Author + information
- Received March 9, 2014
- Revision received May 26, 2014
- Accepted June 30, 2014
- Published online October 21, 2014.
- Sung-Whan Kim, PhD∗,†,‡,§,‖,
- Mackenzie Houge, BS∗,
- Milton Brown, BS∗,¶,
- Michael E. Davis, PhD∗,¶ and
- Young-sup Yoon, MD, PhD∗,¶∗ ()
- ∗Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia
- †Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Incheon, South Korea
- ‡International St. Mary’s Hospital, Incheon, South Korea
- §Department of Anatomy and Cell Biology and Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan, South Korea
- ‖Department of Cardiology, The Fourth Hospital of Harbin Medical University, Harbin, China
- ¶Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, Georgia
- ↵∗Reprint requests and correspondence:
Dr. Young-sup Yoon, Department of Medicine, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Drive, WMRB 3309, Atlanta, Georgia 30322.
Background Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31+ cells are a promising cell source owing to their high angiovasculogenic and paracrine activities.
Objectives This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31+ cells, and to determine whether these cultured CD31+ cells are effective for cardiac and vascular repair.
Methods CD31+ cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms.
Results The CD31+ cells cultured in endothelial cell medium (EC-CD31+ cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31+ cells. When implanted into mouse MI or HLI models, EC-CD31+ cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31+ cells. Histologically, injected EC-CD31+ cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year.
Conclusions Short-term cultured EC-CD31+ cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31+ cells may be a promising source for treating ischemic cardiovascular diseases.
Cell therapy has emerged as a promising new strategy for regenerating damaged ischemic tissue. Experimental studies and pilot clinical trials with various bone marrow (BM) cells, BM-mononuclear cells (MNCs), early endothelial progenitor cells (EPCs), or mesenchymal stem cells (MSCs) have shown favorable effects on cardiac repair after myocardial infarction (MI) (1,2). Mechanistically, paracrine actions are now known to be the main mechanism underlying ischemic tissue repair (3–6).
Recent meta-analyses of clinical trials for cardiac cell therapy with BM cells showed that left ventricular ejection fraction improved only ∼4% (7). Interestingly, selected populations such as CD34+ and CD133+ (also known as prominin-1) cells did not show significant therapeutic advantages over controls; rather, BM-MNCs and EPCs were more effective than controls. These results are not surprising given that paracrine (rather than transdifferentiation) effects are the main mechanism for BM cell therapy, and further suggest that selection of stem or progenitor cells may not be necessary when using BM-derived cells (5,6). We recently reported that BM-derived or peripheral blood-derived MNCs that express CD31 (also known as platelet endothelial cell adhesion molecule-1) on the surface are a specific cell population enriched with angiovasculogenic properties (8,9). Although they include a small stem cell population (<2%), the majority of CD31+ cells are lineage-committed and constitute ∼25% of total MNCs. We found that these cells are more effective than BM-MNCs or BM-CD31- cells for repairing limb ischemia.
However, collective data have shown that there is still much room for improvement in therapeutic efficacy. Specifically, low cell retention in vivo is a major limiting factor for cardiac cell therapy (10), and vessel-forming capability also needs improvement. Moreover, despite its importance, the need to reduce inflammation is relatively underestimated and thus underdeveloped (11).
Accordingly, this study was designed to improve the function of newly identified CD31+ cells by cell culture. Specifically, we sought to find culture conditions to induce higher adhesive, angiogenic, and vasculogenic, but lower inflammatory, activities. We also aimed to determine the therapeutic capability of the cultured CD31+ cells in the treatment of ischemic heart and vascular disease. In addition to the well-known paracrine or humoral effects of the cells, we also addressed important and long-debated mechanistic issues: endothelial transdifferentiation and long-term fate of the implanted BM cells in tissues (12,13). The present study demonstrated that CD31+ cells cultured under specific endothelial cell media exhibited the augmented cell biological characteristics mentioned in the preceding text and are effective for repairing experimental MI and limb ischemia.
An expanded Methods section is available in the Online Appendix.
Isolation and cultivation of CD31+ cells
Fresh human BM samples were purchased from Lonza (Walkersville, Maryland). Isolation of CD31+ cells was performed using magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) with a CD31 antibody, as we previously described (9). CD31+ cells were plated at a density of 1 × 106 cells/cm2 on plates in EBM-2 basal medium supplemented with EGM-2 SingleQuots (Lonza) and 15% fetal bovine serum (FBS) for the endothelial cell (EC) culture conditions. In the MSC culture conditions (MC), cells were grown in Dulbecco’s Modified Eagle’s Medium with low glucose containing 15% FBS. In the hematopoietic stem cell culture conditions (HC), cells were grown in Iscove’s Modified Dulbecco’s Medium supplemented with 10% FBS, stem cell factor (50 ng/ml), thrombopoietin (20 ng/ml), and Fms-related tyrosine kinase 3 ligand (20 ng/ml). All of the subsequent assays were performed with 10-day cultured CD31+ cells.
Cell adhesion assay and Matrigel network formation assay
See the Online Appendix.
Flow cytometry analysis
See the Online Appendix.
Quantitative RT-PCR (qRT-PCR) assay
See the Online Appendix.
Cell transplantation in myocardial infarction and hindlimb ischemia models
Mice were sacrificed 2, 3 or 4 weeks or 1 year after cell transplantation. Hearts and adductor muscles were harvested and fixed for 4 h in 4% paraformaldehyde and incubated overnight in 15% sucrose solution. The tissues were embedded in OCT compound (Sakura Finetek USA, Torrance, California), snap-frozen in liquid nitrogen, and sectioned at 10 - 20 mm thickness as described previously by our laboratory. For quantification of capillary density, 4 randomly selected frozen sections of the peri-infarct border area and ischemic tissue from the adductor muscles from each group were stained with primary biotinylated ILB4 (1:250, Vector Laboratory Inc., Burlingame, California), secondary streptavidin Alexafluor 488 (1:400, Invitrogen, Carlsbad, California) and Cy2 (1:400, Jackson ImmunoResearch, West Grove, Pennsylvania). Five fields from 4 tissue sections were randomly selected, and the number of capillaries was counted in each field. To identify cardiomyocytes, anti-α sarcomeric actinin antibody (Sigma) was used. Ki-67 expression was evaluated with a rabbit monoclonal anti-Ki-67 antibody (Thermo scientific, Hudson, New Hampshire) as described previously (1). TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed using a fluorescein in situ cell death detection kit (Roche Molecular Biochemical, Indianapolis) as described previously (1). To identify human male CD31+ cells injected into female recipients, fluorescence in situ hybridization (FISH) was performed with a Cy3-conjugated (Cambio, Cambridge, United Kingdom) human Y chromosome probe (3). 4',6-diamidino-2-phenylindole (DAPI) was used to identify nuclei. The fibrosis area was examined by the computer software Image-Pro Plus after Masson's trichrome staining. Pictures were taken using fluorescence inverted microscopy or confocal microscopy.
Flow cytometry analysis of digested tissues
To identify the engraftment and endothelial transdifferentiation of tissue-injected CD31+ Cells, we performed flow cytometric analysis as described previously (2,3). Animals were systemically perfused with Alexa Fluor 647-conjugated ILB4 (50 μg for 10 min, Molecular Probes) before sacrifice. In brief, Dil-labeled CD31+ cells were injected into heart or ischemic hindlimb of nude mice. At predetermined time points, the ischemic hindlimb or heart tissues were harvested, minced, and then digested at 37°C for 45–60 min with an enzyme cocktail (Collagenase A, Elastase, and DNase I, Roche Applied Science, Indianapolis, Indiana). Single cell suspensions were prepared by filtering through a 30-μm strainer. Labeled cell populations were measured by a LSRII flow cytometer (BD Bioscience). Flow cytometry data were analyzed with FlowJo software (Tree Star, Ashland, Oregon). Flow cytometric analysis was performed using a variety of controls including unstained samples and isotype antibodies.
All data are shown as mean ± SEM. Statistical analyses were performed with Student t test for comparisons between two groups and ANOVA with the Turkey test for multiple group comparisons by using SPSS version 11.0. p < 0.05 was considered to denote statistical significance. The incidence of limb loss was analyzed using the chi-square test. Where heterogeneity of variance was identified, results were confirmed with the non-parametric Mann-Whitney U test.
Cell biological characteristics of CD31+ cells cultured under various conditions
To investigate the adhesive, vasculogenic, and proliferative capacity of CD31+ cells, we isolated CD31+ cells from human BM-MNCs using MACS. The purity of CD31+ cells was 97.6 ± 1.2%. First, we compared the expansion profile of CD31+ cells. The same number of CD31+ cells was cultivated under 3 culture conditions, MC, EC, and HC. After 7 days of culture, nonadherent cells were removed in MC and EC. The adherent cells showed heterogeneous morphology, including round and spindle-shaped cells (Figure 1A). MC-CD31+ and EC-CD31+ cells grew quickly from day 7 to 25, showing higher proliferation properties than the HC-CD31+ cells (Figure 1B). CD31+ cells in EC (EC-CD31+) for 10 days expressed endothelial cell proteins von Willebrand factor (VWF), kinase insert domain receptor (KDR), CDH5 (VE-cadherin), and CD31, but MC-CD31+ and HC-CD31+ did not express CDH5 or VWF by immunocytochemistry (Figure 1C).
As adhesion capacity is an important indicator of cell engraftment and survival in vivo, we performed an adhesion assay. The number of cells adherent to extracellular matrix proteins fibronectin, vitronectin, collagen I, and laminin was significantly higher in the MC and EC groups than the uncultured CD31+ cells (UC) (all p < 0.01) (Figure 1D). We performed Matrigel tube formation assays with cultured or uncultured CD31+ cells alone or cocultured with human umbilical vein endothelial cells (HUVECs). Without coculture with HUVECs, only EC-CD31+ cells showed meaningful tube formation. With coculture with HUVECs, all cultured CD31+ cells showed higher branching points and tube lengths compared with the UC (Figures 1E and 1F). When HUVECs were cultured alone on Matrigel under these conditions, the HUVECS cultured under EC conditions (EC-HUVECs) exhibited higher branching points and tube lengths compared with the HC-HUVECs and MC-HUVECs (Figure 1G). These data indicate that CD31+ cells cultured in EC have the highest adhesion and independent tube-forming capability.
Surface protein expression of cultured and uncultured CD31+ cells
To characterize cultured CD31+ cells, we performed flow cytometry analyses. Regardless of cell culture, CD31+ cells maintained expression of the pan-hematopoietic marker PTPRC (protein tyrosine phosphatase, receptor type, C, also known as CD45) as well as CD31 (Figure 2). Cells expressing lineage-committed markers CD3 (T cell) or CD19 (B cell) were decreased during culture. Cells exhibiting stem cell markers CD34, PROM1 (CD133), and KIT (proto-oncogene c-Kit, or CD117) were maintained in HC but generally decreased in the other conditions. Under EC, the cell populations expressing hematopoietic-endothelial markers KDR and TEK (tyrosine kinase, endothelial, or TIE2), monocyte-macrophage markers ITGAM (integrin alpha M, or CD11b) and CD14, and a dendritic cell marker, THBD (thrombomodulin, or CD141), were significantly increased, whereas those expressing ENG (endoglin, or CD105) and MCAM (melanoma cell adhesion molecule, or CD146) were not significantly changed. Although MC cells showed similar trends to EC with less prominent increases in TEK, KDR, THBD, ITGAM, and CD14, HC cells did not change or reduced their expression. These data suggest that CD31+ cells cultured under 3 different conditions have distinct characteristics and the EC conditions in general induced the highest endothelial-monocytic phenotype shift of CD31+ cells.
Expression of angiogenic, integrin, and inflammatory genes
To measure mRNA expression, we conducted qRT-PCR. Expression of angiogenic factors such as basic fibroblast growth factor (FGF2), hepatocyte growth factor (HGF), platelet-derived growth factor beta (PDGFB), placental growth factor (PlGF), and matrix metallopeptidase (MMP)-9 were significantly increased in all culture conditions compared with the UC (Figure 3A). Of note, chemokine (C-C motif) ligand 2 (CCL2) and interleukin (IL)-8, which play important roles in arteriogenesis, were also increased in all conditions (Figure 3A). Expression of cell survival factors insulin-like growth factor (IGF)-1 and Ak strain transforming oncogene (AKT1) were generally increased after culture. We also measured expression of integrin beta-1 (ITGB1), integrin beta-2 (ITGB2), integrin alpha-5 (ITGA5), and integrin alpha-6 (ITGA6), as these molecules mediate cell-to-cell and cell-to-extracellular matrix interactions, regulating adhesion and angiogenesis (15,16). These integrin levels were increased in all culture conditions and were particularly higher in EC compared with UC (Figure 3B). Expression of representative inflammatory genes interleukin-1 beta (IL1B), IL6, and interferon gamma (IFNG) was decreased in all conditions compared with UC (Figure 3C). However, anti-inflammatory factors IL10 (17), transforming growth factor beta-1 (TGFB1) (18), interleukin 1 receptor, type 1 (IL1R1), tumor necrosis factor receptor 1A (TNFRSF1A), and leukemia inhibitory factor (LIF) were 3-fold to 30-fold increased in the cultured CD31+ cells, notably in EC (Figure 3D). Collectively, these findings indicate that cultured CD31+ cells, particularly in EC, are enriched with vessel-formation, cell-survival, and anti-inflammatory factors.
Favorable effects of cultured CD31+ cells on repair of cardiac and hindlimb ischemia
We next investigated the therapeutic effects of cultured CD31+ cells on infarct repair. On the basis of the in vitro data, we selected EC-CD31+ cells cultured for 10 days and compared the effects to UC-CD31+ cells and phosphate-buffered saline (PBS). After induction of MI in nude mice, we injected 1 × 106 cells or the same volume of PBS directly into the peri-infarct area. Echocardiography at 3 weeks after cell transplantation demonstrated that left ventricular end-diastolic dimension and left ventricular end-systolic dimension were the lowest in the EC group, and left ventricular fractional shortening was the highest in the EC group followed by the UC and PBS groups (Figures 4A and 4B). Compared with the PBS group, both UC and EC groups showed significantly lower circumferential fibrosis area examined at 4 weeks, with the smallest in the EC group (Figures 4C and 4D). These results indicate that intracardiac implantation of uncultured or cultured CD31+ cells can attenuate cardiac dysfunction and remodeling, with the EC-CD31+ cells having higher potency.
We next evaluated the therapeutic potential of cultured CD31+ cells in HLI. After creating HLI in nude mice, we injected 10-day cultured EC-CD31+ cells, MC-CD31+ cells, or HC-CD31+ cells, UC-CD31+ cells, or PBS into the ischemic hindlimbs (1 × 106 cells per mouse). Laser Doppler perfusion imaging was performed weekly to monitor the ischemic hindlimb’s blood flow. Laser Doppler perfusion imaging analysis revealed a greater recovery of blood perfusion in the EC group compared with each of the other 4 groups (Online Figures 1A and 1B). Although limb loss was frequently observed in the PBS control group, the EC group showed a significantly lower limb loss score than the PBS, UC, or MC groups (Online Figures 1C and 1D). Capillary density in the hindlimb muscle was significantly higher in the EC group compared with that of the PBS, UC, and MC groups (p < 0.01 vs. PBS; p < 0.05 vs. UC or MC) (Online Figure 2A). These results suggest that among all the groups, EC-CD31+ cells have better therapeutic potential for ischemic limb recovery.
Neovascularization, cardiac proliferative and protective effects, and anti-inflammatory effects of cultured CD31+ cells
In the MI model, the capillary density at the peri-infarct area at 4 weeks was also highest in the EC group, followed by the UC and PBS groups (Figure 5A). We determined cardiomyocyte proliferation and apoptosis with cardiac samples harvested at 2 weeks using double staining for Ki67 and sarcomeric α-actinin or double staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and sarcomeric α-actinin, respectively. The EC group showed significantly higher numbers of Ki67+ cardiomyocytes compared with the UC and PBS groups (Figure 5B). Apoptotic cardiomyocytes at the peri-infarct area measured by TUNEL assay were lowest in the EC group, followed by the UC and PBS groups (Figure 5C). These results suggest that EC-CD31+ cells have robust cardiac proliferative and protective effects.
To determine other humoral effects of cell transplantation on ischemic hearts, MI mice were sacrificed and cardiac tissues were collected at 1 week. The expression levels of vascular endothelial growth factor A (VEGFA), angiopoietin-1 (ANGPT1), PDGFB, IGF1, and CD31 were significantly increased in the EC group compared with the PBS or UC groups (Figure 5D). FGF2 was the only factor more highly expressed in the UC group compared with the EC or PBS groups. Compared with the other groups, the expression of anti-inflammatory factors IL10 and TGFB1 was significantly higher and that of the proinflammatory factors IL1B and tumor necrosis factor (TNF) was lower in the EC group (Figures 5E and 5F). These data indicate that direct cardiac injection of EC-CD31+ cells augmented multiple biological factors associated with vascularization and anti-inflammation and reduced proinflammatory factors.
Cultured CD31+ cells showed higher engraftment and endothelial transdifferentiation potential
We determined engraftment and transdifferentiation potential in tissue samples harvested from the heart and the limb muscles at 4 weeks. Histologic analyses demonstrated that injected 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled EC-CD31+ cells or UC-CD31+ cells were engrafted in both tissues and mainly localized in the pericytic or perivascular areas of the hearts (Figure 6A) and limb muscles (Online Figure 3A). We also found that a portion of CD31+ cells was incorporated into vessels and exhibited endothelial marker isolectin B4 (ILB4) (Figure 6B, Online Figure 3B). By histomorphometric analyses, the numbers of engrafted cells (DiI+ cells) and endothelially transdifferentiated cells (DiI+, ILB4+ cells) were significantly higher in the EC group compared with the UC group (Figure 6B, Online Figure 3B, right panels). To more precisely quantify the rate of engraftment and transdifferentiation of CD31+ cells, we conducted flow cytometric analysis on enzymatically digested hearts after systemic injection of ILB4 as described (8). This analysis again demonstrated that the EC group exhibited ∼2-fold higher engraftment and ∼3-fold higher endothelial transdifferentiation rates compared with the UC group (Figures 6C and 6D). Similar results were found in the histologic and flow cytometric analyses of the HLI model (Online Figures 3C and 3D). Collectively, these findings suggest that, compared with uncultured CD31+ cells, EC-CD31+ cells have superior potency for engraftment and endothelial transdifferentiation.
Long-term engraftment and endothelial transdifferentiation potential of CD31+ cells
We further quantified the long-term engraftment and endothelial transdifferentiation of cultured CD31+ cells using tissues harvested 1 year after cell transplantation. Contrary to the prevailing notion that injected BM cells in tissues survive only for a short term, histological analyses of the heart and hindlimb muscles showed engrafted CD31+ cells, as well as those expressing ILB4, within the vascular structure (Figure 7A, Online Figure 4A). Quantitative analyses with flow cytometry after perfusion of ILB4 and enzymatic digestion of heart and hindlimb muscle revealed an ∼0.6% engraftment rate (DiI+ cells per total cells) (Figure 7B) and ∼2.1% endothelially transdifferentiated cells (DiI+ILB4+ cells) among total endothelial cells in heart tissues (Figure 7C), and ∼0.4% engrafted cells (Online Figure 4B) and ∼3.7% endothelially transdifferentiated cells in hindlimb muscle (Online Figure 4C). We also confirmed these results with fluorescence in situ hybridization. Y chromosome signals derived from human CD31+ cells were detected in nuclei of endothelial cells as well as in other myocardial cells (Online Figures 5A and 5B). These findings confirmed the long-term survival and endothelial cell transdifferentiation capabilities of EC-CD31+ cells in vivo.
To improve cardiovascular cell therapy, continuous efforts have been made to identify more potent cells and to improve their function. This study demonstrated that specific culture conditions enhance the tissue reparative function of BM-derived hematopoietic CD31+ cells. In modified endothelial culture conditions, CD31+ cells expressed the highest levels of adhesion, pro-angiogenic, and anti-inflammatory factors, and the lowest levels of inflammatory factors compared with uncultured CD31+ cells or CD31+ cells that were cultured under other conditions. These EC-CD31+ cells, when implanted in vivo, showed robust neovascularization and cell retention, and induced cardiac and ischemic limb repair to a greater extent than uncultured CD31+ cells. Of note, mice receiving EC-CD31+ cells after MI had a significantly higher 7-month survival rate, implying additional clinical benefits of EC-CD31+ cell therapy for heart failure. Although low, cell survival and endothelial transdifferentiation capacity was confirmed for up to a year. This is the first report to demonstrate such long-term engraftment of any injected BM cells and maintenance of the endothelially transdifferentiated phenotype.
The original goals of culturing CD31+ cells were to enhance their adhesion and angiogenic-paracrine capacities. As expected, cultures in the EC conditions augmented both activities in CD31+ cells. The most notably increased factors were integrins, which are cell adhesion receptors that interact with extracellular matrices (19) and play a critical role in cell adhesion, cell survival, endothelial cell migration and proliferation, and matrix metalloproteinase activation. Integrins also interact with receptors for 2 main angiogenic factors, VEGF and FGF, to facilitate angiogenesis (16,20). Under EC conditions, multiple angiogenic, arteriogenic, antiapoptotic, and chemoattractant factors essential for vessel formation, cell survival, and tissue repair were significantly increased. These higher cell adhesion and angiogenic activities in the EC-CD31+ cells synergistically functioned to enhance cell survival, neovascularization, and tissue regeneration in vivo. They further increased cardiomyocyte proliferation and reduced cardiomyocyte apoptosis. It is also likely that the enriched paracrine factors, such as IGF1 and HGF, could have activated resident cardiac stem cells to induce myocardial regeneration (21,22).
To our surprise, key proinflammatory cytokines were suppressed and anti-inflammatory cytokines were increased in the EC-CD31+ cells. These anti-inflammatory properties are beneficial for acute cardiovascular tissue repair. After MI, inflammatory cells infiltrate the ischemic heart tissue and secrete inflammatory cytokines (23). Excessive or prolonged inflammation in MI leads to cardiac dysfunction. Studies also reported that unselected BM cell transplantation caused calcification in hearts or ulceration in limbs, which are associated with proinflammatory activities of the injected cells (24,25). The EC-CD31+ cells highly expressed anti-inflammatory factors IL10 and TGFB1. IL10 is a well-known anti-inflammatory cytokine, a representative suppressor of proinflammatory mediators, and has auspicious effects on cardiac repair (17). TGFB1 is another major regulator controlling inflammation (18). Anti-inflammation factors, such as IL1R1, TNFRSF1A, and LIF, were also more highly expressed compared with uncultured CD31+ cells (26,27).
We also observed improved cell engraftment in the cultured CD31+ cells. At 4 weeks, there were twice as many surviving EC-CD31+ cells as uncultured CD31+ cells in both the MI and HLI models. Given our prior studies showing 4.5 times higher survival of uncultured CD31+ cells compared with uncultured CD31- cells (8,9), EC-CD31+ cells have more than 9 times higher cell engraftment potential than the uncultured CD31- cells that comprise 75% of the most commonly used MNCs for cell therapy. Another important observation is the endothelial transdifferentiation or vasculogenic potential of EC-CD31+ cells. Although the transdifferentiation potential of BM-derived cells has been under debate, this study showed that EC-CD31+ cells retained even higher endothelial transdifferentiation potential than uncultured CD31+ cells (8,9). Compared with uncultured CD31+ cells, EC-CD31+ cells had a 3-fold (MI model) to 4-fold (HLI model) higher transdifferentiation rate at 1 month. The hindlimb muscle showed a substantially higher rate of transdifferentiated ECs than myocardium (8% vs. 1.5%). These engraftment and transdifferentiation analyses were extended up to a year. To date, no studies have addressed the long-term fate of direct tissue-injected BM cells in vivo. Histologic examination of >1,000 sections and flow cytometry analyses demonstrated durable survival and endothelial transdifferentiation of EC-CD31+ cells in both heart and limb muscle at 1 year.
Although EC-CD31+ cells were the most effective for ischemic limb recovery, CD31+ cells cultured under other conditions also were more effective than control cells. Compared with uncultured CD31+ cells, all cultured CD31+ cells displayed increased angiogenic and decreased inflammatory properties. However, EC-CD31+ or HC-CD31+ cells have higher proangiogenic factors and activities than MC-CD31+ cells. The proinflammatory and anti-inflammatory profiles are similar between HC-CD31+ cells and MC-CD31+ cells, but are more favorable in EC-CD31+ cells. However, it remains to be determined whether these cultured cells have different effects on the repair of acute MI according to the culture conditions.
Although meta-analyses demonstrated relatively small effects in improvement of ejection fraction overall for BM cell therapy (7), recent clinical trials with MSCs for ischemic cardiomyopathy demonstrated various beneficial effects, including enhanced heart failure score and reduction of scar size and cardiac volume indices (28,29). The current study also attempted long-term follow-up of cardiac function for a year, but all animals in the UC and PBS groups died by 7 months, whereas those treated with the EC-CD31+ cell group all survived. Together, these studies suggested that adult cell therapy remains a viable therapeutic option, and various parameters other than left ventricular ejection fraction are needed to appropriately address the advantages of clinical cell therapy.
We demonstrated for the first time that culture-expanded CD31+ cells induced enhanced adhesion, vessel formation, cardiomyocyte proliferation, and anti-inflammatory effects, and are effective for repairing cardiovascular damage (Central Illustration). These cells may be a novel and advanced therapeutic option for treating ischemic cardiovascular disease.
COMPETENCY IN MEDICAL KNOWLEDGE: Cell therapy holds promise as a treatment strategy to regenerate damaged ischemic tissue, but low engraftment of administered cells and modest therapeutic effects are important limitations. Short-term cultured bone marrow–derived CD31+ cells yielded higher cell engraftment, vessel formation, cardiomyocyte proliferation, and anti-inflammatory effects in mouse models of ischemic cardiac and vascular diseases.
TRANSLATIONAL OUTLOOK: The clinical efficacy and safety of bone marrow–derived CD31+ cells for tissue salvage and recovery in patients with ischemic limbs and for restoration of ventricular function and survival after myocardial infarction should be investigated in future studies.
For supplemental tables, figures, and an expanded Methods section, please see the online version of this article.
This work was supported in part by NIH grants DP3DK094346, UL1 RR025008, and NIH contract HHSN268201000043C; by NSF-EBICS grant, CBET-0939511 to Dr. Yoon; and by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MOE) (No. NRF-2013R1A1A2059998), and Korean government (MSIP, 2013 041811) to Dr. Kim. The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- bone marrow
- 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
- endothelial cell culture conditions
- endothelial progenitor cell
- fetal bovine serum
- hematopoietic stem cell culture conditions
- hindlimb ischemia
- human umbilical vein endothelial cell
- magnetic activated cell sorting
- MSC culture conditions
- myocardial infarction
- mononuclear cell
- mesenchymal stem cell
- phosphate-buffered saline
- uncultured cells
- von Willebrand factor
- Received March 9, 2014.
- Revision received May 26, 2014.
- Accepted June 30, 2014.
- American College of Cardiology Foundation
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