Stem Cells for Myocardial Regeneration

Donald Orlic, Jonathan M. Hill and Andrew E. AraiStem Cells for Myocardial Regeneration

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This Review is part of a thematic series on Stem Cells, which includes the following articles:

Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes

Derivation and Potential Applications of Human Embryonic Stem Cells

Stem Cells for Myocardial Regeneration

Neural Stem Cells: An Overview

Mesenchymal Stem Cells

Myocyte Death, Growth, and Regeneration in Cardiac Hypertrophy and Failure

Therapeutics and Use of Stem Cells Toren Finkel, Roberto Bolli, Editors

Stem Cells for Myocardial RegenerationDonald Orlic, Jonathan M. Hill, Andrew E. Arai

Abstract—Stem cells are being investigated for their potential use in regenerative medicine. A series of remarkable studiessuggested that adult stem cells undergo novel patterns of development by a process referred to as transdifferentiationor plasticity. These observations fueled an exciting period of discovery and high expectations followed by controversythat emerged from data suggesting cell-cell fusion as an alternate interpretation for transdifferentiation. However, datasupporting stem cell plasticity are extensive and cannot be easily dismissed. Myocardial regeneration is perhaps the mostwidely studied and debated example of stem cell plasticity. Early reports from animal and clinical investigationsdisagree on the extent of myocardial renewal in adults, but evidence indicates that cardiomyocytes are generated in whatwas previously considered a postmitotic organ. On the basis of postmortem microscopic analysis, it is proposed thatrenewal is achieved by stem cells that infiltrate normal and infarcted myocardium. To further understand the role of stemcells in regeneration, it is incumbent on us to develop instrumentation and technologies to monitor myocardial repairover time in large animal models. This may be achieved by tracking labeled stem cells as they migrate into myocardialinfarctions. In addition, we must begin to identify the environmental cues that are needed for stem cell trafficking andwe must define the genetic and cellular mechanisms that initiate transdifferentiation. Only then will we be able toregulate this process and begin to realize the full potential of stem cells in regenerative medicine. (Circ Res. 2002;91:1092-1102.)

Key Words: stem cells � plasticity � ischemia � infarction � myocardial regeneration

In this review, we address the concept of whether stem cellscan repair injured tissues,1–4 with emphasis on ischemic

heart disease. Ischemic heart disease accounts for 50% of allcardiovascular deaths and is the leading cause of congestiveheart failure as well as premature permanent disability inworkers.5 With �1.1 million myocardial infarctions and�400,000 new cases of congestive heart failure each year,cardiovascular disease severely impacts men and women aswell as various ethnic groups. For patients diagnosed with

congestive heart failure, a consequence of chronic heartdisease, the 1-year mortality rate is 20%.

Myocardial Infarction and the Consequencesof Ischemic Heart Disease

Myocardial infarction is, by nature, an irreversible injury.6

Regional systolic function and regional metabolism decreasewithin a few heartbeats of a sudden decrease in myocardialperfusion.7 In some patients, impaired diastolic relaxation

Original received July 12, 2002; revision received October 25, 2002; accepted October 28, 2002.From the Genetics and Molecular Biology Branch (D.O.), National Human Genome Research Institute, NIH, Bethesda, Md; Laboratory of Molecular

Biology (J.M.H.), Cardiovascular Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Md; and Laboratory of Cardiac Energetics (A.E.A.),National Heart, Lung, and Blood Institute, NIH, Bethesda, Md.

Correspondence to Donald Orlic, PhD, Associate Investigator, Genetics and Molecular Biology Branch, National Human Genome Research Institute,NIH, 49 Convent Dr-4442, Bethesda, MD 20892-4442. E-mail [email protected]

© 2002 American Heart Association, Inc.

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may precede global systolic abnormalities. Irreversible car-diomyocyte injury begins after �15 to 20 minutes of coro-nary artery occlusion.8 The subendocardial myocardium hashigh metabolic needs and thus is most vulnerable to ische-mia.9 The extent of the infarction depends on the duration andseverity of the perfusion defect.10 However, the extent ofinfarction is also modulated by a number of factors includingcollateral blood supply, medications, and ischemic precondi-tioning.11 Beyond contraction and fibrosis of myocardial scar,progressive ventricular remodeling of nonischemic myocar-dium can further reduce cardiac function in the weeks tomonths after the initial event.12

Many of the therapies available to clinicians today cansignificantly improve the prognosis of patients with acutemyocardial infarction.13 Although angioplasty andthrombolytic agents can relieve the cause of the infarction,the time from onset of occlusion to reperfusion determinesthe degree of irreversible myocardial injury.14 No medicationor procedure used clinically has shown efficacy in replacingmyocardial scar with functioning contractile tissue. There isneed for new therapeutics to regenerate normal cardiomyocytes.

Recent attempts to repair experimentally induced acutemyocardial infarctions have provided encouraging but limitedsuccess in a number of animal models. The most promisingresults have been obtained after transplantation and mobili-zation of bone marrow cells to the area of infarction. We willreview the emerging literature in the nascent field of stem cellplasticity and describe new instrumentation to deliver andmonitor stem cell activity in myocardial therapy.

Embryonic and Fetal Stem CellsThe most primitive of all stem cell populations are theembryonic stem cells (ES cells) that develop as the inner cellmass at day 5 after fertilization in the human blastocyst. Atthis early stage, ES cells have vast developmental potential.They give rise to cells of the three embryonic germ layers.When isolated and transferred to appropriate culture media,mouse and human ES cells can undergo an undeterminednumber of cell doublings while retaining the capacity todifferentiate into specific cell types, including cardiomyocytes.15,16

Common teaching suggests that stem cells emerging dur-ing late embryonic and fetal development are restricted to theproduction of tissue-specific cell types. Specific gene expres-sion patterns are imprinted and, although stem cells continueto self-renew in adult life, their ability to differentiate islimited to the tissue in which they reside. We will examinethis dogma in light of recent remarkable data that indicate thatadult stem cells retain a high degree of developmentalplasticity. If this challenge to the traditional developmentalparadigm of adult stem cell commitment is sustained, we areabout to enter a revolutionary period in stem cell biology andregenerative medicine.

Adult Mesenchymal Stem Cells (MSCs)MSCs can be derived from adult bone marrow and in vitroappear to have multilineage differentiation capacity.17 Inculture, MSCs can maintain an undifferentiated, stable phe-notype over many generations. However, controversy stillexists regarding their precise phenotype, and there are no

adequate markers to allow selection of purified cell popula-tions. The DNA demethylating agent 5-azacytidine has beenused to induce multiple new phenotypes,18 including cardio-myocytes.19 Additional unexpected differentiation pathwaysinvolving MSCs have been described for the formation ofneural cells20,21 identified on the basis of neural cell–specificmarkers.22

Preclinical models have shown the ability of undifferenti-ated human MSCs to undergo site-specific differentiation intoa functional cardiac muscle phenotype after injection intosheep.23 Thus, they seem to avoid detection by the hostimmune system.24 Allogeneic bone marrow MSCs may there-fore have potential clinical utility because of their lack ofimmunogenicity and relative ease of culture. As such they canbe harvested and cryopreserved ready for infusion immedi-ately after myocardial infarction.

Another subset of bone marrow stromal cells referred to asmesodermal progenitor cells or multipotent adult progenitorcells has been described.25–27 Multipotent adult progenitorcells copurify with MSCs. They proliferate extensively anddifferentiate in vitro into cells of all three germ layers. Wheninjected in vivo they reconstitute bone marrow, liver, gut,lungs, and endothelium.

Adult Hematopoietic Stem/Progenitor CellsTwo categories of blood-forming stem cells exist in adultbone marrow. One population can provide permanent long-term reconstitution of the entire hematopoietic system. Thesecells, referred to as hematopoietic stem cells (HSCs), are rare,perhaps as few as 1:10 000 bone marrow cells.28 However,utilizing the specificity of monoclonal antibodies, HSCs canbe enriched by flow cytometry to near purity on the basis ofsurface markers. As few as 20 to 100 highly purified mousebone marrow HSCs can reconstitute the entire lymphohema-topoietic system in myeloablated adult mice.29–31 They canself-renew and can differentiate into the more mature progen-itor cells in bone marrow.

The progenitor cells of bone marrow have a limitedcapacity for self-renewal and differentiation. They can onlysustain hematopoiesis for 1 to 2 months and therefore areconsidered to be short-term repopulating stem cells. It isproposed that one subclass of mouse progenitor cells, thecommon lymphocytic progenitors (CLP), Lin� c-kit� Sca1�

IL7Ra�, is restricted to the generation of B and T lympho-cytes,32 whereas another subclass, the common myelocyticprogenitors (CMP), Lin� c-kit� Sca1� IL7Ra�, is restricted tothe generation of myelocytic cells.33 These CLP and CMPinitiate hematopoietic activity by giving rise to precursor cellsresponsible for the formation of each blood cell lineage.Although in vivo assays for human CLP and CMP are notestablished, it is clear from in vitro studies that theseprogenitors exist in human bone marrow. In the treatment ofblood disorders, it is now routine clinical practice to isolateand transplant CD34� stem cells. These include progenitorcells and HSCs that provide short-term and long-term hema-topoietic reconstitution. Although several reports demonstratethe presence of HSCs in the Lin-CD34� cell population inmouse34 and human bone marrow,35 the role of CD34� HSCsin hematopoietic reconstitution is not well understood.

Orlic et al Stem Cells for Myocardial Regeneration 1093




Hence, CD34� HSCs are not used in bone marrow transplan-tation. We will use the term bone marrow stem cells(BMSCs) to signify the inclusion of both short- and long-termreconstituting stem cells in donor cell populations.

Short-term repopulating progenitor cells of bone marrowcannot self-renew indefinitely. Other tissue-specific stem/progenitor cells may also need to be continually replenishedby a more primitive stem cell population. Emerging datasuggest that BMSCs have the ability to differentiate into stemand progenitor cells that mature into functional cells in avariety of tissues including myocardium.

Adult Stem Cells Engage in NormalTissue Regeneration

Stem cells are the ancestors of the specialized cells thatimpart function to tissues and organs. Throughout postnatallife, stem cells regenerate tissues that continually lose cellsthrough maturation and senescence. These include the epithe-lial layers in skin; intestinal and pulmonary mucosal linings;and connective tissues such as bone, cartilage, muscle, blood,and bone marrow. Although previously considered to bepostmitotic organs, recent evidence is persuading us toinclude brain36 and heart37–39 on the list of adult tissues withregenerative capacity. The extremely low rate of neural andmyocardial cell turnover may explain why renewing cellswere not previously detected in these organs.

The origin of stem cells in regenerating adult tissues is nowbeing called into question. It can no longer be assumed thattissue-specific stem cells are self-sustaining throughout life orthat they are responsible for regenerating tissues damaged byradiation or chemotherapy treatments. As reported in anumber of recent papers, bone marrow cells appear to havethe capacity to repopulate many nonhematopoietic tis-sues.20,40–43 Thus, bone marrow may serve as a centralrepository for the primitive stem cells that can repopulatesomatic tissues.

Adult Mouse BMSC PlasticityIn a series of reports, it has been suggested that adult BMSCsretain the capacity to produce cells of unrelated tissues. Basedon evidence from several mouse models, tissues of all threegerm layers can be derived from adult BMSCs (Figure 1).These include skeletal muscle,44 hepatocytes,45 neuralcells,46,47 vascular endothelium,48,49 and epithelium of skinand several internal organs.50 Plasticity of transplantedBMSCs has been established by identifying specific cellsurface markers or by fluorescence in situ hybridizationidentification of Y-positive nuclei in donor-derived cells thathave acquired the capacity to synthesize specific protein inregenerating tissues.

In one study, a single male BMSC was transplanted byintravenous injection into lethally irradiated adult mice.50 At11 months, Y-positive cells were identified in the liver,kidneys, skin, and epithelial lining of several internal organsincluding lung and small intestine. The highest percentage ofY-positive cells occurred in epithelial tissues. A commondifficulty with this technique is the possibility that thepercentage of Y-positive cells in any tissue will be underes-timated. This inherent error relates to sampling, because aportion of the nucleus will often be excluded from the planeof the section. Hence, the Y chromosome in many malenuclei will not be recorded. Another difficulty involves thepossibility that the Y-positive nucleus of a nearby blood cellmay be recorded as belonging to the cell in question. Thisproblem can be largely overcome by careful analysis usingappropriate confocal microscopy techniques. Endogenousstem cells in these rapidly renewing tissues are likely to suffersevere depletion during the preconditioning total body radi-ation exposure leading to a critical need for recruitment oflarge numbers of exogenous stem cells. Thus, transplantedself-renewing Y-positive BMSCs may provide a new supplyof stem/progenitor cells for epithelial and other somatictissues in need of regeneration. Studies such as this mark an

Figure 1. Proposed developmental pat-tern for adult BMSC transdifferentiationinto multiple lineages. Exact identity ofcandidate BMSC (single or multiple) hasnot been established. There is evidencesuggesting that marrow-derived HSCs,mesenchymal (stromal) stem cells,and/or cells with potential of embryonichemangioblasts may be involved intransdifferentiation.

1094 Circulation Research December 13/27, 2002




important beginning to our understanding of adult BMSCplasticity.

Can Stem Cell Plasticity Be Explained byCell Fusion?

The enthusiasm generated by findings on stem cell plasticityhas been countered by a degree of skepticism in manyresearch centers. Several recent papers demonstrate in vitrocell-cell fusion of transgenic female-derived neural51 or bonemarrow52 cells with male-derived ES cells. Cell fusionoccurred at an estimated frequency of 1:10 000 or 1:100 000cells, and the hybrid cells displayed a dual phenotype. Theypossessed a large nucleus that contained numerous nucleoliand a tetraploid number of chromosomes. The identificationof XXXY-positive nuclei provided the strongest argument forthe authors’ contention that cell fusion in animal studies maybe a legitimate alternative to the concept of stem cellplasticity. In mouse studies that demonstrate 0.02% to 0.5%donor-derived cells,36,40,50 the cell fusion theory cannot bedismissed. In contrast to these reports of low-level reconsti-tution, wild-type BMSCs injected into genetically defectiveadult mice with a metabolic liver disorder resulted in theregeneration of significant liver mass.45 Also, in an experi-mental retinopathy study in mice, an extensive retinal capil-lary network was regenerated from BMSCs.49 Our ownstudies of myocardial regeneration demonstrated the forma-tion of a new band of myocardium from BMSCs.53,54 It isunlikely that infrequent cell fusion events could explain thesignificant regeneration observed in liver, eye, and heart inthese studies. Nevertheless, the possibility of cell fusion andtetraploidy must be ruled out in these studies as well.

Are Environmental Factors Involved in StemCell Migration?

The homing of stem cells to areas of tissue injury maypotentially occur via two or more distinct scenarios. Onehypothesis suggests that cell necrosis following an injurysuch as myocardial infarction may cause the release of signalsthat circulate and induce mobilization of stem cells from thebone marrow pool. The injured tissue may express appropri-ate receptors or ligands to facilitate trafficking and adhesionof stem cells to the site of injury where initiation of adifferentiation cascade results in the generation of cells of theappropriate lineage. An alternative hypothesis suggests thatstem cells are continually circulating with constant traffickingthrough all tissues, but only at the time of injury do they exitthe blood and begin to infiltrate the site of injury. Bothconcepts support the view that there are circulating stem cellsthat could originate from a common pool in bone marrow.This is further supported by findings that show that stem cellsisolated from skeletal muscle retain hematopoietic activityand are itinerant cells derived from bone marrow.55–57

It is still unclear what environmental cues initiate mobili-zation and homing of adult BMSCs to normal and injuredtissue. Likewise, we know little about the factors that inducethese stem cells to differentiate along the appropriate organ-specific lineage. Below are described several receptor-ligandinteractions that may regulate BMSC trafficking and that arethe subject of intense investigation.

Stem Cell Factor (SCF) and c-kitHSCs, neural crest cells, and germ line cells express c-kit, atyrosine kinase receptor. The migration of these cells duringembryonic development may be regulated by the c-kit ligand,SCF. SCF mRNA is expressed in fetal and neonatal hearts58

and by adult myocardial fibroblasts and macrophages.59,60 Intheory, SCF may provide signals for stem cell migration inresponse to myocardial injury.

We hypothesize that there is an insufficient local stem cellpool to provide acute myocardial regeneration. Withoutaugmentation of the number of circulating BMSCs throughcytokine mobilization,53 the response to ischemia favors scarformation rather than cardiomyocyte regeneration. Recentexciting evidence has shown the central role of SCF, c-kit,and matrix metalloproteinase-9 in the mobilization of stemand progenitor cells from the bone marrow niche within hoursof onset of myocardial necrosis.61

The intense inflammatory reaction that initiates healingafter a left ventricular (LV) myocardial infarction causes alocal accumulation of mast cells62,63 that are positive forCD117, the human equivalent of c-kit. They may migratelocally in response to macrophage secretion of SCF. Thisreinforces the idea that homing signals are released soon aftermyocardial injury.

CXCR4 and Stromal Cell–DerivedFactor-1 (SDF-1)CXCR4 is important for lymphocyte trafficking and recruit-ment at sites of inflammation, eg, after myocardial infarc-tion.64 It appears that CXCR4 serves as a chemokine receptorand together with its ligand, SDF-1, plays an important role invasculogenesis65 and hematopoiesis.66–68 These changes mayoccur in response to a disrupted interaction between SDF-1and CXCR4. This hypothesis is supported in part by studiesthat show that SCF upregulates CXCR4 expression on humanCD34� stem/progenitor cells and enhances their migration inresponse to SDF-1.69 Migration of bone marrow CD34� cellsacross endothelial barriers is modulated by a wide variety ofchemokines, but the largest response is seen with � and �SDF-1.70

Granulocyte Colony-Stimulating Factor (G-CSF)The cytokine G-CSF is widely used to mobilize stem/progenitor cells that are harvested by leukapheresis, stored,and subsequently reinfused to support hematopoietic recov-ery in patients after chemotherapy or radiation treatment.How G-CSF mobilizes stem cells and progenitor cells fromthe bone marrow into the circulation is not clear becauseBMSCs do not generate G-CSF receptor.71 This suggests thatan indirect mechanism may exist. For example, a single doseof G-CSF can induce a downregulation in SDF-1 levelswithin 24 hours and an upregulation of CXCR4 expression onhematopoietic cells. Accordingly, G-CSF stimulation poten-tiates the homing abilities of cytokine-stimulated BMSCs, anaction that can be inhibited by pretreatment with anti-CXCR4antibodies.72

Vascular Endothelial Growth Factor (VEGF)/Flk-1The roles of the VEGF isoforms and the tyrosine kinaseVEGF receptors in endothelial cell proliferation and differ-





entiation are well described.73 However, evidence is emerg-ing that VEGF receptor expression, most notably VEGFreceptor-2 (KDR/flk-1), may define the point of divergence inthe differentiation pathway of bone marrow–derived stemcells along a vascular progenitor lineage.24,74

BMSCs Regenerate Cells in Animal Models ofSeveral Human Diseases

Hepatocyte RegenerationThe capacity of highly enriched mouse long-term repopulat-ing BMSCs to regenerate functional hepatocytes was tested inadult knockout female recipients deficient in fumarylaceto-acetate hydrolase (FAH�/�) synthesis.45 At 7 months after anintravenous injection of male BMSCs, donor Y-positive cellsinfiltrated the liver parenchyma and gave rise to nodules thatcomprised 30% to 50% of the liver mass. These wild-typedonor-derived hepatocytes synthesized FAH, resulting inimproved liver function and survival. This rescue of FAH�/�

mutants demonstrated the efficacy of BMSC-induced repairof a genetic disease.

Endothelial RegenerationGFP� cells were isolated from transgenic mouse bone mar-row on the basis of a Lin� Sca-1� c-kit� phenotype. A singletransplanted GFP� cell was injected into each of a largenumber of mice, and in several recipients the bone marrowwas reconstituted within 6 months. The investigators thenused an Argon Green laser system to induce photocoagulationin the retinal vasculature as a model for retinopathy. Dam-aged vessels were replaced within 3 weeks with a new,developing GFP� capillary network. Because blood cells andendothelium were both derived from the same single GFP�

cell, the authors attributed this activity to adult bone marrowcells comparable to the embryonic hemangioblasts.49

Myocardial RegenerationOur data on regeneration in an adult mouse model ofmyocardial infarction demonstrate the ability of BMSCs todifferentiate into cardiac myocytes, endothelial cells, andvascular smooth muscle cells.53,54 Ischemic injury to themyocardium of the left ventricle was produced by ligation ofthe descending branch of the left coronary artery (LCA)without reperfusion. The infarcts occupied as much as 70% ofthe free wall of the left ventricle with loss of myocytes andcoronary vessels. When male BMSCs (phenotype, Lin�

c-kit�) that carried the gene encoding enhanced green fluo-rescent protein (eGFP) were injected within 5 hours aftercoronary ligation (Figure 2), a band of regenerating myocar-dium was seen at 9 days after surgery (Figure 3A). This bandconsisted of Y-positive, eGFP� cardiac myocytes and smallcoronary vessels.54 Regeneration was not observed (Figure3B) in hearts that were transplanted with the subpopulation ofbone marrow cells (phenotype, Lin� c-kit�) known to bedevoid of stem cells.31 Cardiomyocytes (Figures 3C through3E), smooth muscle cells, and endothelial cells were all eGFPpositive.54 Early-acting cardiac-specific transcription factorsGATA-4, Csx/Nkx2.5, and MEF-2 were expressed in thedeveloping cardiomyocytes as well as cardiac myosin, sarco-meric �-actin, and connexin 43. The immature myocytes

were arranged into what appeared to be an early form of anintegrated syncytium with some connexin 43 at the lateralborder of adjacent cells (Figures 3F and 3G). LV end-diastol-ic pressure and LV developed pressure improved 30% to 40%in hearts transplanted with BMSCs compared with negativecontrol mice.

Myocardial regeneration was also examined in a mousemodel in which BMSCs marked with the �-galactosidasegene were used to create chimeric bone marrow in adultmice.40 Subsequently, myocardial infarcts were induced, as inour study, by ligation of the LCA. �-Galactosidase–positiveBMSCs were found to migrate to the site of injury and to giverise to new cardiomyocytes and endothelium. Although thelevel of �-galactosidase–positive cells was only 0.02% of thetotal myocardial cells counted, this study demonstrated anatural but very inefficient response by the BMSCs to repairthe damaged myocardium.

We were able to demonstrate extensive regeneration in themouse myocardial infarction model with cytokine-mobilizedautologous BMSCs.53 After 5 daily injections of recombinantrat SCF and recombinant human G-CSF, the wave of circu-lating BMSCs reached a peak.75 Myocardial infarctionsproduced by ligation of the LCA showed a new band ofmyocardium. The new myocytes resembled fetal cardiacmyocytes in size and gene expression. Their failure to matureand their continued proliferation remain unresolved issues.Numerous developing capillaries and arterioles were ob-served, and some contained red blood cells in their lumen.This suggested that anastomosis had occurred with the sparedcoronary vessels. Cytokine therapy improved hemodynamicfunctions, including ejection fraction, LV end-diastolic pres-sure, and LV end-systolic pressure, and resulted in markedlyincreased survival at 27 days.

These experiments demonstrated the capacity of adultBMSCs to give rise to new myocytes, endothelial cells, andsmooth muscle cells in ischemic myocardium. However, theydid not define whether one or more BMSC populations wereresponsible for the generation of these several myocardial celltypes. Nor did they exclude the possibility that some stem

Figure 2. Myocardial infarction is induced in adult female miceby ligation of LCA. BMSCs from adult eGFP transgenic malemice are enriched by flow cytometry on the basis of lineagedepletion and c-kit expression (Lin� c-kit�). Lin� c-kit� subpopu-lation consists of both stem and progenitor cells. Drawing byDarryl Leja (National Human Genome Research Institute, NIH,Bethesda, Md).





cells with regenerative capability may have originated inother organs, including the heart.

Adult Human BMSC PlasticityY-Positive Cells InfiltrateNonhematopoietic OrgansTherapeutic transplants of sex-mismatched bone marrow andorthotopic organ transplants in male recipients have beenused to study human BMSC plasticity. Archival samples ofliver and heart obtained from female recipients of a male bonemarrow transplant and male patients who had received a liveror heart transplants from female donors were positive forY-chromosomes.76,77 Although differing widely in percentageof Y-positive myocytes detected, several reports38,39 concludethat recipient cells repopulate the myocardium in transplantedhearts. Taken together, these data provide evidence thatcirculating human BMSCs traffic to nonhematopoietic or-gans, where they give rise to cells of a completely differentorigin and phenotype.

Clinical Trials to Regenerate Myocardium inIschemic Heart DiseaseIn 10 patients with acute myocardial infarction, autologousmononuclear bone marrow cells were transplanted via theinfarct-related artery after angioplasty. At 3 months aftertransplant, the infarct area had decreased significantly com-pared with 10 patients not given cell therapy. The treatedpatients also showed improved LV end-systolic volume andcontractibility. This is the first report78 to demonstrate clinicalfeasibility of intracoronary infusion of bone marrow cells formyocardial repair.

Seiler et al79 recently reported the effects of intracoronary andsystemic administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with coronary arterydisease. GM-CSF is a cytokine with effects on bone marrowsimilar to the more commonly utilized G-CSF, albeit with lesspotency for BMSC mobilization. Twenty-one patients who werenot amenable to or refused coronary bypass surgery participatedin this randomized, double blind, placebo-controlled study. Tenindividuals received GM-CSF via intracoronary infusion into thevessel believed to subserve ischemic myocardium, followed bysystemic administration of GM-CSF daily for 2 weeks. Analysisof mobilization of BMSCs was not performed in this study, butbecause the leukocyte counts were only twice the baselinevalues, it was suggested that only modest BMSC mobilizationwas achieved.

An invasive measure of collateral artery blood flow (esti-mated by coronary artery pressure distal to balloon occlusion)before and after administration of GM-CSF or placeboindicated improved collateral flow in the GM-CSF group at 2weeks, but not in the placebo group, with reduced ECG signsof myocardial ischemia during coronary balloon occlusion.Because the quantity of BMSCs mobilized with GM-CSFwas probably low, the coronary vascular benefit determinedin this study may have resulted from direct effects of thiscytokine on angiogenesis or on collateral vascular dilator tonewith improved regional blood flow. No clinically relevant endpoints (eg, exercise-induced myocardial ischemia or LVcontractile response to stress) were assessed in this study.

Figure 3. BMSCs regenerate infarcted myocardium. Lin� c-kit�

eGFP� cells from transgenic mice were injected into myocardi-um near the site of an acute infarction. A, After 9 days, partialregeneration of structure and function were observed. Asteriskindicates necrotic myocytes; red, cardiac myosin; and green,nuclei labeled with propidium iodide. B, Lin� c-kit� bone marrowcells are devoid of stem cells and do not regenerate myocardi-um. Original magnification, �50. C, Cardiac myosin (red). D,eGFP (green). E, Overlay of cardiac myosin (red) and eGFP(green). Propidium iodide–stained nuclei (blue). EN indicatesendocardium; EP, epicardium; and arrows at subendocardium,area of myocardial infarction not regenerated. Original magnifi-cation, �250. F, Adult control heart shows myocytes positive forconnexin43 at intercalated disks (arrows) and at lateral marginsof adjacent cells. G, Newly formed young myocytes in a Lin�

c-kit�–treated heart show connexin43 in their cytoplasm withsome distribution at lateral cell margins (arrows). Arrowheadsindicate spared myocytes in the epicardium. First published inOrlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B,Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, AnversaP. Bone marrow cells regenerate infarcted myocardium. Nature.2001;410:701–705.





These studies represent the first clinical attempts to regen-erate myocardium after infarction. However, the empiricalnature of these observations emphasizes the need for contin-ued preclinical testing to determine the phenotype of the bonemarrow cells involved in myocardial repair and to define thesignaling required for their migration and differentiation intomyocardial cells. When these questions are resolved, we canlook to a time when transplanted or cytokine-mobilized stemcells may provide a new modality for the treatment of heartdisease.

Role of Imaging in Assessing the Success ofStem Cell Therapy

Imaging will play an important role in assessing the myocardialresponse to stem cell therapy. In preclinical trials, analysis oftissue specimens allow detailed cellular characterization ofgenetic and cell surface markers. However, the need to euthanizethe animal at fixed time intervals raises the complementary needfor serial noninvasive imaging of myocardial function, perfu-sion, viability (Figure 4), and cell tracking.

Echocardiography assessment of myocardial function iswell established. Doppler tissue imaging now provides real-time quantitative measurements of regional contractile func-tion that have higher temporal resolution80 than any measure-ment scheme other than implanted ultrasonic crystals.81

Similarly, single photon emission computed tomography(SPECT)82 and positron emission tomography (PET) imagingcan determine myocardial perfusion and viability. Develop-ments in cardiovascular MRI warrant further discussionbecause this technology is well suited to serial studies.

MRI is generally accepted as a gold standard method forevaluating cardiac anatomy and volumes. Cine MRI providesexcellent contrast between the myocardium and blood83 andperforms with diagnostic accuracy comparable to that of

dobutamine stress echocardiography.84–86 The programmablenature of the imaging planes allows reproducible and volu-metric coverage of the heart. This leads to markedly smallersample size requirements for clinical trials87 than can beachieved with echocardiography. The technology also scaleswell with subject size from mouse to human (Figure 5). Thereare several MRI-specific ways to quantitatively assess re-gional function including myocardial tagging,88 velocity-encoded imaging,89–91 and displacement-encoding methods.92

Myocardial perfusion imaging using contrast-enhancedfirst-pass MRI can now obtain whole-heart coverage at aresolution double that of a PET scanner and 4 times theresolution of SPECT.93 Perfusion can be evaluated semiquan-titatively in 250-�L samples of myocardium with highstatistical certainty.94 This has translated to excellent diag-nostic accuracy in patients with possible coronary arterystenosis95,96 when compared with gold standards of quantita-tive coronary angiography or PET scans.

New MRI myocardial viability methods97 are now wellvalidated. Gadolinium hyperenhancement correlates closelywith infarcted myocardium defined by triphenyltetrazoliumchloride.98,99 Gadolinium distributes in a pattern similar tothat of sodium as demonstrated by electron probe x-raymicroanalysis100 and by imaging.100 The gadolinium chelatesused clinically equilibrate rapidly in the extracellular volume.Because of membrane rupture, the myocytes of acutelyinfarcted myocardium fail to exclude sodium and gadoliniumfrom the intracellular space resulting in substantial contrastenhancement relative to normal myocardium. The volume ofdistribution remains high in chronic myocardial infarc-tion.101–103 Furthermore, gadolinium enhancement differenti-ates stunned from infarcted myocardium with high specific-ity.98,99,104,105 The main advantage of this technique overnuclear methods is the high image resolution achievable.106

There is a relationship between the transmural extent of

Figure 4. Serial MRI scans to evaluate ventricular remodelingassociated with acute myocardial infarction. Short-axis imagesare shown at end diastole (top row) and end systole (bottomrow) before infarction (first column), 1 week after infarction (mid-dle column), and 6 weeks after infarction (third column). Scale isthe same on all images. In addition to the reduction in wallthickening in the anterior septum and anterior wall, there is pro-gressive dilatation of the ventricle from 17 to 22 to 25 mm onthe end-systolic images over this series of examinations. Goodimage quality and ability to reproducibly image the heart allowquantification of ventricular remodeling at multiple time pointsbefore and after interventions.

Figure 5. Cine MRI from human to mouse. Short-axis imagesare shown at end diastole (top row) and end systole (bottomrow) for a 50-kg human (left column), a 5-kg rhesus monkey(middle column), and a 25-g mouse (right column). White barindicates a 1-cm scale on each end-systolic image. Inset inupper left of end-systolic images shows hearts displayed at thesame scale as the human heart. Mouse images were providedby T.C. Hu and A.P. Koretsky (Laboratory of Functional andMolecular Imaging, National Institute of Neurological Disordersand Stroke, NIH, Bethesda, Md).





infarction and clinical definitions of viability107,108 (Figure 6).MRI assessment of viability correlates with PET except thatMRI appears to detect subendocardial infarction missed bythe lower-resolution nuclear techniques.109

Recent developments have taken MRI beyond traditionalanatomic and functional imaging. Real-time visualizationnow allows identification of regions of myocardial infarctionand precise MRI-guided delivery of therapeutic agents. Fur-thermore, the injection sites can be identified using contrastagents.110 It is also feasible to label cells with iron particlesand detect their distribution in the body noninvasively.111

Gadolinium can be attached to antibodies for specific labelingof compounds in vivo.112 New contrast agents have allowedMRI visualization of gene expression at a cellular resolution(�10 �m).113 Apoptotic cells have been detected with tar-geted MRI contrast agents.114 Labeling and detection of stemcells is anticipated to enable MRI to trace their distribution invivo.115

Noninvasive imaging can monitor the response to stem celltherapy at approximately the level of gross pathology. Thereis still a need to develop techniques that can detect the cells

introduced into the myocardium and to follow their divisionover time. There are important subtleties of cellular function,growth, and proliferation that cannot be imaged with thekinds of noninvasive methods described. Thus, there willremain a substantial need for detailed physiological andpathological methods to understand the myocardial responseto stem cell therapy.

Predicted Role for BMSCs inRegenerative MedicineIt is now routine practice for patients about to undergomyeloablation to be treated with G-CSF to mobilize CD34�

BMSCs into the circulation for the purpose of collection andsubsequent use for bone marrow reconstitution. If BMSCplasticity resides in the primitive CD34� population assuggested in a recent study,48 this approach may havepotential clinical utility in cardiac patients. In patients withrefractory myocardial ischemia, safety feasibility studies havealready begun.116 These studies are designed to determinewhether BMSCs can traffic to the heart and develop intocardiomyocytes and coronary vessels.116

Existing data indicate that BMSCs continually proliferateand enter the circulation. These circulating BMSCs appear tohave an engraftment phenotype that fluctuates with the phaseof cell cycle.117 Thus, BMSCs with different engraftmentcapabilities are continually passing through capillary net-works in all tissues. Data showing production of a variety ofbone marrow–derived cell types including hepatocytes andcardiomyocytes suggest that circulating BMSCs seed anddifferentiate into tissue-specific cells. It remains to be estab-lished whether this plasticity is attributed to a single subpopu-lation of BMSCs or to multiple subpopulations each beingtissue restricted. At present, we can only acknowledge theability of BMSCs to colonize skeletal muscle,55–57 skin,50

bone,117 liver,45 retina,49 and heart.48,53,54

SummaryWe propose, without much evidence, that the differentiationof BMSCs that seed peripheral organs is regulated byexposure to local environmental factors. Thus, a BMSC or itsprogeny that in bone marrow would give rise to granulocytes,erythrocytes, and platelets will give rise to lymphocytes in thethymus, hepatocytes in the liver, and cardiac myocytes in theheart. This has exciting clinical potential because BMSCs areself-renewing and can be easily harvested from bone marrowand peripheral blood. Furthermore, clinical experience showsthat BMSC transplantation does not lead to neoplasia as mayoccur with other stem cell populations. The need to expandthe scope of investigations using embryonic and fetal stemcells is of paramount importance and cannot be overstated,but adult BMSCs may offer the best near-term promise fortissue repair.

Although not conceived 3 to 4 years ago, tissue regenera-tion using adult BMSCs is now openly discussed among eventhe most conservative scientists and clinicians. If additional,encouraging preclinical data can be obtained, the next decadeis likely to witness clinical trials aimed at testing the capacityof BMSCs to regenerate damaged tissues.

Figure 6. MRI infarct characterization can measure the transmu-ral extent of infarction. A normal heart is characterized by uni-formly dark myocardium on inversion recovery images 20 min-utes after injection of gadolinium (top row). Areas of myocardialinfarction accumulate more gadolinium than normal tissue andthus appear brighter on these images (hyperenhanced). Middleimage is from a patient with chronic subendocardial infarction;bottom image, transmural infarction.





AppendixGlossary of Terms

Stem cells: primitive cells that have the capacity for extensiveself-renewal and the ability to differentiate into multiple cell types.

Embryonic stem cells: pluripotent cells derived from the inner cellmass of the blastocyst; they give rise to cells of all three germlayers.

Hemangioblasts: primitive embryonic cells that give rise to bothHSCs and endothelial progenitor cells; they may also exist in adultbone marrow.

Adult stem cells: present in all renewing tissues; these cells dividefor self-renewal and differentiate into multiple progenitor celltypes.

Hematopoietic stem cells: rare adult stem cells present in blood andbone marrow; they give rise to several distinct populations ofblood-forming progenitor cells.

Progenitor cells: multipotential intermediate stem cells that serve asthe direct precursors for tissue-specific mature cells.

Endothelial progenitor cells: cells that are present in blood and bonemarrow; they are involved in angiogenesis and postnatalneovasculogenesis.

Mesenchymal stem cells: also referred to as marrow stromal cells;these cells differentiate in vitro along multiple pathways thatinclude cardiac myogenesis.

Plasticity or transdifferentiation: the capacity of adult stem cells thatreside in one tissue to differentiate into mature cells of an unrelatedtissue.

AcknowledgmentsThis work was supported entirely by intramural funding from theNational Human Genome Research Institute and the National Heart,Lung, and Blood Institute, NIH.

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Stem Cells for Myocardial Regeneration

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