Mesenchymal Stem Cells: Future Source for Reparative Medicine
Rinky Bhatia, MD; Joshua M. Hare, MD 

CHF.  2005; 11 (2): 87-91.  ?2005 Le Jacq Communications, Inc.

Abstract and Introduction

Abstract

Current treatments for ischemic cardiomyopathy are aimed toward minimizing the deleterious consequences of damaged myocardium. The possibility of treating heart failure by generating new myocardium and vascular structures has provided major impetus for recent stem cell research. Mesenchymal stem cells (MSCs), also referred to as marrow stromal cells, differentiate into a wide variety of lineages, including myocardial smooth muscle and possibly endothelial cells. The multilineage potential of MSCs, their ability to elude detection by the host's immune system, and their relative ease of expansion in culture make MSCs a very promising source of stem cells for transplantation. This paper reviews animal and human trials studying the role of MSCs in cardiomyogenesis and vasculogenesis in postinfarct myocardium, factors that stimulate MSC differentiation, routes of MSC delivery, and methods of detecting MSC engraftment.

Introduction

Coronary artery occlusion leads to irreversible cardiomyocyte injury within 15.20 minutes. In the postinfarct ventricular remodeling process that ensues, cardiomyocytes are replaced by fibrous tissue, contractile function deteriorates, and the left ventricle dilates.[1] This loss of contractile tissue often leads to ischemic cardiomyopathy, with mortality rates exceeding 50% within 5 years of initial diagnosis.

Conventional therapeutic modalities are targeted toward minimizing the deleterious consequences of diseased myocardium. The possibility of treating heart failure by generating new myocardial and vascular structures has spurred intense interest in exploring cell-based therapeutics. Here we review potential roles for mesenchymal stem cells in the repair of postinfarct myocardium.

What Are Mesenchymal Stem Cells?

Stem cells are immature tissue precursor cells that are able to self-renew and differentiate into multiple cell lineages.[2] Due to the multiple ethical concerns surrounding the use of embryonic stem cells, recent studies have focused on various sources of adult stem cells, including hematopoietic, mesenchymal, cardiac, neural, hepatic, pancreatic, and skeletal muscle satellite stem cells.[3?11]

Mesenchymal stem cells (MSCs), also referred to as marrow stromal cells, are located in the bone marrow (BM) and peripheral blood. MSCs support hematopoiesis by expressing various cytokines, growth factors, integrins, and adhesion molecules. MSCs can also differentiate into a wide variety of lineages. In vivo studies detected injected MSCs in host adipose tissue, lung, articular cartilage, perivascular areas of the central nervous system, cardiac muscle, skeletal muscle, liver, BM, endothelium, spleen, and thymus.[5?13]

BM-derived stem cells include both hematopoietic and mesenchymal cells. Unlike hematopoietic stem cells, MSCs are CD34? and CD45?. Other cell-surface markers characteristic of MSCs include CD29, CD44, CD71, CD90, CD106, CD120a, CD124, SH2, SH3, and SH4.[12,13]

Allogeneic MSCs do not stimulate graft vs. host reaction. MSCs show minimal expression of MHC class II and lack B-7 costimulatory molecules required to illicit a T-cell.mediated immune response.[14] BM cells cultured with allogeneic dendritic cells or peripheral blood lymphocytes suppress proliferation of CD4 and CD8 (65%?5% and 75%?15%, respectively) by producing transforming growth factor β1 and hepatocyte growth factor.[15] Moreover, injection of allogeneic MSCs into baboons does not cause rejection.[16]

In summary, the multilineage potential of MSCs, their ability to allude detection by the host's immune system, and their relative ease of expansion in culture make MSCs a very promising source of stem cells for transplantation. Ideally, MSCs can be harvested, expanded, and cryopreserved, ready for injection into patients following myocardial infarction.

Animal Studies

Early in vitro studies show after 2 weeks of treatment with 5-azacytidine, murine BM stromal cells form myotube-like structures expressing myosin, α-actin, GATA 4, MEF-2, TEF-1, Nkx2.5, atrial natriuretic peptide, and brain natriuretic peptide. After 3 weeks, they express action potentials similar to those in the sinus node.[17] These data suggest that stromal stem cells, including MSCs, differentiate into cardiomyocytes under appropriate culture conditions.

Subsequent in vivo studies investigating the role of MSCs in myocardial regeneration and vasculogenesis will be briefly reviewed. In a pivotal study by Orlic et al.,[11] Lin?/c-kit+ cells (presumably containing both MSCs and hematopoietic stem cells) expressing enhanced green fluorescent protein from male mice were injected into the infarcted myocardium of female mice. Nine days after stem cell injection, newly formed cardiomyocytes containing Y chromosome and green fluorescent protein occupied 68% of the infarct area. Left ventricular (LV) enddiastolic pressure was 36% lower in treated mice compared with controls. These Lin?/c-kit+ cells differentiated into myocardial, endothelial, and smooth muscle cells. These new myocytes expressed myocyte enhancer factor-2 (MEF-2), cardiac specific transcription factor GATA- 4, and connexin.[43]

Similarly, lacZ-labeled human MSCs were injected into the infarcted myocardium of adult mice.[18] These lacZ cells contained desmin and troponin T within 14 days, and α-actinin and phospholamban within 60 days. Two weeks after Di-I labeled autologous MSCs were surgically injected into infarcted swine myocardium,[19] cardiomyocyte-specific proteins were expressed. Within 4 weeks, contractility improved (5.4%?12.2% vs. ?3.37%?2.7% in controls) while wall thickening increased in treated swine. In summary, these data suggest that MSCs can differentiate into cardiomyocytes and improve hemodynamic function.

In addition to cardiomyocyte regeneration, MSCs are also involved in vasculogenesis. CD34?, c-kit+, lacZ+ male stem cells were injected into irradiated female mice before infarct. Four weeks postinfarct, 0.02% of cardiomyocytes and 3.3% of endothelial cells were found to be lacZ+.[9]

Similarly, MSCs labeled with 99Tc and lacZ were delivered into rats via IV infusion or LV cavity injection.[20] LacZ+ donor cells localized in the infarct border zone and expressed genes which facilitate vasculogenesis.

When autologous BM cells were trans-endocardially injected into the infarcted myocardium of swine, contractility and collateral flow significantly improved.[21] In vitro studies showed that these stem cells secreted vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein, a mechanism by which BM cells could increase collateral flow.

Thirty days after syngenic, DAPIlabeled mesenchymal progenitor cells were injected into the infarcted myocardium in rats, some DAPI-labeled cells stained for desmin and á-SM actin, whereas others stained for the endothelial marker CD31.[22] Ejection fraction (EF), fractional shortening, and vascular density were significantly higher in treated animals, whereas LV systolic dimension and LV dilatation were significantly lower. This implies that mesenchymal progenitor cells can differentiate into both cardiomyocytes and endothelial cells when transplanted into infarcted myocardium.

Similarly, Kamihata et al.[23] noted a 5.7-fold increase in the number of collateral vessels 3 weeks after BM mononuclear cells were injected into the infarcted zone in rats. These BMderived endothelial cells expressed fibroblast growth factor, VEGF, angiopoietin, interleukin-1β, and tumor necrosis factor-á, which further upregulate vasculogenesis. EF improved by 48% in the stem cell-treated group, compared with no change in controls.

In summary, these studies show that MSCs are involved in both cardiomyogenesis and vasculogenesis. However, these studies used BM stem cells, which contain both MSCs and hematopoietic stem cells. Therefore, the significant improvement in hemodynamics cannot be attributed to MSCs alone.

Human Studies

Similarly, BM stem cells containing both MSCs and hematopoietic stem cells have been studied in myocardial regeneration in humans. In the Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) trial,[24] autologous CD34+/45+ hematopoietic stem cells were delivered to patients 4.3?1.5 days postinfarction via intracoronary infusion. After 4 months, EF increased from 51.6?9.6% to 60.1?8.6% and end-systolic LV volumes decreased (56.1?20 mL to 42.2?15.1 mL) in treated patients. Wall motion in the infarct zone and myocardial viability also improved. No malignant arrhythmias or inflammatory responses were noted.

Another study treated 10 patients 5?9 days postinfarct with intracoronary infusion of autologous mononuclear BM cells.[25] After 3 months, a significant increase in stroke volume index, myocardial perfusion, infarct wall motion, and LV contraction were observed. When eight patients with refractory stable angina had autologous BM cells injected into the myocardium, they had 16.4% less anginal episodes after 3 months.[26] Magnetic resonance imaging (MRI) showed an 11.6% increase in regional wall thickening, 5.5% increase in regional wall motion, and a 3.9% reduction in the percent mass of hypoperfused myocardium in treated patients.

Perin et al.[27] delivered BM mononuclear cells via trans-endocardial injection into patients with end-stage ischemic cardiomyopathy. After 2 months, there was a significant improvement in New York Heart Association (NYHA) class, end-systolic volume, and total percent of reversible defect on single proton emission computed tomography (SPECT). After 4 months, EF improved from baseline of 20%?9% to 29%?13% in treated patients.

In the recent Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST) study,[28] 60 patients with ST elevation myocardial infarction were randomized to receive optimal medical therapy or medical therapy with intracoronary injection of autologous BM cells 4.8 days postinfarct.[28] After 6 months, left ventricular ejection fraction did not change in the medical therapy group (51.3%?9.3% to 52.0%?12.4%), whereas left ventricular ejection fraction increased from 50.0%?10.0% to 56.7%?12.5% in the group treated with BM cells. Despite this improvement in left ventricular ejection fraction, infarct size as measured by cardiac MRI did not decrease in the group that received BM cells. Arrhythmias or significant in-stent restenosis did not occur.

In summary, these data suggest that BM-derived stem cells containing MSCs improve cardiac function and hemodynamics when transfused postinfarct.

Route of MSC Delivery

The most efficacious route of MSC delivery needs to be established. Theoretically, IV infusion would be the easiest route. After human MSCs were systemically infused into mice, human DNA was found in mice marrow, cardiac muscle, and teeth.[29] As cells localize to other organs, the number settling in the myocardium is reduced. In fact, when MSCs were IV infused in baboons and rats, MSCs localized in the liver, spleen, BM, and lungs, precluding significant myocardial engraftment. Other studies have also shown that most IV infused MSCs settle in capillary beds of the liver and lungs.[30,31] No significant toxicities have been associated with IV infusion.

Rombouts et al.[32] showed that 24 hours of in vitro culture decreased homing of MSCs to BM and spleen. This could partly be explained by the decrease in chemokine receptors and adhesion molecules such as SH3, ICAM-1, and integrin-β1 that have been noted in the in vitro expansion of MSCs.[33] This loss of homing ability, combined with the significant entrapment of MSCs into the lungs and liver, make IV infusion of MSCs peri-infarct a suboptimal option.

Conversely, Bensidhoum et al.[34] showed MSCs expressing the Stro-1+ antigen had a six-fold higher engraftment rate into muscles and lower-lung entrapment compared with Stro-1- MSC when injected IV. This raises the possibility that selected Stro-1+ MSCs could engraft successfully when infused IV.

Most studies reviewed in this paper delivered MSCs to infarcted myocardium via intracoronary or intramyocardial injection. Delivery into the LV cavity resulted in increased uptake of stem cells into the myocardium and significantly lower entrapment of donor cells in the lungs compared with IV injection (0.9?0.32 vs. 0.2?0.02) measured using 99mTc labeled cells.[20] Although a greater proportion of stem cells are directly delivered to the infarcted region, these modalities are technically difficult and carry the risk of any invasive procedure, including infection, perforation, or bleeding. Interventricular delivery could injure the myocardium, inducing ventricular arrhythmias.

Despite these risks, intracoronary or intramyocardial delivery of MSCs peri-infarct offers greater homing and seeding potential compared with IV infusion. Therefore, in acute coronary syndromes when cardiac catheterization is being considered, intracoronary or intramyocardial injection would theoretically provide greater benefit than IV infusion. In chronic coronary ischemia, the less invasive route of IV injection could be considered first. However, to the extent that homing signals emanating from the heart may be absent in the chronic scar, direct injections could be invaluable.

Factors That Simulate MSC Differentiation

In vitro studies showed BM stem cells cocultured with rat cardiomyocytes expressed cardiac-specific proteins. However, stem cells did not express transcription factors for cardiac proteins when cultured in media alone. These observations support the importance of cell-to-cell signals and other environmental factors in promoting stem cell migration and differentiation.

Ischemia and hypoxia may release cytokines and promote expression of adhesion molecules that facilitate the migration of stem cells to infarcted myocardium. For example, matrix metalloproteinase-9 is elevated in the postinfarcted myocardium. Matrix metalloproteinase-9 upregulates soluble kit, which facilitates stem cell mobilization from BM.[35]

Stromal cell-derived factor and chemokines of the CC, CXC, and CX(3)C classes stimulate transmigration of BM-derived stem cells across the endothelium. Interestingly, the migratory response to stromal cellderived factor-1 and VEGF was significantly reduced in BM stem cells derived from patients with ischemic cardiomyopathy compared with those from healthy controls. This implies that dysfunction of BM stem cells in patients with ischemic cardiomyopathy may limit their therapeutic potential for clinical cell therapy.[36]

Granulocyte colony stimulating factor (G-CSF) also facilitates stem cell homing and differentiation. Mice treated with G-CSF and stem cell factor 5 days before and 3 days postinfarct were found to have a 250-fold increase in circulating Lin?/c-kit+ cells (including MSCs).[37] Newly formed myocardium reduced infarct size from 64% to 39%, and EF was 114% higher in treated mice.

In another study, G-CSF mobilized human BM-derived angioblasts were injected into rats postinfarction.[38] After 2 days, stem cells were found in the infarcted myocardium. After 15 weeks, the percentage of infarcted myocardium fell from 36% to 12%. Cardiac output fell 26% in treated rats vs. 48% in controls. Capillary formation was also three-fold higher in treated animals.

Krumwieh et al.[39] injected granulocyte- macrophage colony stimulating factor (GM-CSF) directly into the coronary artery serving ischemic myocardium, then injected GM-CSF through peripheral veins for another 2 weeks. Improved collateral blood flow and fewer electrocardiographic signs of ischemia were noted. Pretreatment with GM-CSF augmented endothelial progenitor cell mobilization from BM in mice with hind-limb ischemia.[40] These studies suggest that G-CSF augments stem cell recruitment to ischemic myocardium.

MSCs can facilitate vasculogenesis by increasing VEGF levels. After MSCs are intramyocardially injected into the infarct zone, local VEGF levels rise, vascular density and regional blood flow increases, and contractility improves.[41]

In summary, stimuli that mobilize stem cells from BM, chemokines that facilitate transendothelial migration of stem cells, and the cell-to-cell signals and local growth factors which lead to differentiation of MSCs require more detailed analysis.

Cell Fusion?

The data reviewed above support the idea of MSC trans-differentiation. Several studies have shown that these differentiated stem cells were euploid. However, other investigations focused on the role of cell fusion or the presence of additional primitive progenitors copurifying with MSCs in stem cell differentiation. Terada et al.[42] demonstrated that when female BM cells were cocultured with male embryonic cells contained polyploid DNA content (4n- 6n). Polymerase chain reaction analysis revealed a hybrid genotype of BM cells and embryonic cells. However, the frequency of spontaneous fusion was very low (2?11 clones/106 BM cells). In addition, tetraploid hybrid cells were observed when mouse brain cells were cocultured with embryonic stem cells.[43] The frequency of hybrid isolation was 10?4 to 10?5 per brain cell. In conclusion, these studies suggest that cell fusion is not a frequent event.

Methods of Detecting MSC Engraftment

Potential mechanisms of detecting stem cell engraftment in vivo include echocardiography, SPECT, MRI, and positron emission tomography scanning.

Recent studies have shown that SPECT successfully localizes MSC distribution and documents total reversible defect and global LV function in patients injected with MSCs.[44] Similarly, positron emission tomography has been used to document changes in myocardial flow after stem cell recruitment in the postinfarct baboon model.

MRI also localizes MSCs in the swine infarct zone.[45] MRI has the ability to achieve higher image resolution than echocardiography, positron emission tomography, or SPECT. Furthermore, MRI-guided catheter injection of stem cells was successful in the swine postinfarct model.[46]

Future Research

Although recent work shows great potential for MSCs in cardiomyogenesis, many questions remain to be answered. Future work should focus on any deleterious long-term effects of MSC transplantation postinfarction. For example, embryonic stem-cell derived cardiomyocytes demonstrated spontaneous activity, prolonged action potential duration, and easily inducible triggered arrhythmias in vitro.[47] However, animal and human studies with MSCs did not show increased arrhythmias in postinfarct transplanted models when subjects were followed for less than 6 months. Future studies should assess the long-term arrhythmic potential of stem cell-derived cardiomyocytes. The potential of stem cell-derived cardiomyocytes to promote tumorigenesis should also be assessed.

Many issues regarding the use of MSCs must be clarified. Future work is needed to define the exact subpopulation of cells that induce cardiomyogenesis and vasculogenesis. Determination of the optimal number of stem cells that must be injected and the optimal time course of delivery to maximize recovery of cardiac function postinfarct is needed. Safe, reproducible, and efficient techniques to deliver MSCs should be developed. Enhancement of imaging modalities used to detect stem cell engraftment would be greatly beneficial. In addition, detailed identification of cell-to-cell signals and other local factors that promote trans-differentiation will enhance our understanding of the mechanisms leading to differentiation. Long-term studies assessing the deleterious consequences of stem cell infusion and randomized clinical control trials evaluating the ability of MSCs to improve morbidity and mortality are also needed.

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Funding Information

This work was supported by grants from The Johns Hopkins University School of Medicine Institute for Cell Engineering (ICE), The Donald W. Reynolds Foundation, NIH R21 HL-72185 (to JMH), and Osiris Therapeutics, Baltimore, MD.

Reprint Address

Joshua M. Hare, MD, Johns Hopkins Medical Institutions and Institute for Cell Engineering (ICE), Cardiology Division, 720 Rutland Avenue, Ross 1059, Baltimore, MD 21205 E-mail: jhare@mail.jhmi.edu
Rinky Bhatia, MD, and Joshua M. Hare, MD. Department of Medicine, Division of Cardiology and Institute for Cell Engineering (ICE), Johns Hopkins University School of Medicine, Baltimore, MD