S1P–S1PR2 Axis Mediates Homing of Muse Cells Into Damaged Heart for Long-Lasting Tissue Repair and Functional Recovery After Acute Myocardial InfarctionNovelty and Significance
Rationale: Multilineage-differentiating stress enduring (Muse) cells, pluripotent marker stage-specific embryonic antigen-3+ cells, are nontumorigenic endogenous pluripotent-like stem cells obtainable from various tissues including the bone marrow. Their therapeutic efficiency has not been validated in acute myocardial infarction.
Objective: The main objective of this study is to clarify the efficiency of intravenously infused rabbit autograft, allograft, and xenograft (human) bone marrow-Muse cells in a rabbit acute myocardial infarction model and their mechanisms of tissue repair.
Methods and Results: In vivo dynamics of Nano-lantern–labeled Muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected GFP (green fluorescent protein)-Muse cells estimated to be engrafted into the heart at 3 days. The migration and homing of the Muse cells was confirmed pharmacologically (S1PR2 [sphingosine monophosphate receptor 2]–specific antagonist JTE-013 coinjection) and genetically (S1PR2-siRNA [small interfering ribonucleic acid]–introduced Muse cells) to be mediated through the S1P (sphingosine monophosphate)–S1PR2 axis. They spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-I, sarcomeric α-actinin, and connexin-43, and vascular markers. GCaMP3 (GFP-based Ca calmodulin probe)-labeled Muse cells that engrafted into the ischemic region exhibited increased GCaMP3 fluorescence during systole and decreased fluorescence during diastole. Infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. These effects were partially attenuated by the administration of GATA4-gene–silenced Muse cells. Muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression.
Conclusions: Muse cells may provide reparative effects and robust functional recovery and may, thus, provide a novel strategy for the treatment of acute myocardial infarction.
Acute myocardial infarction (AMI) is a common cause of morbidity and mortality worldwide.1 Severe ischemia leads to extensive tissue damage and heart failure because of functional cardiomyocyte loss. Stem/progenitor cell therapy to replenish cardiomyocytes is a potential treatment, and many stem cell types have been studied intensively. Clinical trials have demonstrated the safety of treatment with bone marrow (BM)-mesenchymal stem cells (MSCs) and BM-mononucleated cells. Although their efficacy is under scrutiny, some of the trials produced clinically beneficial results.2,3
Editorial, see p 1036
Meet the First Author, see p 1034
Multilineage-differentiating stress enduring (Muse) cells are collectable as cells positive for the pluripotent surface marker stage-specific embryonic antigen-3 (SSEA-3).4–7 They locate in the BM (≈0.03% of the mononucleated fraction), adipose tissue, dermis, and connective tissue of various organs and circulate in the peripheral blood.4,5,8–10 Approximately 30 mL of fresh human BM aspirate yields ≈0.15 million Muse cells, which reach ≈1 million after 3 days in culture, suggesting their practical use.5,9
Muse cells are stress tolerant and secrete prosurvival factors that play a key role in regulating the cell response to DNA damage after internal or external injury and reduce cellular stress and subsequent apoptosis.4,9 They express pluripotency markers such as Sox2, Oct3/4, and Nanog and are able to differentiate into cells of all 3 germ layers from a single cell. Notably, these properties are recognized in Muse cells directly collected from BM aspirates, indicating that their characteristics are not newly acquired by in vitro manipulation nor are they modified under culture conditions.4
Muse cells are reported to repair skin ulcers of animal models of diabetes mellitus by replenishing new dermal and epidermal cells and in stroke models by replenishing new neuronal cells that extend neurites and incorporate into the pyramidal tract and sensory circuit to deliver robust functional recovery when locally transfused.8,11,12 By intravenous injection, they selectively home to damaged tissue and spontaneously differentiate into new tissue-specific cells and repair tissues in various animal models of liver, muscle, kidney, and skin injury.4,8,13–15
The therapeutic efficiency of Muse cells, however, has not yet been validated in AMI. Using a rabbit AMI model, we clarified the therapeutic efficiency of Muse cells and the mechanisms by which they migrate to the damaged site and repair the AMI tissue.
The authors declare that all data that support the findings of this study are available within the article and its Online Data Supplement.
Preparation of Muse Cells
Male Japanese white rabbits (2.0–2.5 kg) were used. Rabbit BM-MSCs cultured from BM aspirate of each animal were transfected with lentivirus-GFP (green fluorescent protein) as described previously,4 and then, SSEA-3+ fraction (named Muse cells) and SSEA-3− fraction (named non-Muse cells) cells were isolated from BM-MSCs by fluorescence-activated cell sorting (Online Figure IB).6 For human cells, human BM-MSCs (Lonza, Basel, Switzerland) were introduced with lentivirus-GFP, and Muse cells were collected as SSEA-3+ cells. The Ethics Committee of Gifu University School of Medicine, Gifu, Japan, approved the study protocol (permit number: 23–24).
AMI Model and Injection of Cells
Only healthy male Japanese white rabbits (weighing ≈2 kg) were used, and animals that appeared sick or died during the experiment were excluded from the analyses. We used only male rabbits to avoid hormonal effects. The investigators evaluating the outcomes were blinded to the protocols and treatments, and the AMI rabbits were randomly assigned by sealed envelopes to groups for the experiments. The AMI model was created as previously described.16,17 Rabbits were anesthetized, and myocardial ischemia was induced by occluding the coronary artery for 30 minutes and then reperfusing it. Twenty-four hours after coronary artery ischemia–reperfusion, the rabbits were injected into the ear vein with autograft 3×105 cells/2 mL saline (Muse cells [Muse group], non-Muse cells [non-Muse group], or BM-MSCs [MSC group]). The vehicle group was injected with 2 mL saline. Each group was followed for 2 weeks (n=10 per group) or 2 months (n=7 per group). The Muse group was also examined at 3 days (n=8). Allograft Muse cells (n=7) and human xenograft Muse cells (n=5) were injected in the same manner and followed for 2 weeks and 2 months without immunosuppression. For the 6-month experiment, allograft Muse and vehicle groups (n=5 for each group) were prepared. The effect of an S1PR2 (sphingosine monophosphate receptor 2) antagonist on the integration of allograft Muse cells after injection (n=5) was also evaluated. The physiological aspects of integrated Muse cells were evaluated by intramuscular injection of GCaMP (GFP-based Ca calmodulin probe)-labeled human Muse cells or human fibroblasts into the ischemic area and followed for 2 weeks (n=3 in each). The effect of intravenous injection of GATA4-gene–silenced Muse cells was also evaluated at 2 weeks (n=5).
Normally distributed data are presented as the mean±SE, and non-normally distributed data are presented as the median with interquartile range. The normality of the data distributions was tested using the Kolmogorov–Smirnov test. The significance of differences among groups for variables that were normally distributed was determined by ANOVA combined with Fisher method (Stat View; J5.0 software). Otherwise, a Kruskal–Wallis test was used to compare the differences among groups. Power and sample size calculations were performed to determine the sample size required to detect 10% reduction of the infarct size with an SD of 5% based on previous studies.16,17 A sample size of at least 4 subjects in each group was required to provide 80% power at an α of 0.05 to demonstrate a 10% difference.
Values of P<0.05 (*) were considered significant, and values of P<0.01 (**) and P<0.001 (***) were considered highly significant.
Additional Materials and Methods are provided in the Online Data Supplement.
Characterization of Muse Cells
Rabbit BM-MSCs contained ≈0.5% SSEA-3+ Muse cells (Online Figure I). The characteristics of the rabbit Muse cells (SSEA-3+ in BM-MSCs) were essentially the same as described previously for human Muse cells4,6,18 (Online Figure I). Cardiac-lineage differentiation was also assessed; Muse cells exhibited a higher potential for cardiac-lineage differentiation than non-Muse cells (Online Figure II).
Engraftment of Muse Cells Into AMI Heart After Infusion
On day 3, GFP-positive (GFP+) Muse cells were detected mainly in the border area and to a lesser extent in the infarct area (Figure 1A, A-1). The estimated total cell number engrafted into the heart was 43 555±11 992, corresponding to 14.5±4.0% of the total number of injected cells. Of the total engrafted GFP+ cells, 9.9±0.8% were positive for Ki67, a marker of proliferating cells at day 3 (Figure 1B). GFP+ cells with cardiomyocyte-like morphology were observed in left ventricular (LV) sections at 2 weeks in the Muse group (542±89 cells/per transverse LV sectional slice; Figure 1A, A-2), but few GFP+ cells were detected in the non-Muse group (0.9±0.2 cells per transverse LV sectional slice, P<0.001; Online Figure III). When GFP+ cardiomyocyte-like cells were plotted and compared with adjacent sections stained with hematoxylin–eosin in the Muse group, they were abundant within the myocardium but scarce in the fibrotic scar region (Figure 1A). The estimated number of GFP+ cells in the whole LV at 2 weeks was 65 140±3312 cells, and still 61 740±3990 at 2 months.
In vivo dynamics of Muse and non-Muse cells was evaluated using Nano-lantern–introduced cells.19 The signal of homing was recognized in the heart and lung, but not in other organs, at day 3, and that in the lung was higher than that in the heart in both the Muse and non-Muse groups (Figure 1C; Online Figure IV). Focusing on the heart, however, the signal was substantially higher in the Muse group than in the non-Muse group. At 2 weeks, the signal in the heart became more intense in the Muse group compared with day 3, whereas that in the non-Muse group declined to the under the detection level at this time point (Figure 1D).
Ki67 positivity among GFP+ Muse cells at 2 weeks was ≈1.5%, lower than that at 3 days (Online Figure V).
Effect of Muse Cells on Infarct Size
The blood was collected from the ear artery 24 hours after ischemia–reperfusion, and the plasma troponin-T, an indicator of the infarct size,20 was measured. All 4 groups subjected to AMI contained a substantial troponin-T level (≈4 ng/mL), and there were no statistical differences among the 4 groups (Online Figure VI).
At 2 weeks, infarct size (% infarct area to LV area) in the Muse group was drastically reduced compared with the other 3 groups, and less than half (≈44%) that in the vehicle group (Muse versus vehicle in infarct size; 14.1±1.3% versus 31.7±1.3%; P<0.001, corresponding to ≈56% reduction in infarct size; Figure 1E and 1G). This tendency continued for up to 2 months, and the infarct size in the Muse group was also drastically reduced compared with the other 3 groups, ≈49% of that of the vehicle group (Muse versus vehicle in infarct size; 15.3±1.5% versus 31.3±1.8%; P<0.001, corresponding to ≈51% reduction in infarct size), with an infarct size reduction ≈2.5-fold greater in the Muse group than in the MSC group (Figure 1F and 1H).
Marker Expression in Engrafted Autograft Muse Cells
At 2 weeks, 38.2±5.5% of the integrated GFP+ cells in the Muse group expressed ANP (atrial natriuretic peptideFigure 2A), 14.4±3.3% expressed cardiac troponin-I (Figure 2B), and 17.7±2.5% expressed sarcomeric α-actinin (Figure 2C). Connexin-43 was observed between the host and GFP+ cells (Figure 2D). At 2 months, GFP+ cells expressed lower levels of ANP (6.5±0.1%; Figure 2E) and higher levels of cardiac troponin-I (41.3±2.1%; Figure 2F) and sarcomeric α-actinin (45.7±1.0%; Figure 2G and 2H) compared with that at 2 weeks, suggesting that differentiation progressed after homing. Some of the GFP+ cells that were negative for cardiac markers were positive for vascular endothelial markers CD31 and α-smooth muscle actin at both 2 weeks (Figure 2M and 2N) and 2 months (data not shown). At 2 months, 25.4±6.1% of the GFP+ cells were positive for α-smooth muscle actin.
To investigate whether the differentiation was dependent on fusion, fluorescence in situ hybridization was performed using a 2-week AMI heart with GFP+-human Muse cell infusion. Most GFP+/sarcomeric α-actinin+ cells with striations reacted to the human fluorescence in situ hybridization probe but not to the rabbit probe in an adjacent section (Figure 2I and 2J). Only a small proportion of GFP-human Muse cells (0.33±0.06%) was positive for both the human and rabbit probes. Analysis by quantitative polymerase chain reaction of the human-specific Alu sequence at 2 weeks revealed that 7 pg of the Alu sequence was detected per nanogram of rabbit heart tissue DNA, confirming the integration of human Muse cells into the rabbit heart.
Microvessels positive for CD31 were significantly more abundant in regions of the border area in the Muse group compared with the other groups (Figure 2K and 2L), and GFP+ cells were incorporated into vessels and expressed vascular markers, suggesting that Muse cells contribute to neovascularization (Figure 2M and 2N).
Cardiac Function and LV Remodeling
Indicators of cardiac remodeling, LV end-systolic diameter, and LV end-diastolic diameter at 2 weeks were ≈23% and ≈14% lower, respectively, in the Muse group than in the vehicle group (Figure 3A and 3B). Functional parameters, that is, ejection fraction (EF), fractional shortening, and LV systolic function (+dP/dt) and LV diastolic function (−dP/dt), were substantially higher (≈28%, ≈35%, and ≈31% and ≈31%, respectively) in the Muse group than in the vehicle group (Figure 3C through 3E). These tendencies were maintained at 2 months (Figure 3F through 3J), and EF was ≈38% higher in the Muse group (61.1±2.7%) than in the vehicle group (44.2±2.2%) with a rate of increase ≈2.0-fold higher than that in the MSC group. The non-Muse and MSC groups also showed moderate recovery compared with the vehicle group in all parameters at 2 weeks and 2 months.
Allograft and Xenograft Muse Cell Infusion Without Immunosuppression
We examined the therapeutic potency of allograft and xenograft human Muse cells without immunosuppression at 2 weeks. Although the estimated number of total engrafted GFP+ Muse cells in the allograft (51 732±2526 cells) and xenograft (42 285±1275 cells) groups was lower than that in the autograft group (Figure 3K), the infarct size and EF were significantly improved in the allograft and xenograft groups compared with the vehicle group, and no significant differences were detected among autograft, allograft, and xenograft groups (Figure 3L and 3M). Particularly in the allograft group, compared with the vehicle group, infarct size corresponded to ≈51%, and EF increased ≈32%. The allografts (Online Figure VII) and xenografts (Figure 3N through 3P) also expressed cardiac markers ANP, troponin-I, and sarcomeric α-actinin.
The infarct size was significantly larger in the GATA4-gene–silenced human Muse group compared with that in the naive human Muse group, but still smaller than that in the vehicle group (Figure 3Q; Online Figure VIII). EF was significantly lower in the GATA4-gene–silenced human Muse group than in the naive Muse group, but still greater than that in the vehicle group (Figure 3R).
In Vivo Imaging of Integrated GCaMP3-Labeled Muse Cells
To distinguish GCaMP3 fluorescence in the host AMI tissue, human GCaMP3-labeled Muse cells were locally injected into the ischemic myocardium after ischemia–reperfusion. At 2 weeks, GCaMP3 fluorescence was observed in the myocardial layer of the beating heart. In the Muse cell group, the intensity of the GCaMP3 fluorescence was higher during systole and lower during diastole (Figure 4A; Online Movie I). The fluorescence signal showed 1:1 graft–host coupling during spontaneous beating (Figure 4B). GCaMP3-labeled Muse cells in the host tissue expressed troponin-I and connexin-43 (Figure 4C and 4D). The control group that received local injection of human GCaMP3-labeled fibroblasts did not exhibit the fluctuation of GCaMP3 fluorescence intensity synchronous with the heart beat in the injected area (Online Figure IX; Online Movie II).
Mechanism of Muse Cell Migration Toward the Postinfarction Heart
With respect to the high capacity of Muse cells to migrate to and engraft into damaged tissue, we confirmed in vitro the higher migration capacity of Muse cells compared with non-Muse cells toward AMI tissue. The number of rabbit/human Muse cells migrating toward the AMI heart was nearly 3 (rabbit)-fold and 8 (human)-fold higher than that of non-Muse cells (Figure 5A; Online Figure X). The number of migrated rabbit/human non-Muse cells did not differ between normal and AMI heart (Figure 5A; Online Figure X). Thus, in contrast to non-Muse cells, Muse cells exhibited potent migration toward AMI heart.
Several G-protein–coupled receptors play a key role in cell migration and homing, and the S1P–S1PR axis mediates the migration of immune cells and some stem/progenitor cells.21–24 Among 5 S1PR1~S1PR5 subtypes,22–24 expression of S1PR2 was substantially higher in Muse cells than in non-Muse cells as assessed by quantitative polymerase chain reaction and Western blot (Figure 5B and 5C). Indeed, Muse cells, but not non-Muse cells, migrated toward an S1PR2-specific agonist (SID46371153) in a dose-dependent manner, suggesting tight involvement with the S1P–S1PR2 axis (Figure 5D). Compared with normal heart tissue, the S1P level in the AMI heart was significantly greater in the border area at 6 hours (≈1.55-fold) and at 24 hours (≈1.56-fold) after AMI (Figure 5E). The infarct area exhibited the same tendency, but the degree of increase was lower than that in the border area (Figure 5E). JTE-013, an antagonist particularly selective for S1PR2,21 inhibited the migration of Muse cells, but not non-Muse cells, toward the AMI heart in a dose-dependent manner (Figure 5F; Online Figure X). When JTE-013 was coinjected, integration of Nano-lantern-Muse cells was substantially attenuated, and the signal became under the detection level at 2 weeks (Figure 5G). Consistently, the total estimated number of GFP+ Muse cells that engrafted into the whole LV at 2 weeks decreased to 3600±853, substantially lower than the number that integrated without JTE-013 (51 732±2526; Figure 5H). Compared with allografts without JTE-013, the reduction of the infarct size by allograft Muse cells coinjected with JTE-013 was lower (25.5±2.5%; Figure 5I) and recovery of EF (53.1±0.8%; Figure 5J) was substantially attenuated. Furthermore, the knockdown of S1PR2 in the Muse cells was confirmed by polymerase chain reaction and Western blot (Figure 5K and 5L). Although Muse cells without silencing migrated toward the S1PR2-specific agonist SID46371153 (Figure 5M) and AMI cardiac tissue (Figure 5N), S1PR2 gene–silenced Muse cells exhibited attenuated migration toward SID46371153 (Figure 5M) and AMI cardiac tissue (Figure 5N), suggesting that Muse cells migrate via the S1P–S1PR2 axis, which is an on-target effect. These findings support the central role of the S1P–S1PR2 axis in the specific homing of Muse cells into the postinfarct heart.
Paracrine Effect and Suppression of Fibrotic Scar Formation by Muse Cells
VEGF (vascular endothelial growth factor) and HGF (hepatocyte growth factor) production in vitro were significantly higher in Muse cells than in non-Muse cells and MSCs (Figure 6A). Both MMP (matrix metalloproteinase)-2 and MMP-9 were produced by Muse cells, non-Muse cells, and MSCs. Although Muse cells produced the highest levels of MMP-2 in vitro, MSCs produced the highest levels of MMP-9 levels (Figure 6B). Despite the high production ability of these factors by Muse cells in vitro, Western blot of the border area heart tissue at day 3 revealed that the amount of these factors in the tissue was not statistically different among the 3 groups, except that VEGF was significantly higher in the Muse group than in the other groups, consistent with the ELISA results (Figure 6C).
The paracrine effects of Muse cells on apoptosis and fibrosis were examined. On day 3 after AMI, terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive myocytes were observed in the peri-infarct areas, and double staining for terminal deoxynucleotidyl transferase dUTP nick-end labeling and antimyoglobin showed that apoptotic cardiomyocytes were significantly reduced in the Muse group (5.9±1.1%) compared with the control group (11.8±1.5%; P<0.05; Figure 6D).
At 2 weeks, the Sirius Red–positive area in the infarct area was significantly smaller in the Muse group (12.6±1.1%) than in the control group (20.1±4.3%), suggesting a fibrosis-suppressing effect of Muse cells (P<0.05; Figure 6E).
Immunotolerance and Immunomodulatory Effect of Muse Cells
Because the xenografts showed integration and EF recovery for up to 2 weeks without immunosuppression, we validated the human Muse cells. All Muse cells expressed human leukocyte antigen (HLA)-ABC (similar to classical major histocompatibility complex class I cells), whereas none of them expressed HLA-DR on surface (major histocompatibility complex class II; Figure 7A through 7D; Online Figure XI). Coculture of Muse cells and naive human T cells led to an upregulation of regulatory T-cell factors, interleukin-10 and CD2525 (Figure 7E), and coculture of monocyte-Muse cells or monocyte-dendritic cell progenitors-Muse cells significantly suppressed the differentiation of monocytes into monocyte-dendritic cell progenitors (P<0.05) and into monocyte-dendritic cells (P<0.001; Figure 7F). Notably, ≈88% of Muse cells expressed HLA-G, an immunotolerance factor expressed in the placenta during pregnancy (Figure 7G).26 Furthermore, in a rabbit AMI model, GFP-labeled allograft Muse cells engrafted to the infarct border area expressed HLA-G on day 3 after AMI (2 days after intravenous injection of Muse cells; Figure 7H).
Long-Term Effects of Muse Cells
Compared with the vehicle group at 6 months after AMI (Figure 7I), the allograft group still exhibited a substantial reduction in infarct size; the infarct size was ≈45% of the vehicle group, thus maintaining the same tendency as that seen at 2 weeks and 2 months in the autograft. Attenuation of remodeling in LV end-systolic diameter and LV end-diastolic diameter (30% and 16.5% lower than the vehicle group, respectively) and functional recovery in EF, fractional shortening, and +dP/dt and −dP/dt (59.7%, 75%, and 30.2% and 27.1% higher, respectively) was more impressive than that observed at 2 weeks and 2 months, because the allograft group maintained improvement of functional parameters, whereas the vehicle group exhibited deterioration at 6 months compared with 2 weeks and 2 months (Figure 7J). GFP quantitative polymerase chain reaction confirmed the integration of allografts in the heart at 6 months, whereas that in the lung and spleen remained under the limits of detection (Figure 7K). GFP+ allografts maintained cardiac marker expression (Figure 7L).
The main effects of intravenously injected Muse cells on AMI were tissue repair accompanied by an infarct size reduction and functional recovery. These effects depended on the preferential migration and homing of Muse cells to the infarct region. In vitro, Muse cells exhibited the potential for cardiac- and vascular-lineage differentiation and the ability to produce factors relevant to the paracrine effect. In vivo, Muse cells exhibited the potential for spontaneous differentiation into cardiac- and vascular-lineage cells and delivered neovascularization, antifibrosis/fibrolytic, and antiapoptotic effects after homing to the AMI tissue.
In a rat AMI model, intramyocardial injection of 2 million human BM-MSCs led to neovascularization and recovery of LV function but did not regenerate cardiomyocytes.27 In another rat AMI model, intravenous injection of 5 million rat BM-MSCs led to a ≈24% reduction in infarct size compared with the untreated group.28 Although data observed in the present and other studies must be cautiously compared because of differences in the models, animal species, number of cells transplanted, and approach used, the present findings of a ≈55% and ≈48% reduction in infarct size by autografts and allografts, respectively, at 2 weeks, that did not diminish for up to 6 months (55% reduction) in allografts is remarkable compared with the previous reports. The beneficial effects of Muse cells are considered to be delivered by multiple mechanisms.
Preferential Homing of Muse Cells to the Infarct Region
In contrast to non-Muse cells, the organ distribution of Muse cells was highly specific to the AMI heart compared with the other organs, including the lung and spleen. The engraftment ratio of Muse cells into AMI heart was estimated to be ≈14.5% at 3 days, which is high compared with that of MSCs; a few or no engraftment of MSCs into the AMI heart when intravenously injected.28,29
We clarified in this study that the preferential homing of Muse cells into the infarct tissue is mainly mediated by the S1P–S1PR2 axis, in contrast to homing of MSCs, which is mediated by SDF-1 (stromal cell–derived factor 1)–CXCR4 (C-X-C chemokine receptor type 4) system.30 The contribution of the S1P–S1PR2 axis to Muse cell migration and homing was confirmed by coinjection of JTE-013 and gene silencing of S1PR2 in Muse cells. CXCR4 antagonist AMD3100 could suppress Muse cell migration toward the liver-damaged animal serum, although only partially, and unlike the S1P–S1PR2 axis, its suppression effect did not differ significantly between Muse and non-Muse cells,14 suggesting that the contribution of the SDF-1–CXCR4 system, the mediator of MSC migration,30 is smaller than that of the S1P–S1PR2 axis. On the basis of these findings together, the S1P–S1PR2 axis may explain the difference of migratory activities between Muse and non-Muse cells.
The results of the JTE-013 coinjection experiment suggested that efficient homing of Muse cells to the postinfarct heart is a key point for delivery of the beneficial effects of Muse cells because the reduction of infarct size and recovery of the EF were significantly smaller after the injection of JTE-013. Findings from experiments in which suicide gene–introduced Muse cells were injected also suggested the importance of the presence of Muse cells in the postinfarct tissue for the delivery of beneficial effects (Online Figure XIII).
The infarction might not have fully developed into fibrotic scar tissue at 24 hours after 30-minute ischemia–reperfusion, the time point at which the Muse cells were infused.16 As shown in Figure 5E, the S1P level in the border and infarct areas was significantly higher at 6 and 24 hours after ischemia–reperfusion than that in the noninfarct area. Muse cells migrated to both the infarct and border areas and more abundantly in the border area at day 3 as shown in Figure 1A. It is possible that Muse cells in the severely infarcted area could not survive for a long period because of the poor blood supply and harsh environment, even though they are stress tolerant, whereas Muse cells in the other areas were able to survive and exert their reparative effects.
The degree of infarct size reduction and functional recovery in non-Muse and MSC groups was not remarkable compared with the Muse group (Figure 1G and 1H). Concomitantly, homing of non-Muse cells to the postinfarct heart was low, and majority of these cells were trapped in the lung (Figure 1C and 1D). The less remarkable effects in these groups compared with the Muse group might be because of the low homing rate of the cells, as non-Muse cells and MSCs also have salutary effects represented by paracrine effects (Figure 6A through 6C). We, therefore, examined whether the direct delivery of non-Muse cells by intramyocardial injection induces clear tissue repair effects and functional recovery. The beneficial effect observed in the non-Muse group was greater than that in the vehicle group, whereas that in the Muse group was still higher than that in the non-Muse group (Online Figure XIV).
Differentiation Potential of Muse Cells
Muse cells demonstrate a commitment to cardiac and vascular lineages after homing. Neovascularization is indispensable for maintaining tissue repair, and in this sense, vascular differentiation of Muse cells is considered one of the mechanisms contributing to the observed tissue repair.
Engrafted Muse cells expressed cardiac markers, ANP, cardiac troponin-I, sarcomeric α-actinin, and connexin-43, without fusing with host cardiomyocytes. They also exhibited GCaMP3 activity synchronous with systole and diastole, suggesting physiological activity. These results suggest the potential of Muse cells to spontaneously differentiate into cardiomyocytes after homing to the infarcted tissue. Even if engrafted Muse cells differentiated into cardiomyocytes, the contribution of the differentiated cells to tissue repair and functional recovery remains speculative (Online Discussion I). Intravenous injection of GATA4-gene–silenced Muse cells attenuated the beneficial effects, that is, infarct size reduction and functional recovery, observed after intravenous injection of naive Muse cells, but it did not completely abolish the effects of the Muse cells. One possible explanation for the substantial attenuation of the beneficial effect of Muse cells by GATA4 silencing is suppression of the paracrine effects of Muse cells by GATA4 silencing. In fact, GATA4 is reported to support the paracrine effects of cardiac stem cells.31
Furthermore, considering the number of Muse cells estimated to be integrated at 2 weeks (≈65 000 cells) and 2 months (≈62 000 cells), hypothetical Muse cell–derived cardiomyocytes might represent a small proportion of the total number of myocytes present in the heart, or even in the peri-infarct region. Therefore, evidence demonstrating cardiac differentiation and its contribution to beneficial effects should be carefully interpreted.
Paracrine and Immunomodulatory Effects of Muse Cells
In vitro, Muse cells produced antifibrosis/fibrolytic factors MMP-2 and -9 and trophic factors HGF and VEGF, relevant to antiapoptosis, stimulation of endogenous cardiac progenitors, and neovascularization.17,32–34 Concomitantly, the paracrine effect of Muse cells in vivo was suggested by higher neovascularization in the border area, smaller fibrotic area, and low number of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive cells compared with the vehicle group.
Even when the homing of Nano-lantern–labeled Muse cells was almost completely abolished by JTE-013, the reduction in infarct size and improvement of EF by Muse cells were not completely abolished, but instead, some recovery was still recognized (Muse+JTE-013 in Figure 5I and 5J), although not as remarkable as that seen without JTE-013 (Muse in Figure 5I and 5J). The remnant recovery might be because of factor(s) other than the presence of Muse cells in the infarct region, such as paracrine effects.
A recent publication using mixed lymphocyte proliferation and indoleamine 2, 3 dioxygenase activity assays suggested that Muse cells have immunosuppressive effects.15 In this study, Muse cells were newly shown to activate regulatory T cells and suppress dendritic cell differentiation. Interestingly, they also expressed HLA-G, which is present in immune-privileged organs such as the placenta, thymus, ovary, and testis and is associated with reduced inflammation and immune responses, as well as tolerogenic properties, through interactions with inhibitory receptors on dendritic cells, natural killer cells, and T cells.35 Although not yet identified, the rabbit homologue of HLA-G may exist because MHC (major histocompatibility complex) class Ib, in which HLA-G belongs to, exists generally in mammals.35 Muse cells engrafted to the infarct border area expressed HLA-G on day 3 after AMI in rabbits (Figure 7H). The expression ratio in Muse cells was remarkably high compared with that of other stem cells; undifferentiated human embryonic stem and induced pluripotent stem cells do not express HLA-G,36,37 and <20% of adult BM-MSCs express HLA-G.38 Because HLA-G is suggested to promote graft tolerance in heart transplantation,39 the high expression of HLA-G together with the immunomodulatory effects of allograft and xenograft Muse cells may contribute to their escape from immunologic attack in the early phase of integration. The mechanism of how these cells could survive in the host tissue after differentiation into cardiomyocytes at the late phase (6 months) of AMI remains to be investigated.
To deduce the equivalency of myocardial infarction among the 4 groups, we measured plasma troponin-T levels in all animals at 24 hours after AMI immediately before the intravenous administration of cells, based on the report that plasma troponin-T levels are highly linearly correlated with infarct size at 24 hours after AMI.20 Plasma troponin-T levels did not differ significantly among the 4 groups. Because, however, plasma troponin levels are not a definitive measure of infarct size, the possibility that the Muse cell–treated rabbits had smaller infarcts at the initial stage cannot be completely excluded.
Although the investigators were blinded to the protocol and the AMI rabbits were randomized in this study, a potential bias cannot be completely excluded. Therefore, these data should be cautiously interpreted. Furthermore, the ability of Muse cells to cause functionally significant regeneration of myocytes seems unlikely because of the low numbers of these cells engrafting in the myocardium when intravenously administered—a finding also demonstrated when other types of stem cells are injected intravenously.28,29
Muse cells injected intravenously drastically reduced the infarct size and improved LV function and remodeling, and allogenic Muse cells remained cardiac marker–positive in the host tissue at 6 months; the remarkable improvement did not diminish compared with that at 2 weeks.
Cell therapy should be implemented in the early stages of AMI before the infarcted tissue becomes a fibrotic scar. For autologous cell therapy of Muse cells, however, a certain period of time is necessary for a series of processes, including collection of BM aspirate from the patient, selection, and expansion of Muse cells on a clinically relevant scale. Therefore, autologous Muse cell therapy may not always prove realistic for AMI patients. Rather, allogenic cell transplantation may prove more practical. BM-Muse cells from healthy donors that are expanded may be applied to AMI patients immediately after percutaneous coronary intervention.
We thank Yukihiro Akao (Gifu University) and Dr Gregorio Chazenbalk (University of California, Los Angeles) for helpful discussion.
Sources of Funding
This study was supported by Grants-in-Aid from the New Energy and Industrial Technology Development Organization and the Japan Agency for Medical Research and Development.
Y. Yamada, Shingo Minatoguchi, and Shinya Minatoguchi are affiliated with Department of Cardiology, Gifu University Graduate School of Medicine (Gifu, Japan) and S. Wakao, Y. Kushida, and M. Dezawa are affiliated with the Department of Stem Cell Biology and Histology at Tohoku University Graduate School of Medicine (Sendai, Japan), which are parties to a codevelopment agreement with Life Science Institute, Inc (LSII; Tokyo, Japan). S. Wakao and M. Dezawa have a patent for Muse cells, and the isolation method thereof licensed to LSII. The other authors report no conflicts.
In January 2018, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.18 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.311648/-/DC1.
- Nonstandard Abbreviations and Acronyms
- acute myocardial infarction
- atrial natriuretic peptide
- bone marrow
- ejection fraction
- green fluorescent protein
- hepatocyte growth factor
- human leukocyte antigen
- left ventricle
- matrix metalloproteinase
- mesenchymal stem cells
- multilineage-differentiating stress enduring
- sphingosine monophosphate
- sphingosine monophosphate receptor 2
- stromal cell–derived factor 1
- stage-specific embryonic antigen-3
- vascular endothelial growth factor
- Received July 3, 2017.
- Revision received February 8, 2018.
- Accepted February 22, 2018.
- © 2018 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Beneficial effects of stem cell therapy using bone marrow (BM)-mononucleated cells and BM-mesenchymal stem cells (MSCs) have been reported in animal models of acute myocardial infarction and in some clinical trials.
Multilineage-differentiating stress enduring (Muse) cells, collectable as stage-specific embryonic antigen-3+ cells, are pluripotent-like stem cells located in the BM (≈0.03% of the mononucleated fraction), peripheral blood, and connective tissue of various organs. They also correspond to ≈1% of MSCs.
After administration, Muse cells preferentially home to damaged tissue and spontaneously differentiate into new tissue-specific cells in stroke, liver cirrhosis, and chronic kidney disease models.
What New Information Does This Article Contribute?
Intravenously injected autograft, allograft, and human xenograft Muse cells reduced infarct size, left ventricular function, and attenuated left ventricular remodeling in a rabbit acute myocardial infarction model, and the allografts remained engrafted and sustained functional recovery for up to 6 months without immunosuppression.
The S1P (sphingosine monophosphate)–S1PR2 (sphingosine monophosphate receptor 2) axis mediated the preferential homing of Muse cells to the infarct and the border areas.
The Muse cells had pleiotropic effects, and beneficial effects are mediated by their paracrine effects.
Muse cells exhibited the potential to spontaneously differentiate into cardiac and vascular lineages after homing, and GCaMP3 (GFP-based Ca calmodulin probe)-Muse cells exhibited GCaMP3 activity synchronous with ECG recordings after engraftment.
Among stem cells, BM-mononucleated cells and BM-MSCs are currently used in clinical trials for acute myocardial infarction. These cells are nontumorigenic, safe, and considered to deliver beneficial effects mainly by paracrine effects. Although Muse cells are a small subpopulation of BM-mononucleated cells and BM-MSCs, they demonstrate several unique features. First, the organ distribution of intravenously injected Muse cells, which is mediated mainly by the S1P–S1PR2 axis, in contrast to MSC homing, which is mediated by SDF-1 (stromal cell–derived factor 1)–CXCR4 (C-X-C chemokine receptor type 4) system, was highly specific to the infarcted heart, rather than the lung or spleen. Second, Muse cells engrafted to the infarcted heart for a longer period, and allograft Muse cells, in particular, remained engrafted and promoted sustained functional recovery for up to 6 months without immunosuppression. Third, after homing, Muse cells exhibited the potential to spontaneously differentiate into cardiac- and vascular-lineage cells. The estimated number of cardiac marker–positive Muse cells represented only a small proportion of the total number of myocytes in the left ventricle; therefore, the contribution of differentiation to their beneficial effects remains uncertain. Muse cells may be a viable candidate cell source for stem cell therapy for acute myocardial infarction.