivedHSCs


to regenerate the damaged myocardiumwas demonstrated by Orlicand colleagues who injected HSCs into the peri-infarcted region ofthe left ventricle inmicewithmyocardial infarction. Nine days afterimplantation, they detected newly formed myocardium occupying68% of the infarcted portion of the ventricle. Moreover, this tissueconsisted of proliferating myocytes and vascular structures [53].Enhancing controversy in the field, three subsequent studiesreported inability to reproduce their results [54–56]. Nonetheless, afollow up study by the same group demonstrated that cytokineinduced increase in mobilization of c-kit+ cells in mice with acutemyocardial infarction resulted in increased ejection fraction, asignificant degree of tissue regeneration as well as decreasedmortality and infarct size [57].

Sharing several antigenic determinants with HSCs, EPCs arebelieved to be derived from a common precursor, thehemangioblast [58]. Unlike the mature endothelial cell, EPCsexpress CD133 and are mobilized from the BM into theperipheral blood in response to tissue ischemia [59,60]. Hill andcolleagues measured EPCs in the peripheral blood samples of 45men with varying degrees of cardiovascular risk and nocardiovascular risk. They reported an inverse correlationbetween cardiovascular risk and the number of circulatingEPCs [61]. Moreover, in one of the largest multicenter stem celltrials documented, Schachinger et al. injected enriched progeni-tor cells into the coronary arteries of 101 patients who had acutemyocardial infarction (REPAIR-AMI trial). After 4 months, thetreated patients had a greater improvement in the ejectionfraction than the control group (5.5% vs 3.0%), and thisimprovement was seen more so in the patients with the worstejection fraction at baseline. At 1 year follow up these patientshad a reduction in the combined outcomes of death, recurrenceof the myocardial infarction and any revascularization procedure[62].

MSCs provide the environment for HSCs to differentiate andhave the capacity to differentiate into osteoblasts, chondroblasts,fibroblasts, myoblasts and adipocytes [63,64]. These cells ad-here to plastic in vitro and are known to express several markersincluding CD29, CD44, CD 71, CD73, CD 90, CD105 and CD166 while being negative for CD34 and CD45, known markersfor hematopoeitic stem cells [65]. The potential of MSCs todifferentiate into cardiomyocytes has been reported by severalinvestigators [66–68]. In our laboratory, we injected MSCs intodamaged porcine myocardium three days after acute myocardialinfarction and demonstrated a significant improvement in cardiacfunction, reduced infarct size and myocardial regeneration [69].Additionally, in a recent clinical trial, Chen and colleagues ran-domly assigned sixty-nine patients who underwent primary per-cutaneous coronary intervention within 12 h after onset of acutemyocardial infarction to receive intra-coronary injection of auto-logous bone marrow mesenchymal stem cell or standard saline.They demonstrated a significant improvement in left ventricularfunction after infusion of the stem cells [70]. Our group hasrecently conducted a 53 patient randomized clinical trial of allo-geneic MSCs administered to patients following acute MI. Thistrial met its primary endpoint, demonstrating an excellent safetyprofile, and importantly showed provisional evidence for efficacyin terms of arrhythmia suppression, improved pulmonary func-

tion, ejection fraction recovery, and patient global health status[71]. Together these early clinical studies set the stage for sub-sequent efficacy trials in this and other patient populations.

2.1.2. Skeletal myoblastsAs one of the first stem cell therapies utilized in clinical trials

for cardiac regeneration, skeletal myoblasts or satellite cells usu-ally reside under the basal membrane of skeletalmuscle fibres andmediate regeneration of skeletal muscle following injury [72].Although several clinical trials have demonstrated improvementin heart function following implantation, it is apparent that thesecells do not differentiate into cardiomyocytes nor do they cou-ple electromechanically with the native myocardium [73–78].Unfortunately, prior to this cell type being used clinically, themechanisms by which these myoblasts improve function must beelucidated and their potential for inducing ventricular arrythmiasmust be nullified [73].

2.1.3. Embryonic stem cellsEmbryonic stemcells are derived from the inner cell mass of the

preimplantation embryo. Unlike other stem cell sources, these cellsare totipotent and capable of differentiating into endoderm, meso-derm and ectoderm. Given the totipotency of these cells, and thefact that several investigators had demonstrated the differentiationof mouse embryonic stem cells into beating cardiomyocytes invitro, their potential in regenerating cardiac tissue was muchanticipated [79–81]. Unfortunately, the use of embryonic stem cellfor heart disease was tempered by ethical concerns, the possibilityof teratoma formation, immune rejection, as well as arrythmo-genicity. Moreover, while the differentiation of mouse embryonicstem cells into cardiac tissue was first reported more than twodecades ago, it was in only in 2001, that Kehat et al. demonstratedthe derivation of cells with phenotypic properties of cardiomyo-cytes from human embryonic stem (ES) cells [79,82]. Human EScells were cultivated in suspension and plated to form aggregatestermed embryoid bodies. Spontaneously contracting areas ap-peared in 8% of the embryoid bodies and cells from the spon-taneously contracting areas within the embryoid bodies stainedpositively with anti-cardiac myosin heavy chain, anti-α-actinin,anti-desmin, anti-cardiac troponin I, and anti-ANP antibodies [82].The potential of human embryonic stem cell derived cardiomyo-cytes to regenerate the heart has been reported in only a fewstudies. Kehat and colleagues transplanted human embryonic stemcell derived cardiomyocytes into the ventricular wall of swinewithcomplete atrio-ventricular block. The investigators demonstratedthat the transplanted cells could successfully pace the heart [83]. Inanother report, Laflamme and colleagues injected humanembryonic stem cell-derived cardiomyocytes into the uninjuredleft ventricle of athymic rats. They demonstrated the successfulengraftment of the human myocardium into the rat hearts withinfour weeks following injection. Additionally, although the initialgrafts had non-cardiac elements, by four weeks the grafts weremainly cardiomyocytes expressing sarcomeric actins andmyosins.Unexpectedly, they also found that the engrafted cells had atremendous degree of proliferative activity [84].

Purified embryonic stem cell-derived cardiomyocytes clearlymay be an excellent stem cell source for cardiac regeneration.


Yet, obtaining purified cardiomyocytes is still difficult despiteseveral techniques being used to optimize the yield. Unfortu-nately, lessons learnt from cardiogenesis have been useful butstill not optimal. Further large animal studies are necessary toexplore the safety and efficacy of these cells in heart disease.

2.2. Stem cell delivery

The ideal route for stem cell delivery to the diseased heart isunclear. Within the context of ischemic cardiomyopathy, severalroutes have been explored. Direct intra-myocardial injection hasbeen utilized successfully in patients with myocardial infarctionand is advantageous in allowing precise delivery to the injuredarea in the myocardium. However in the setting of global diseaseas seen in the pediatric population this would not be ideal andmoreover local injection of cells into an injuredmyocardium couldcreate small areas of cells that may not couple electrically with thenative myocardium and thereby precipitate arrhythmias [85]. Theintravenous route is clearly least invasive however native homingmechanisms must be intact. Intra-coronary injection allows thestem cells to be administered adjacent to the injured territory, butthere may be impairment of coronary flow during the infusion andthere may be the risk of re-stenosis [86–88].

Given the heart pathology seen in the pediatric population,perhaps other more novel routes may be relevant. The follow-ing sections will explore intrauterine stem delivery and tissueengineering.

2.2.1. Intrauterine stem cell therapyGiven that most heart diseases in infants have a familial com-

ponent, and the tremendous improvement in prenatal diagnosis, thefuture of stem cell regenerative therapy in pediatric heart diseasemaylie in intrauterine therapeutic interventions. Furthermore, prenatalstem cell therapies have several advantages over postnatal therapies.These include (1) the immunologic tolerance of the fetus whichwould avoid the need forHLAmatched donor as partial or non-HLAmatched donor cells would be recognized as ‘self’ (2) the relativelylower fetal size which would allow for a large “dosage” of stem cellsper kilogram bodyweight (3) the fetus is expanding severalcompartments at an early gestational age and several stem cells aremigrating and repopulating tissues and (4) the early treatment ofdisease may relieve postnatal morbidity and mortality [89].

The clinical success of in utero hematopoeitic stem celltherapy has been demonstrated in congenital immunodeficientdisorders, and transplantation of human mesenchymal cells intoearly gestation sheep resulted in engraftment in several tissuesincluding the heart [90].

The potential of in utero stem cell therapy for the treatment ofcardiac disease was elegantly demonstrated by Fraidenraich andcolleagues. Id Knockout mouse embryos are known to havemultiple cardiac defects and die by mid-gestation. This groupdemonstrated that injection of wild type embryonic stem cells intothe Id Knock out blastocysts corrected the cardiac defects andrescued embryonic lethality. Moreover, they demonstrated thatinjection of the embryonic stem cells into pregnantmothers of the idmutants partially corrected the cardiac defects. Since the embryonicstem cells did not cross the placental barrier, it was shown that

insulin-like growth factor as well as WNT5a may have beenresponsible for this partial correction of the cardiac phenotype.These data suggest that intrauterine delivery of embryonic stemcells may be useful in the prevention of congenital heart disease, bysecreting factors that are necessary for cardiac formation [91].

Another disease that intra-uterine therapy may theoretically behelpful is muscular dystrophy. In a recent report, Chan and col-leagues transplanted human fetal mesenchymal cells into pregnantmdx mice (a model of human duchenne muscular dystrophy) anddemonstrated long-term engraftment of the transplanted humancells in multiple organs and myogenic differentiation in skeletaland cardiac muscle [92]. Dystrophin was however only observedin the heart in very small clusters and the level of chimerismdemonstrated in this study would not have been clinically useful.

Indeed, the low level of chimerism has been one of the factorsthat have limited this field. In principle it would have appearedthat this therapy would have been ideal for congenital car-diomyopathies secondary to metabolic disease, but this has notbeen the case. Although fetal tolerance was thought to be one ofthe advantages of this therapy, it is apparent that engraftmentmay still be limited in the fetus secondary to an immunologicrejection mechanism. Strategies to improve prenatal tolerancewill be necessary before this therapy is clinically applicable.

2.2.2. Engineered heart tissueUnlike adult heart disease inwhich theremay bemainly regional

ischemic damage, congenital pediatric heart disease may necessi-tate complete replacement of themyocardium or repair of structuraldefects. While heart transplantation and pediatric cardiovascularsurgery has improved outcomes, the waiting list for transplantsseems insurmountable and there remains significant morbidity andmortality even after these therapeutic interventions. Several in-vestigators have explored the concept of using engineered hearttissue (EHT) to regenerate the heart. Zimmermann et al. createdEHTs from neonatal rat heart cells and implanted them on myo-cardial infarcts in immune-suppressed rats. These EHTs were madefrom heart cells, liquid collagen,Matrigel and growth supplements,and reconstituted into circular molds. Matrigel is a porous liquidcollagen which solidifies at room temperature. Twenty eight daysafter implantation, thus group demonstrated improved cardiacfunction and electrical coupling of the ECT to the native myo-cardium [93]. Unfortunately, the utilization of postnatal cardio-myocytes is perhaps not a good strategy as these cells do notmultiply sufficiently. To circumvent this problem, enriched embry-onic stem cell-derived cardiomyocytes mixed with liquid collagenand supplemented with Matrigel was used to construct EHT. Thistissue could beat synchronously, respond to pharmaceutical sti-mulation and vascularized with no evidence of teratoma formationafter implantation into nudemice [94]. In a proof of concept model,Kofidis and colleagues injected a liquid compound consisting ofembryonic stem cells and growth factor-free Matrigel into theinfarcted mouse myocardium. Two weeks after injection, theydemonstrated significant improvement in fractional shortening andretention of ventricular as well as septal wall thickness in the treatedgroup as compared to controls.Moreover, the graft/infarct ratio wassignificantly higher in the mice which received the Matrigel withstem cells as compared to those which only received the stem cells


[95]. Though they did not precisely count the number ofcardiomyocytes that were obtained from their graft, this interven-tion could offer exciting prospects for the treatment of childrenwithseptal defects or single ventricles, as it is possible that the EHTderived patch would grow with development. In a descriptiveclinical study, investigators seeded autologous bone marrow cellsonto a scaffold and created patches and conduits for the treatment ofcongenital heart defects [96]. They studied 42 patients, ages 1–24 years and followed them for 1 to 32months. They demonstratedno evidence of thrombosis, aneurysmal formation or calcification.While they could not account for the fate of the seeded cells or thepotential durability, they suggested that utilizing this techniquemaydecrease the need for re-intervention in growing patients. None-theless, there are several problems which must be solved prior thisbeing accepted for clinical use. As reviewed by Zimmermann et al.,EHTs need to generate sufficient force to withstand circumferentialwall stress during the cardiac cycle, be vascularized sufficiently toallow nutrient delivery, have the capability to structurally andelectrically incorporate into the damaged heart and resist rejectionafter implantation [97].

2.2.3. Valvular heart diseaseYet, for the treatment of congenital valvular heart disease, tissue

engineered valves with stem cells may be a means to prolong theviability of implanted valves as they may grow and remodel as thechild develops. The concept of in vitro heart valve tissueengineering involves harvesting autologous stem cells from thepatient, expanding them in vitro and seeding them onto abiodegradable valve scaffold. The constructs are then placed in abioreactor and conditioned for implantation [98].

Sutherland and his colleagues created autologous living heartvalves from bone marrow derived mesenchymal cells in combina-tion with a synthetic biodegradable scaffold [99]. They implantedthem into the pulmonary position of sheep on cardiopulmonarypass and evaluated the valves at implantation, at 4 months in vivoand demonstrated little or no change in the gradients at 4 months offollowup. Eightmonths after implantation, the valves had a cellularphenotype and architecture which was reminiscent of that seen innative valves, with three distinct layers equivalent to the zonaventricularis, the zona spongiosa, and the zona fibrosa. Thesevalves were however thicker than normal valves and there was nodata on long-term followup.Using a similar concept, Cebotari et al.reported the clinical application of tissue engineered valves usingautologous endothelial progenitor cells in two pediatric patientswhowere followed for 3.5 years. They demonstrated an increase inengineered pulmonary valve annulus diameter and only trivial ormild regurgitation, with no evidence of valve degeneration in bothpatients [100]. Yet, the utility of these engineered valves in theneonatal period may be limited by the lag time for harvesting andisolation of the cells to processing of the tissue engineered valveleaflets. With this in mind, Schmidt and colleagues isolated humanfetal mesenchymal progenitors from routinely sampled prenatalchorionic villus specimens expanded them in vitro and seeded themonto biodegradable scaffolds [101]. They demonstrated that thevalves had mechanical profiles similar to native valves andmorphologically resembled their native counterparts. In as muchas this therapy has the potential to provide a novel approach to

reducing some of the adverse complications associated with pros-thetic valves in children, further long-term studies are important todetermine if there may be any tumor formation from the seededstem cells.

3. Stem cells and pediatric cardiomyopathy

The efficacy of stem cell therapy for ischemic cardiomyo-pathy has been demonstrated in several clinical trials [102–105]. Unfortunately, the data on cardiomyopathy with etiologiessimilar to those seen in the pediatric population is sparse [106].

In one of the first published reports evaluating stem cells innon-ischemic cardiomyopathy, Agbulut and colleagues adminis-tered allogeneic unpurified bone marrow cells intra-myocardiallyto mice with doxorubicin induced cardiomyopathy. Two weeksafter transplantation they demonstrated that a small number of thebone marrow cells appeared phenotypically close to cardiomyo-cytes however given the small number of cells, the functionalefficacy of the stem cells in this study was questionable [107].

In order to evaluate the efficacy of stem cells in non-ischemicheart disease, Wang and colleagues injected embryonic stemcells into the tail vein of mice with viral myocarditis [108].Thirty days after injection, there was significantly less necrosisand inflammatory cell infiltration in the embryonic stem celltreated animals (Fig. 6). Moreover, the mortality of the embry-onic stem cell group was significantly lower than the controlanimals but cardiac function was not evaluated.

Nagaya et al. evaluated the cardiac function of rats with non-ischemic dilated cardiomyopathy who had received mesench-ymal stem cells intra-myocardially [109]. Four weeks after celltransplantation, the treatment group had a significant improve-ment in left ventricle dP/dt, decreased left ventricular end-diastolic pressure, decrease in collagen volume fraction in themyocardium, as well as induction of angiogenesis evidenced byincreased vascular structures. Moreover, Guarita-Souza et al.demonstrated that simultaneous autologous transplantation ofco-cultured skeletal myoblasts and mesenchymal cells into ratswith cardiomyopathy secondary to Chagas disease resulted inan improvement in ventricular function [110].

Yet, in pediatric patients with cardiomyopathy secondary tometabolic disease, regeneration of the heart tissue may need amore global stem cell approach as multiple organ systems areaffected. Varied improved outcome has been documented fol-lowing hematopoeitic stem cell transplantation in patients withHurler Syndrome and Maroteaux-Lamy Syndrome [111–113].Braunlin and colleagues followed a patient with Hurler Syn-drome who had received bone marrow transplantation 14 yearsprior to her death. They demonstrated improved or stable leftventricular function ten years after transplantation and minimalthickening of the coronary artery intima which sharply contraststhat seen in untreated patients [114]. In addition, Krivit et al.described normal cardiopulmonary function two years follow-ing bone marrow transplantation in a patient with Maroteaux-Lamy Syndrome [115]. Given these anecdotal reports, there iscurrently a phase II non-randomized clinical trial recruitingpatients who have in born errors of metabolism, includingHurler and Maroteaux-Lamy Syndromes. While cardiac

Fig. 6. Photomicrographs (light microscopy) of H and E stained myocardial sections from ES cell treated myocarditis mice. In virus control hearts, there wasinflammatory cell infiltration and necrosis at day 7 (a). At days 14 (c) and 30 (e), extensive infiltration as well as fibrosis and calcification was observed.Photomicrographs of stem cell treated myocarditis hearts at days 7 (b), 14 (d), and 30 (f) are shown. The arrows point to infiltration and necrosis (Reproduced withpermission from Wang et al., Cell Transplant 2002).


function is not a primary outcome, the study will seek todetermine the safety and efficacy of hematopoeitic stem celltransplantation in these diseases, using a specific conditioningregimen. Primarily, they will evaluate donor cell engraftment upto one year and secondarily, survival will be determined up tothree years (Clinical Trials. Gov identifier NCT00176917).

Within the context of cardiomyopathy associatedwithmusculardystrophies, although gene therapy has lead to some improve-ments in skeletal muscle disease, there has been little advancementin gene therapy for heart disease. Klug and colleagues injectedembryonic stem cell-derived cardiomyocytes into the leftventricular wall of mdx mice. They detected stable embryonicstem cell-derived cardiomyocytes which expressed dystrophin inthe recipient heart up to 7 weeks after injection but these were notquantified [116]. Similarly, Payne and colleagues transplantedskeletal muscle derived stem cells into the heart of dystrophindeficient mdx mice and demonstrated several dystrophin positive

myocytes but only few expressed a cardiac phenotype [117]. Ina recent report, investigators administered mesangioblasts (vesselassociated stem cells) to dogs with golden retriever musculardystrophy. This canine model reproduces the full disease pa-thology evident in human duchenne muscular dystrophy. Thetreated animals exhibited restoration of dystrophin in the skeletalmuscle as well as normal muscle morphology and function.Unfortunately, cardiac function was not reported. Future studiesare necessary to evaluate the stem cell population which will bemost efficacious in treating the skeletal muscle disease as well asthe cardiomyopathy evidenced in this disorder.

4. Stem cells and pulmonary hypertension

Pulmonary hypertension remains a significant cause of mor-bidity and mortality in neonates who have been perinatallyexposed to hypoxia. Prevailing theories into the pathogenesis of


pulmonary hypertension suggests mainly the involvement ofresident pulmonary vascular cells, however the existence ofcells with stem cell-like characteristics in the normal maturevascular media raises the possibility that pathological lesions inthe arteries could also originate from stem cells. Davie andcolleagues reported that c-kit+ cells were increased in the pul-monary artery adventitia of hypoxic calves as compared to thenormoxic animals and while it is still unclear whether these cellswere resident stem cells, or cells which migrated to the lungfrom the bone marrow, or mature cells which expressed c-kit, itraises the question of whether stem cells contribute to pulmo-nary vascular remodeling [118]. Moreover, in another study bythis group, circulating mesenchymal precursors, so called fibro-cytes were shown to contribute to pulmonary adventitial remo-deling during hypoxia-induced pulmonary hypertension anddepletion of these cells appeared to attenuate remodeling [119].On the other hand, intra-tracheal administration of mesenchy-mal stem cells attenuated monocrotaline-induced pulmonaryhypertension in a rodent model [120]. Clearly, the differencein these studies may be due to the difference in the modelsof pulmonary hypertension however, most recently Rochefortet al. failed to demonstrate significant retention of infusedmesenchymal cells in pulmonary artery media of hypoxic rats[121].

Endothelial progenitor cells have been shown to contribute tophysiological neovascularization in the normal lung, suggesting arole for these cells in normal lung maintenance [122]. In addition,recent data demonstrated that umbilical cord-derived endothelialprogenitor cells attenuate monocrotaline-induced PH [123–125].In the first published clinical trial using endothelial progenitor cellsin patients with idiopathic pulmonary hypertension, Wang andcolleagues administered stem cells or placebo intravenously tothirty one patients with idiopathic pulmonary hypertension [126].Twelveweeks after administration, they demonstrated a significantimprovement in 6-min walk distance, mean pulmonary vascularresistance and cardiac output, with no severe adverse effects.Although the population was small and the follow up time wasshort, it provides one of the first clinical evidence of the usefulnessof stem cells in pulmonary hypertension. There is currently anotherclinical trial recruiting patients with idiopathic pulmonaryhypertension. This study is seeking to assess the safety of auto-logous progenitor cell-based gene therapy of heNOS in patientswith severe Pulmonary Arterial Hypertension refractory to con-ventional treatment (PHACeT).

5. Conclusion

Although data on stem based therapies for pediatric cardio-pulmonary disease is sparse, recent clinical reports of successfultreatment of heart disease in adults provide new hope that novelinterventions for the pediatric population will soon be obtained.The discovery of embryonic progenitors which are committed to acardiac fate as well as the molecular pathways which direct theirspecification should provide a firm foundation for new studiesdirected at increasing the yield and purity of cells for regenerativetherapy. However, given that the pediatric heart disease may be apart of a global disease entity, unique stem cell delivery methods

must be developed for this population. Furthermore, since severecardiopulmonary disease may be associated with decreased neu-rodevelopmental outcomes, early and possibly preventative cell-based strategies should be investigated.

Finally, the promise of cell-based therapies for pediatric heartdisease is great and open for exploration but as with any newtherapy, the risk to benefit ratio of stem cell regenerative treat-ment in the pediatric population must be clarified. Clearly, thereis still tremendous morbidity in children with congenital heartlesions and the financial burden is great, yet in a population thatis still developing several organs long-term safety issues mustbe addressed.

References

[1] Hoffman JI, Kaplan S. The incidence of congenital heart disease. J AmColl Cardiol 2002;39:1890–900.

[2] Bancalari E, Claure N, Sosenko IR. Bronchopulmonary dysplasia: changes inpathogenesis, epidemiology and definition. Semin Neonatol 2003;8:63–71.

[3] Kelly RG, Brown NA, Buckingham ME. The arterial pole of the mouseheart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev Cell2001;1:435–40.

[4] Mjaatvedt CH, Nakaoka T, Moreno-Rodriguez R, et al. The outflow tractof the heart is recruited from a novel heart-forming field. Dev Biol2001;238:97–109.

[5] Waldo KL, Kumiski DH, Wallis KT, et al. Conotruncal myocardiumarises from a secondary heart field. Development 2001;128:3179–88.

[6] Buckingham M, Meilhac S, Zaffran S. Building the mammalian heartfrom two sources of myocardial cells. Nat Rev Genet 2005;6:826–35.

[7] Cai CL, Liang X, Shi Y, et al. Isl1 identifies a cardiac progenitor populationthat proliferates prior to differentiation and contributes a majority of cells tothe heart. Dev Cell 2003;5:877–89.

[8] Christoffels VM, Mommersteeg MT, Trowe MO, et al. Formation of thevenous pole of the heart from an Nkx2-5-negative precursor populationrequires Tbx18. Circ Res 2006;98:1555–63.

[9] Rawles M. The heart-forming areas of the early chick blastoderm. PhysiolZoo 1943;16:22–42.

[10] Redkar A, Montgomery M, Litvin J. Fate map of early avian cardiacprogenitor cells. Development 2001;128:2269–79.

[11] Stalsberg H, DeHaan RL. The precardiac areas and formation of thetubular heart in the chick embryo. Dev Biol 1969;19:128–59.

[12] Eisenberg CA, Eisenberg LM.WNT11 promotes cardiac tissue formationof early mesoderm. Dev Dyn 1999;216:45–58.

[13] Brandon C, Eisenberg LM, Eisenberg CA. WNT signaling modulates thediversification of hematopoietic cells. Blood 2000;96:4132–41.

[14] Kattman SJ, Huber TL, Keller GM. Multipotent flk-1+ cardiovascularprogenitor cells give rise to the cardiomyocyte, endothelial, and vascularsmooth muscle lineages. Dev Cell 2006;11:723–32.

[15] Meilhac SM, Esner M, Kelly RG, Nicolas JF, Buckingham ME. Theclonal origin of myocardial cells in different regions of the embryonicmouse heart. Dev Cell 2004;6:685–98.

[16] Wu SM, Fujiwara Y, Cibulsky SM, et al. Developmental origin of abipotential myocardial and smooth muscle cell precursor in the mammalianheart. Cell 2006;127:1137–50.

[17] Moretti A, Caron L, NakanoA, et al. Multipotent embryonic isl1+ progenitorcells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell2006;127:1151–65.

[18] Laugwitz KL, Moretti A, Lam J, et al. Postnatal isl1+ cardioblasts enterfully differentiated cardiomyocyte lineages. Nature 2005;433:647–53.

[19] Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells aremultipotent and support myocardial regeneration. Cell 2003;114:763–76.

[20] Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adultmyocardium: homing, differentiation, and fusion after infarction. Proc NatlAcad Sci U S A 2003;100:12313–8.

[21] Matsuura K, Nagai T, Nishigaki N, et al. Adult cardiac Sca-1-positive cellsdifferentiate into beating cardiomyocytes. J Biol Chem 2004;279:11384–91.


[22] Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett2002;530: 239–43.

[23] Martin CM, Meeson AP, Robertson SM, et al. Persistent expression of theATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in thedeveloping and adult heart. Dev Biol 2004;265:262–75.

[24] Messina E, De Angelis L, Frati G, et al. Isolation and expansion of adultcardiac stem cells from human and murine heart. Circ Res 2004;95:911–21.

[25] Schultheiss TM, Xydas S, Lassar AB. Induction of avian cardiacmyogenesis by anterior endoderm. Development 1995;121:4203–14.

[26] Gupta M, Gupta MP, Arcilla RA. Molecular regulation of cardiacmyogenesis and morphology during development. Prog Pediatr Cardiol1998;9:155–70.

[27] Foley A, Mercola M. Heart induction: embryology to cardiomyocyteregeneration. Trends Cardiovasc Med 2004;14:121–5.

[28] Brade T, Manner J, Kuhl M. The role of Wnt signalling in cardiacdevelopment and tissue remodelling in the mature heart. Cardiovasc Res2006;72:198–209.

[29] Brand T. Heart development: molecular insights into cardiac specificationand early morphogenesis. Dev Biol 2003;258:1–19.

[30] Bittner RE, Schofer C, Weipoltshammer K, et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophicmdx mice. Anat Embryol (Berl) 1999;199:391–6.

[31] Rafii S, Shapiro F, Rimarachin J, et al. Isolation and characterization ofhuman bone marrow microvascular endothelial cells: hematopoieticprogenitor cell adhesion. Blood 1994;84:10–9.

[32] Eglitis MA, Mezey E. Hematopoietic cells differentiate into bothmicroglia and macroglia in the brains of adult mice. Proc Natl AcadSci U S A 1997;94: 4080–5.

[33] Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain:expression of neuronal phenotypes in adult mice. Science 2000;290: 1775–9.

[34] Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turningblood into brain: cells bearing neuronal antigens generated in vivo frombone marrow. Science 2000;290:1779–82.

[35] Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potentialsource of hepatic oval cells. Science 1999;284:1168–70.

[36] Theise ND, Badve S, Saxena R, et al. Derivation of hepatocytes from bonemarrow cells in mice after radiation-induced myeloablation. Hepatology2000;31:235–40.

[37] Theise ND, Nimmakayalu M, Gardner R, et al. Liver from bone marrowin humans. Hepatology 2000;32:11–6.

[38] Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stemcells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–34.

[39] Gussoni E, SoneokaY, StricklandCD, et al. Dystrophin expression in themdxmouse restored by stem cell transplantation. Nature 1999;401: 390–4.

[40] Mason RJ, Williams MC, Moses HL, Mohla S, Berberich MA. Stem cellsin lung development, disease, and therapy. Am J Respir Cell Mol Biol1997;16:355–63.

[41] Theise ND, Henegariu O, Grove J, et al. Radiation pneumonitis in mice: asevere injury model for pneumocyte engraftment from bone marrow. ExpHematol 2002;30:1333–8.

[42] Kotton DN, Ma BY, Cardoso WV, et al. Bone marrow-derived cells asprogenitors of lung alveolar epithelium. Development 2001;128:5181–8.

[43] Ortiz LA,Gambelli F,McBrideC, et al.Mesenchymal stemcell engraftmentin lung is enhanced in response to bleomycin exposure and ameliorates itsfibrotic effects. Proc Natl Acad Sci U S A 2003;100:8407–11.

[44] Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation ofglucose-competent pancreatic endocrine cells from bone marrow withoutevidence of cell fusion. J Clin Invest 2003;111:843–50.

[45] Hess D, Li L, Martin M, et al. Bone marrow-derived stem cells initiatepancreatic regeneration. Nat Biotechnol 2003;21:763–70.

[46] Okamoto R, Yajima T, Yamazaki M, et al. Damaged epithelia regeneratedby bone marrow-derived cells in the human gastrointestinal tract. NatMed 2002;8:1011–7.

[47] Brittan M, Hunt T, Jeffery R, et al. Bone marrow derivation of pericryptalmyofibroblasts in the mouse and human small intestine and colon. Gut2002;50:752–7.

[48] Pereira RF, Halford KW, O'Hara MD, et al. Cultured adherent cells frommarrow can serve as long-lasting precursor cells for bone, cartilage, andlung in irradiated mice. Proc Natl Acad Sci U S A 1995;92:4857–61.

[49] Epperly MW, Guo H, Gretton JE, Greenberger JS. Bone marrow origin ofmyofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell MolBiol 2003;29:213–24.

[50] Grove JE, Lutzko C, Priller J, et al. Marrow-derived cells as vehicles fordelivery of gene therapy to pulmonary epithelium. Am J Respir Cell MolBiol 2002;27:645–51.

[51] Trotman W, Beckett T, Goncz KK, Beatty BG, Weiss DJ. Dual Ychromosome painting and in situ cell-specific immunofluorescencestaining in lung tissue: an improved method of identifying donor marrowcells in lung following bone marrow transplantation. Histochem Cell Biol2004;121:73–9.

[52] Kondo M, Wagers AJ, Manz MG, et al. Biology of hematopoietic stemcells and progenitors: implications for clinical application. Annu RevImmunol 2003;21:759–806.

[53] Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerateinfarcted myocardium. Nature 2001;410:701–5.

[54] Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells donot transdifferentiate into cardiac myocytes in myocardial infarcts. Nature2004;428:664–8.

[55] Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL,Robbins RC. Haematopoietic stem cells adopt mature haematopoieticfates in ischaemic myocardium. Nature 2004;428:668–73.

[56] Nygren JM, Jovinge S, Breitbach M, et al. Bone marrow-derivedhematopoietic cells generate cardiomyocytes at a low frequency throughcell fusion, but not transdifferentiation. Nat Med 2004;10:494–501.

[57] Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repairthe infarcted heart, improving function and survival. Proc Natl Acad SciU S A 2001;98:10344–9.

[58] Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM.Origin of endothelial progenitors in human postnatal bone marrow. J ClinInvest 2002;109:337–46.

[59] Tepper OM, Capla JM, Galiano RD, et al. Adult vasculogenesis occursthrough in situ recruitment, proliferation, and tubulization of circulatingbone marrow-derived cells. Blood 2005;105:1068–77.

[60] Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivoexpanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci U S A 2000;97:3422–7.

[61] Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells,vascular function, and cardiovascular risk. N Engl J Med 2003;348:593–600.

[62] Schachinger V, Erbs S, Elsasser A, et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med2006;355:1210–21.

[63] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietictissues. Science 1997;276:71–4.

[64] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adulthuman mesenchymal stem cells. Science 1999;284:143–7.

[65] Haynesworth SE, Baber MA, Caplan AI. Cell surface antigens on humanmarrow-derived mesenchymal cells are detected by monoclonal anti-bodies. Bone 1992;13:69–80.

[66] Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generatedfrom marrow stromal cells in vitro. J Clin Invest 1999;103:697–705.

[67] Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Humanmesenchymal stem cells differentiate to a cardiomyocyte phenotype in theadult murine heart. Circulation 2002;105:93–8.

[68] Shake JG, Gruber PJ, Baumgartner WA, et al. Mesenchymal stem cellimplantation in a swine myocardial infarct model: engraftment andfunctional effects. Ann Thorac Surg 2002;73:1919–25 discussion 1926.

[69] Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair withintramyocardial injection of allogeneic mesenchymal stem cells after myo-cardial infarction. Proc Natl Acad Sci U S A 2005;102:11474–9.

[70] Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function ofintracoronary transplantation of autologous bone marrow mesenchymalstem cell in patients with acute myocardial infarction. Am J Cardiol2004;94:92–5.


[71] Hare J, Dib N, Traverse JH, et al. A double-blind, randomized, placebocontrolled clinical trial of allogeneic mesenchymal stem cells for thetreatment of patients with acute myocardial infarction. J Am Coll Cardiol2007;49:2405–3A.

[72] Menasche P. Skeletal muscle satellite cell transplantation. Cardiovasc Res2003;58:351–7.

[73] Menasche P, Hagege AA, Vilquin JT, et al. Autologous skeletal myoblasttransplantation for severe postinfarction left ventricular dysfunction.J Am Coll Cardiol 2003;41:1078–83.

[74] Leobon B, Garcin I, Menasche P, Vilquin JT, Audinat E, Charpak S.Myoblasts transplanted into rat infarcted myocardium are functionallyisolated from their host. Proc Natl Acad Sci U S A 2003;100:7808–11.

[75] Siminiak T, Fiszer D, Jerzykowska O, et al. Percutaneous trans-coronary-venous transplantation of autologous skeletal myoblasts in the treatmentof post-infarction myocardial contractility impairment: the POZNANtrial. Eur Heart J 2005;26:1188–95.

[76] Dib N, Michler RE, Pagani FD, et al. Safety and feasibility of autologousmyoblast transplantation in patients with ischemic cardiomyopathy: four-year follow-up. Circulation 2005;112:1748–55.

[77] Reinecke H, Minami E, Poppa V, Murry CE. Evidence for fusion betweencardiac and skeletal muscle cells. Circ Res 2004;94:e56–60.

[78] Reinecke H, Poppa V, Murry CE. Skeletal muscle stem cells do nottransdifferentiate into cardiomyocytes after cardiac grafting. J Mol CellCardiol 2002;34:241–9.

[79] Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitrodevelopment of blastocyst-derived embryonic stem cell lines: formationof visceral yolk sac, blood islands and myocardium. J Embryol ExpMorphol 1985;87:27–45.

[80] Fijnvandraat AC, van Ginneken AC, de Boer PA, et al. Cardiomyocytesderived from embryonic stem cells resemble cardiomyocytes of theembryonic heart tube. Cardiovasc Res 2003;58:399–409.

[81] Maltsev VA, Wobus AM, Rohwedel J, Bader M, Hescheler J.Cardiomyocytes differentiated in vitro from embryonic stem cellsdevelopmentally express cardiac-specific genes and ionic currents. CircRes 1994;75: 233–44.

[82] Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cellscan differentiate into myocytes with structural and functional propertiesof cardiomyocytes. J Clin Invest 2001;108:407–14.

[83] Kehat I, Khimovich L, Caspi O, et al. Electromechanical integration ofcardiomyocytes derived from human embryonic stem cells. Nat Biotechnol2004;22:1282–9.

[84] Laflamme MA, Gold J, Xu C, et al. Formation of human myocardium inthe rat heart from human embryonic stem cells. Am J Pathol 2005;167:663–71.

[85] Steele A, Steele P. Stem cells for repair of the heart. Curr Opin Pediatr2006;18:518–23.

[86] Kang HJ, Kim HS, Zhang SY, et al. Effects of intracoronary infusion ofperipheral blood stem-cells mobilised with granulocyte-colony stimulat-ing factor on left ventricular systolic function and restenosis aftercoronary stenting in myocardial infarction: the MAGIC cell randomisedclinical trial. Lancet 2004;363:751–6.

[87] Balsam LB, Robbins RC. Haematopoietic stem cells and repair of theischaemic heart. Clin Sci (Lond) 2005;109:483–92.

[88] Oettgen P, Boyle AJ, Schulman SP, Hare JM. Cardiac stem cell therapy.Need for optimization of efficacy and safety monitoring. Circulation2006;114:353–8.

[89] Flake AW. In utero stem cell transplantation. Best Pract Res Clin ObstetGynaecol 2004;18:941–58.

[90] Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymalstem cells engraft and demonstrate site-specific differentiation after inutero transplantation in sheep. Nat Med 2000;6:1282–6.

[91] Fraidenraich D, Stillwell E, Romero E, et al. Rescue of cardiac defects in idknockout embryos by injection of embryonic stem cells. Science 2004;306:247–52.

[92] Chan J, Waddington SN, O'Donoghue K, et al. Widespread distributionand muscle differentiation of human fetal mesenchymal stem cells afterintrauterine transplantation in dystrophic mdx mouse. Stem Cells2007;25: 875–84.

[93] Zimmermann WH, Melnychenko I, Wasmeier G, et al. Engineered hearttissue grafts improve systolic and diastolic function in infarcted rat hearts.Nat Med 2006;12:452–8.

[94] Guo XM, Zhao YS, Chang HX, et al. Creation of engineered cardiactissue in vitro from mouse embryonic stem cells. Circulation 2006;113:2229–37.

[95] Kofidis T, Lebl DR,Martinez EC, Hoyt G, TanakaM, Robbins RC. Novelinjectable bioartificial tissue facilitates targeted, less invasive, large-scaletissue restoration on the beating heart after myocardial injury. Circulation2005;112:I173–7.

[96] Shin'oka T, Matsumura G, Hibino N, et al. Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.J Thorac Cardiovasc Surg 2005;129:1330–8.

[97] Zimmermann WH, Didie M, Doker S, et al. Heart muscle engineering: anupdate on cardiac muscle replacement therapy. Cardiovasc Res 2006;71:419–29.

[98] Schmidt D, Hoerstrup SP. Tissue engineered heart valves based on humancells. Swiss Med Wkly 2006;136:618–23.

[99] Sutherland FW, Perry TE, Yu Y, et al. From stem cells to viable autologoussemilunar heart valve. Circulation 2005;111:2783–91.

[100] Cebotari S, Lichtenberg A, Tudorache I, et al. Clinical application oftissue engineered human heart valves using autologous progenitor cells.Circulation 2006;114:I132–7.

[101] Schmidt D, Mol A, Breymann C, et al. Living autologous heart valvesengineered from human prenatally harvested progenitors. Circulation2006;114:I125–31.

[102] Assmus B, Schachinger V, Teupe C, et al. Transplantation of ProgenitorCells and Regeneration Enhancement in Acute Myocardial Infarction(TOPCARE-AMI). Circulation 2002;106:3009–17.

[103] Schachinger V, Assmus B, BrittenMB, et al. Transplantation of progenitor cellsand regeneration enhancement in acute myocardial infarction: final one-yearresults of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004;44: 1690–9.

[104] Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomisedcontrolled clinical trial. Lancet 2004;364:141–8.

[105] Meyer GP, Wollert KC, Lotz J, et al. Intracoronary bone marrow celltransfer after myocardial infarction: eighteenmonths' follow-up data fromthe randomized, controlled BOOST (BOne marrOw transfer to enhanceST-elevation infarct regeneration) trial. Circulation 2006;113:1287–94.

[106] Pillekamp F, Reppel M, Brockmeier K, Hescheler J. Stem cells and theirpotential relevance to paediatric cardiology. Cardiol Young 2006;16:117–24.

[107] Agbulut O, Menot ML, Li Z, et al. Temporal patterns of bone marrow celldifferentiation following transplantation in doxorubicin-induced cardio-myopathy. Cardiovasc Res 2003;58:451–9.

[108] Wang JF, Yang Y, Wang G, et al. Embryonic stem cells attenuate viralmyocarditis in murine model. Cell Transplant 2002;11:753–8.

[109] Nagaya N, Kangawa K, Itoh T, et al. Transplantation of mesenchymal stemcells improves cardiac function in a rat model of dilated cardiomyopathy.Circulation 2005;112:1128–35.

[110] Guarita-Souza LC, Carvalho KA, Woitowicz V, et al. Simultaneousautologous transplantation of cocultured mesenchymal stem cells andskeletal myoblasts improves ventricular function in a murine model ofChagas disease. Circulation 2006;114:I120–4.

[111] Vinallonga X, Sanz N, Balaguer A, et al. Hypertrophic cardiomyopathy inmucopolysaccharidoses: regression after bone marrow transplantation.Pediatr Cardiol 1992;13:107–9.

[112] Peters C, Steward CG. Hematopoietic cell transplantation for inheritedmetabolic diseases: an overview of outcomes and practice guidelines. BoneMarrow Transplant 2003;31:229–39.

[113] Gatzoulis MA, Vellodi A, Redington AN. Cardiac involvement inmucopolysaccharidoses: effects of allogeneic bone marrow transplantation.Arch Dis Child 1995;73:259–60.

[114] Braunlin EA, Rose AG, Hopwood JJ, Candel RD, Krivit W. Coronary arterypatency following long-term successful engraftment 14 years after bonemarrow transplantation in the Hurler syndrome. Am J Cardiol 2001;88:1075–7.

[115] Krivit W, Pierpont ME, Ayaz K, et al. Bone-marrow transplantation in theMaroteaux-Lamy syndrome (mucopolysaccharidosis type VI).


Biochemical and clinical status 24 months after transplantation. N Engl JMed 1984;311:1606–11.

[116] Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selectedcardiomyocytes from differentiating embronic stem cells form stableintracardiac grafts. J Clin Invest 1996;98:216–24.

[117] Payne TR, Oshima H, Sakai T, et al. Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells. GeneTher 2005;12:1264–74.

[118] Davie NJ, Crossno Jr JT, Frid MG, et al. Hypoxia-induced pulmonaryartery adventitial remodeling and neovascularization: contribution ofprogenitor cells. Am J Physiol LungCellMol Physiol 2004;286:L668–78.

[119] FridMG,Brunetti JA, BurkeDL, et al. Hypoxia-induced pulmonary vascularremodeling requires recruitment of circulating mesenchymal precursors of amonocyte/macrophage lineage. Am J Pathol 2006;168:659–69.

[120] Baber SR, Deng W, Master RG, et al. Intratracheal mesenchymal stemcell administration attenuates monocrotaline-induced pulmonary hyper-tension and endothelial dysfunction. Am J Physiol Heart Circ Physiol2007;292:H1120–8.

[121] Rochefort GY, Vaudin P, Bonnet N, et al. Influence of hypoxia on thedomiciliation of mesenchymal stem cells after infusion into rats: possibilities

of targeting pulmonary artery remodeling via cells therapies? Respir Res2005;6:125.

[122] Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelialprogenitor cells responsible for postnatal vasculogenesis in physiological andpathological neovascularization. Circ Res 1999;85:221–8.

[123] Nagaya N, Kangawa K, Kanda M, et al. Hybrid cell-gene therapy forpulmonary hypertension based on phagocytosing action of endothelialprogenitor cells. Circulation 2003;108:889–95.

[124] Takahashi M, Nakamura T, Toba T, Kajiwara N, Kato H, Shimizu Y.Transplantation of endothelial progenitor cells into the lung to alleviatepulmonary hypertension in dogs. Tissue Eng 2004;10:771–9.

[125] Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ.Rescue of monocrotaline-induced pulmonary arterial hypertensionusing bone marrow-derived endothelial-like progenitor cells: efficacy ofcombined cell and eNOS gene therapy in established disease. Circ Res2005;96:442–50.

[126] Wang XX, Zhang FR, Shang YP, et al. Transplantation of autologousendothelial progenitor cells may be beneficial in patients with idiopathicpulmonary arterial hypertension: a pilot randomized controlled trial. J AmColl Cardiol 2007;49:1566–71.

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