Integrative Physiology

Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial InfarctionNovelty and Significance

Thomas E. Sharp, Giana J. Schena, Alexander R. Hobby, Timothy Starosta, Remus M. Berretta, Markus Wallner, Giulia Borghetti, Polina Gross, Daohai Yu, Jaslyn Johnson, Eric Feldsott, Danielle M. Trappanese, Amir Toib, Joseph E. Rabinowitz, Jon C. George, Hajime Kubo, Sadia Mohsin, Steven R. Houser
https://doi.org/10.1161/CIRCRESAHA.117.311174
Circulation Research. 2017;121:1263-1278
Originally published September 14, 2017

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Abstract

Rationale: Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients.

Objective: To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model.

Methods and Results: Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia–reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5–ethynyl–2′deoxyuridine (EdU)—a thymidine analog—containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals.

Conclusions: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

Introduction

Ischemic heart disease is the greatest single cause of mortality worldwide, accounting for ≈7 million deaths annually.1 Ischemic heart disease is caused by coronary artery disease that frequently results in myocardial infarction (MI). MI causes the death of myocardial tissue, even when blood flow is restored (ischemia followed by reperfusion [I/R]), resulting in a reduction in the number of cardiac myocytes. Although mortality, after MI, has improved dramatically in the era of prompt reperfusion therapy,2 the infarcted tissue is largely replaced by scar, and the resultant cardiac structural and functional remodeling causes progressive declines in cardiac pump function3 that can lead to heart failure (HF). Novel therapeutic strategies that reduce pathological post-MI structural and functional remodeling and thereby prevent HF development are needed.

Editorial, see p 1210

In This Issue, see p 1205

Meet the First Author, see p 1206

A variety of cell therapies have been studied with the goal to enhance vascularization of the ischemic heart4–6 and to replace lost cardiac myocytes.7–10 Most of these studies have shown small improvements in cardiac structure and function and a few have shown evidence for myogenesis.11,12 However, effects observed to date have been modest, and there is a need for novel cell therapies with a well-documented ability to reduce adverse post-MI remodeling. In addition, issues including cell safety, dosing, time, and route of delivery, in our view, still have not been adequately defined.

Studies in rodent models, using different cell types and multiple delivery techniques, have overwhelmingly suggested that cell therapy can attenuate post-MI remodeling.13–15 However, studies in preclinical large animal models and early phase I and II clinical trials11,16–23 have generally shown more modest effects. Importantly, early-stage clinical trials have demonstrated the safety of cell therapy in post-MI patients21,23–25 or in patients with established cardiac disease.22,26,27

We have identified a novel stem cell population, isolated from cortical bone and have shown that these cells have cardioprotective features, both in vitro28 and in vivo.29 Cortical bone stem cells (CBSCs) were found to be distinct from cardiac-derived stem cells and mesenchymal stem cells, and they possess characteristics that account for their in vivo cardioprotective features.28 CBSCs improved post-MI survival, reduced scar size, reduced ventricular dilation, and improved cardiac function when transplanted into the MI border zone (BDZ) of a mouse model,29 and their effects were superior to those of cardiac-derived stem cells, both in vitro and in vivo.28,29

The present study was designed to test the hypothesis that injection of CBSCs, into the BDZ, in a large animal model (swine) of an I/R-induced MI reduces post-MI structural and functional remodeling and prevents the development of HF. The study examined whether CBSCs reduced initial cardiac injury (cardioprotection), survived in the MI BDZ after transendocardial delivery, and whether they reduced post-MI structural and functional remodeling to improve cardiac pump function. These studies used approaches that could be implemented in patients who have experienced an MI and have undergone reperfusion therapy.

Methods

A full methods section is available in the Online Data Supplement. Figure 1 is a timeline of the experiments performed in the study.

Figure 1.
Figure 1.

Study timeline. Protocol used to perform baseline assessment, induce acute myocardial infarction (MI), therapeutic delivery, EdU minipump implantation and explant, and functionally measurements. CBSC indicates cortical bone stem cell; CNR, could not resuscitate; DPO, died post-operation; and LAD, left anterior descending.

Results

Ischemia–Reperfusion/MI Survival and Arrhythmogenic Events

Twenty-three animals underwent ischemia–reperfusion (IR)/MI (72-hour cohort [n=11] and 3-month cohort [n=12]). Two animals in each group could not be resuscitated because of pulseless electric activity during ischemia. The IR/MI survival rate is 78.3% (Figure 2A). One animal (72 hours, vehicle-treated cohort) died within 2 hours postop because of a lethal arrhythmia. All animals who survived >2 hours post-MI survived the entirety of the study (n=18, total). There was no significant difference in body weight at baseline or during follow-up (Online Tables I and II). Circulating cardiac troponin I (on reperfusion; Figure 2B) was measured to confirm MI induction and define initial injury. All animals were in sinus rhythm before balloon occlusion (Online Figure IA) and showed characteristic ECG changes (ST-segment alterations) after ischemia induction (Online Figure IB and IC). Most animals fibrillated (61%) during the MI and were successfully defibrillated (Methods). There were no ventricular arrhythmias observed during the NOGA mapping and injection procedure. No antiarrhythmic agents were required after animals returned to sinus rhythm after reperfusion (Online Figure IC).

Figure 2.
Figure 2.

Survival and initial injury assessment. Hematologic, structural, and histological assessments of initial injury were performed at 72 h post IR/myocardial infarction (MI). A, Survival curve of all 23 animals put on study. Two animals died during the ischemic period and one during the first 24 h postop. Eighteen survived and completed the study timeline. X=time of MI. B, Circulating cardiac troponin I (cTnI) was analyzed at 2 h post-reperfusion in all animals (n=18). **P value ≤0.01 vs baseline. C, The mean circulating cTnI at 2 h post-reperfusion between treatment groups. D–F, The change in left ventricular end-diastolic (EDV)/systolic (ESV) volume and ejection fraction (LVEF) in each treatment group at 72 h post-IR/MI. G and H, Representative images of mid-myocardial cross-sections stained for TTC (vehicle [VEH] and cortical bone stem cell (CBSC), respectively). Scale bar=10 mm. F, The mean TTCNEG tissue (nonmetabolically active tissue) as a percentage of total ventricular area (n=4, each). J, The mean TUNEL+ (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling) nonmyocytes per mm2 in each group. K, The mean TUNEL+ myocytes per mm2 in each group. Representative confocal images of TUNEL+ myocytes (arrowheads) and nonmyocytes (arrows) in VEH- and CBSC-treated animals at 72 h post-MI. Scale bar=50 μm. *P value ≤0.05 vs VEH-treated. **P value ≤0.01 vs baseline.

Acute Study (72 Hours)

Initial Injury, Apoptosis, Safety and Efficacy of Delivery, and Cell Retention

Circulating cardiac troponin I was elevated at 2 hours post-MI in all animals (Figure 2B), confirming MI induced myocardial necrosis. No differences in 2-hour circulating cardiac troponin I levels between treatment groups were observed in either short- (72 hours) or long-term (3 months) cohorts, showing that the initial IR injury was similar in vehicle- and CBSC-treated animals (Figure 2C). Transthoracic echocardiography (ECHO) was performed to assess structural and functional changes at 72 hours post-IR/MI±CBSCs. There were no differences in volumetric ECHO-derived parameters and cardiac structural dimensions between treatment groups (Figure 2D through 2F; Online Figure II). However, when assessing ECHO speckle-tracking strain (Online Figure III), we observed a significant preservation of the longitudinal strain in CBSC- versus vehicle-treated animals (14.52±0.8% [CBSC] versus 8.7±1.13% [vehicle]; P≤0.005) and a preservation in radial strain as compared with baseline (Online Figure IIIH and IIII).

Cross-sections of cardiac tissue from explanted hearts were treated with triphenyltetrazolium chloride, to differentiate metabolically active from necrotic tissue (Figure 2G and 2H). There were no differences in triphenyltetrazolium chloride negative tissue at 72 hours post-MI±CBSCs (28.1±6.13% [vehicle] versus 27.1±3.73% [CBSC]; Figure 2I; P=ns). These results clearly show that the extent of myocardial damage caused by I/R in vehicle- and CBSC-treated animals was not different. These results also show that CBSC treatment used in these experiments after IR/MI did not cause acute cardioprotection, to reduce the initial infarct size. TUNEL (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling) staining and quantification showed a significant reduction in the number of apoptotic nonmyocytes per mm2 in the CBSC- versus vehicle-treated animals (Figure 2J). However, there was no significant difference in the number of apoptotic myocytes per mm2 between groups at 72 hours post-MI (Figure 2K).

A NOGA mapping system was used to assess electric conductance within the heart and determine the interface (BDZ) between viable and nonviable tissue. The system was also used to guide catheter-based transendocardial injections30 (Online Figure IVC). Injection sites were observed in explanted hearts with gross visualization of fluorescent microspheres within the myocardium in all 18 animals in the 72-hour and 3-month cohorts (Online Figure IVD). Between 50% and 60% of the injection sites were found at the epi- or endocardial surfaces in 72-hour and 3-month post-MI studies (Online Tables I and II). Transendocardial injections induced occasional preventricular contractions, but no sustained or lethal ventricular arrhythmias were observed during delivery of treatment. The viability of the CBSCs after passing through the MYOSTAR injection catheter was confirmed (Online Figure IV).

Tissue from validated injection sites was processed for either molecular or histological analysis. A dual CBSC labeling strategy (GFP [green fluorescent protein] and sex mismatch) was used to identify the CBSCs (Figure 3), and both approaches have been used previously.31–33 Injection sites from all (8) 72-hour post-MI animals were immunostained for GFP, EdU, α-sarcomeric actin, and DAPI. GFP+ CBSCs were identified in all animals at 72 hours post-MI (n=4; Figure 3A through 3E). Interestingly, the GFP+ CBSCs were EdU+, showing that the cells were alive and proliferative (Figure 3–1, –2, and –3). Polymerase chain reaction analysis of tissue from injection sites confirmed the presence of the Y chromosome in CBSC-treated animals (Figure 3F). These studies show that CBSCs injected into the MI BDZ survive and proliferate within the injured tissue but do not prevent the necrotic injury that is characteristic of IR/MI30.

Figure 3.
Figure 3.

Cortical bone stem cell (CBSC) retention and proliferation. GFP+/Y chromosome+ CBSC retention was assessed by immunofluorescence and PCR at 72 h post IR/myocardial infarction (n=4). A, Confocal image (10×) of a transendocardial injection site with GFP+ CBSCs. B, Confocal image (40×) from injection site with GFP+/EdU+ CBSCs from image (A). B1–B4, Single-channel breakdown from 40× images (α-sarcomeric actin, EdU, GFP and DAPI, respectively; arrowheads identify GFP+/EdU+ cells). 1–3 Zoomed confocal images from the 40× image (B) of representative GFP+/EdU+ CBSCs (arrows). C–E, Representative confocal images (40×) of GFP+/EdU+ CBSCs from animals 2 to 4. GFP (green), EdU (red), α-actin (white) and DAPI (blue). Scale bars=50 μm. F, PCR products run on 1.5% agrose gel from vehicle (VEH) and CBSC injection sites. Product size≈300 bp. BL indicates blank; F (−control), female heart; GFP, green fluorescent protein; M (+control), male heart; and ML, molecular weight ladder.

CBSC Treatment Increases Cell Proliferation

EdU was infused into vehicle- and CBSC-treated animals (n=4, each) for the first 3 days after MI to identify cells with newly formed DNA (Figure 4). We imaged and quantified the number of EdU+ cells at 72 hours post-MI±CBSCs. Confocal images from the infarct zone (IZ), BDZ, remote zone (RZ), and septal BDZ of all animals were examined (Figure 4A1 through 4A4 [vehicle] and Figure 4B1 through 4B4 [CBSC]), and EdU+ cells were identified and quantified using Naquantus34—a novel semiautomated quantification software. We calculated the total number of nuclei (DAPI+ [all cells]), EdU+/DAPI+ cells (total EdU+ cells), actin+/DAPI+ (total myocytes), and EdU+/actin+/DAPI+ cells (EdU+ myocytes; Figure 4; Online Figure VA through VD) in each region. A large number of EdU+ cells were observed 3 days after MI+CBSCs, and the GFP+ CBSCs were EdU+. The majority of these EdU+ cells were nonmyocytes (Figure 4 [arrowheads]). EdU+ nonmyocytes were predominately made up of Von Willebrand Factor+ (VWF) cells, CD45+ cells, and smooth muscle actin+ cells (Online Figure VI). There was a significant 2-fold increase in the percentage of EdU+ nuclei (Figure 4A and 4B [arrowheads]) in CBSC- versus vehicle-treated heart in the BDZ (Figure 4C2), with nonsignificant increases in all other regions (Figure 4C1, 4C3, and 4C4). A few EdU+ cardiac myocytes were identified in every heart (Figure 4A4 and 4B2 [arrows]). None of these were GFP+, so they were not derived from injected CBSCs. There was no significant difference in the percentage of EdU+ myocytes in CBSC- versus vehicle-treated hearts in the IZ, BDZ, and septal BDZ (Figure 4D1 and 4D4). Most EdU+ myocytes were found at or near the border of the infarcted regions.

Figure 4.
Figure 4.

Cortical bone stem cells (CBSCs) induce an increase in proliferative nonmyocytes in the myocardial infarction (MI) border zone. EdU+ cells were imaged and quantified at 72 h post-MI in (A) Vehicle- (VEH) and (B) CBSC-treated heart. (A1–A4) Representative confocal images (20×) from the 4 distinct regions of the myocardium post-IR/MI+VEH. B1–B4, Representative confocal images (20×) from the 4 distinct regions of the myocardium post-IR/MI+CBSCs. 40× representative confocal images illustrate EdU+ myocytes and nonmyocytes from hearts±CBSCs. EdU (green), α-actin (red), and DAPI (blue). Arrows=myocytes, arrowheads=nonmyocytes. 20× scale bars=200 μm, 40× scale bars=50 μm, and zoom/channel breakdown scale bars=25 μm. C1–C4, Quantification of total EdU+ cells as a percentage of total DAPI+ in each region. D1–D4, Quantification of total EdU+actin+DAPI+ cells as a percentage of total actin+DAPI+ (myocyte) in each region. N=3, each group. *P value ≤0.05 vs VEH. BDZ indicates border zone; IZ, infarct zone; RZ, remote zone; and sBDZ, septal border zone.

Long-Term Study (3 Months)

Characterization of Calcification, Sarcoidosis, Foreign Bodies, and Allograft Rejection Post-MI±CBSCs

The International Society for Heart and Lung Transplantation provides guidelines for characterizing the immune response because of an allograft and the potential for rejection.35 H&E (hematoxylin & eosin) stained sections were analyzed for the severity of the immune cell infiltrate and the degree to which it encroached on parent myocardium (Online Table III). We used tissue sections from regions of the injection site, as well as noninjection site regions. The vehicle-treated animals had a greater incidence of calcification versus CBSC-treated animals (Online Table III; Online Figure VII). No sarcoidosis was observed in any section. Noncellular foreign bodies were found within the myocardium of both treatment arms consisting of fluorescent microspheres that were injected during the time of therapy delivery (Online Table III; Online Figure VII). Less immune cell infiltrate was observed at 3 months post-MI in animals who received CBSC therapy as compared with vehicle-treated animals (Online Table III).

Preservation of Left Ventricular Structure and Function

Ten animals (vehicle- [n=5] and CBSC-treated [n=5]) were studied for 3 months after IR/MI. Time-dependent changes in cardiac structure and function were evaluated with ECHO and invasive hemodynamic measurements, which were performed before IR/MI and were repeated during terminal studies. ECHO measurements documented increases in left ventricular (LV) end-diastolic volume and LV end-systolic volume in both treatment groups versus baseline. However, CBSC-treated hearts showed significantly less progressive LV-chamber dilation as compared with vehicle-treated hearts. At 3 months post-MI, the LV end-diastolic volume and LV end-systolic volume of CBSC-treated animals were significantly smaller than in vehicle-treated animals (Figure 5A and 5B; LV end-diastolic volume [53.0±3.0 versus 43±2.5 mL]; LV end-systolic volume [38.6±3.4 versus 22.0±2.4 mL]; P≤0.05). LV ejection fraction (LVEF) was reduced in both groups by ≈10% at 1 month post-MI (Figure 5C). During the next 2 months, LVEF fell in the vehicle-treated animals, whereas ejection fraction remained stable in CBSC-treated animals. LVEF was significantly greater in CBSC- versus vehicle-treated animals at 3 months post-MI (Figure 5C). At 3 months post-MI, the stroke volume and cardiac output were both significantly reduced in the vehicle-treated versus CBSC-treated animals (Figure 5E and 5F). Accounting for the growth of the animals by normalizing to the body surface area (m2) did not change the differences between treatment groups (Online Table IV). Chamber dilation was confirmed through structural measurements of the cardiac internal diameter at end-diastole and systole (Figure 5H through 5K); also through a significant reduction in the LV relative wall thickness (Online Table IV). The intraventricular septum dimension was better preserved in CBSC-treated versus vehicle-treated animals (Figure 5G and 5J), whereas the internal diameter of the LV was significantly less dilated at 3 months post-MI in CBCS-treated versus vehicle-treated animals (Figure 5H and 5K). These studies show that CBSC therapy reduces the progression of cardiac structural remodeling during the 3 months after MI.

Figure 5.
Figure 5.

Cortical bone stem cells reduce left ventricular structural remodeling and preserve function post-IR/myocardial infarction (MI) serial transthoracic echocardiography was performed. Volumetric measurements were assessed. A, Left ventricular end-diastolic (LVEDV) and (B) left ventricular end-systolic volumes (LVESV). C, Assessment of left ventricular ejection fraction (LVEF). D, Heart rate, (E) stroke volume, (F) cardiac output, (G–I) 2D m-mode LV wall thickness, and internal diameter at end-diastole. J–L, 2D m-mode LV wall thickness and internal diameter at end systole. n=5 in each group. *P value ≤0.05, **P value ≤0.01, and ***P value ≤0.005 vs baseline within group, #P value ≤0.05, ##P value ≤0.01, and ###P value ≤0.005 vs VEH post-MI. d indicates diastole; IVS, interventricular septum; LVID, left ventricular internal dimension; LVPW, left ventricular posterior wall; and s, systole.

CBSCs Reduce Hemodynamic Deterioration and Preserve Contractile Reserve

Differences in ventricular dilation at 3 months post-MI determined with serial ECHO were confirmed in terminal studies using invasive hemodynamic methods (Figure 6). The pressure–volume loops (Figure 6A) and end-diastolic pressure–volume relationship (Figure 6B), at spontaneous heart rates, were shifted to significantly larger volumes at 3 months post-MI in the vehicle-treated animals. This rightward shift of the pressure–volume relationships at end-diastole confirms ventricular dilation. Significantly smaller changes in these parameters were found in the CBSC-treated animals (Figure 6D and 6E). There was significantly less rightward shift in the end-diastolic pressure–volume relationship in CBSC- versus vehicle-treated animals at 3 months post-MI (Figure 6H). These results confirm that CBSC treatment reduces progressive ventricular dilation and preserves LVEF as compared with vehicle treatment (Figure 6G and 6H, Online Table V). The volumes at dP/dt max and min were not significantly increased in the CBSC-treated animals, further documenting preservation of cardiac chamber size in CBSC-treated animals (Online Table V). Consistent with previous work,3 diastolic function was decreased post-MI. The isovolumic relaxation time constant (τ) was prolonged, and the minimal rate of relaxation (dP/dt min) was decreased in vehicle-treated animals versus baseline, whereas these measurements were not significantly different with CBSCs (Online Table V).

Figure 6.
Figure 6.

Cortical bone stem cells (CBSCs) reduce left ventricular dilation and preserve cardiac functional reserve. Invasive hemodynamics were performed at baseline and 3 mo post-myocardial infarction (MI)±CBSCs±DOB. A, Representative pressure–volume (PV) loops at baseline and 3 mo post-MI (red line) in vehicle- (VEH) treated animal. B and C, EDPVR and ESPVR is shifted rightward 3 mo post-MI vs pre-MI in VEH-treated animals. Pre-MI=open squares, 3 mo post-MI=red squares. D, Representative PV loops from a CBSC-treated animal at baseline and 3 mo post-MI (blue line). E, EDPVR remains unchanged, whereas (F) ESPVR is shifted rightward 3 mo post-MI vs pre-MI in CBSC-treated animals. Pre-MI=open circles, 3 mo post-MI=red circles. G, Representative 3-mo post-MI PV loops±CBSCs, the PV loops are shifted rightward in the VEH group. H, EDPVR is significantly shifted rightward in the VEH-treated animals vs CBSC treatment, whereas (I) shows no significant change in ESPVR between groups 3 mo post-MI. J, Representative PV loops during dobutamine (DOB) challenge (2.5 μg·kg·min) 3 mo post-MI±CBSCs. VEH+DOB=dashed red line/red squares, CBSC+DOB=dashed blue line/blue circles. K, No significant difference in the EDPVR between groups+DOB, whereas (L) demonstrates a significant difference in the ESPVR, indicating preserved contractile reserve in the CBSC group. N=5 in each group.

The rightward shift in the pressure–volume loop in vehicle-treated animals is consistent with a decrease in cardiac systolic function (Figure 6C). The end-systolic pressure–volume relationship—an indicator of systolic performance—is determined by 2 parameters: the end-systolic elastance (ie, slope) and the volume intercept (V0). Changes in both determinants are considered to determine changes in cardiac systolic function (ie, contractility).36,37 The end-systolic pressure–volume relationship was shifted rightward to a greater extent in vehicle- versus CBSC-treated animals after MI (Figure 6I). The end-systolic elastance was reduced in both CBSC- and vehicle-treated animals (5.63±0.40–4.35±0.41 [CBSC] and 5.04±0.39–3.78±0.47 [vehicle]; P>0.05 [between groups 3 months post-MI]). The volume intercept (V0) was significantly increased only in the vehicle-treated animals versus baseline (Online Table V). These studies document a reduction of cardiac function in all MI animals, regardless of treatment, with better preservation of cardiac contractile properties in CBSC-treated animals.

Reduced inotropic reserve is a characteristic feature of the failing heart.38 To explore the idea that CBSC treatment improves post-MI contractile reserve, cardiac hemodynamics were measured in vehicle- and CBSC-treated MI animals before and after dobutamine (Figure and 6I and 6J). Dobutamine (2.5 ug·kg·min) caused a shift in the end-diastolic pressure–volume relationship toward baseline in CBSC-treated but not in vehicle-treated animals (Figure 6J and 6K). In the CBSC cohort, dobutamine treatment created a significant leftward shift in the end-systolic pressure–volume relationship beyond baseline levels (Figure 6I) and caused a 2-fold increase in the end-systolic elastance (4.35±0.41 [3 months post-MI+CBSCs] to 8.09±0.71 [+dobutamine]; P≤0.05) but did not alter V0 (Online Table V). These changes were significantly greater than in dobutamine-treated MI+vehicle animals (Figure 6J through 6L; Online Table V; P≤0.05). Dobutamine only caused a significant increase in end-systolic elastance in CBSC-treated animals (Online Table V). There was also a significant reduction in volume end-systole and an increased ejection fraction with CBSC treatment+dobutamine versus vehicle+dobutamine (Online Table V). Collectively, these data show that CBSC treatment preserves basal contractility and systolic functional reserve.

CBSC Treatment Reduces Scar Size, Inhibits Hypertrophic Remodeling and Apoptosis

Heart weight (HW) and HW-to-body weight ratios were not different in vehicle- and CBSC-treated hearts (Online Table II). However, scar size was significantly smaller in CBSC- versus vehicle-treated animals at 3 months post-MI (8.5±2.2% versus 16.15±1.85%; P≤0.05; Figure 7A). In addition, anterior wall thickness was significantly greater in CBSC- versus vehicle-treated animals (Figure 7B and 7C). At the cellular level, myocyte cross-sectional area (μm2) was significantly smaller in CBSC- versus vehicle-treated hearts (BDZ [565.8±2.9 versus 304±1.1 μm2; P≤0.0001] and RZ [400.6±2.4 versus 276.6±1.2 μm2; P≤0.0001]; Figure 7D and 7F), with a rightward shift in the BDZ and RZ cross-sectional area histograms with vehicle treatment (Figure 7E and 7G). CBSC cross-sectional area was slightly but significantly greater than noninfarcted control animals (Figure 7D and 7F) but significantly smaller than vehicle-treated MI myocytes. Myocyte nuclear density per mm2 was evaluated in the BDZ and RZ. Myocyte nuclear density was significantly greater in the CBSC-treated versus vehicle-treated animals (933.8±85.25 versus 623.1±48.32 myocyte nuclei/mm2; P≤0.05; Figure 7H and 7I) but not significantly different from noninfarcted controls. There was a significant reduction in TUNEL+ nonmyocytes (Figure 7J) and myocytes (Figure 7K) at 3 months in animals that received CBSC treatment compared with vehicle-treated animals in the BDZ and RZ. These data show that CBSC-treated hearts have greater muscle mass because of a reduction in scar with no difference in HW or HW/body weight ratio. CBSC-treated animals had smaller cardiac myocytes as observed by a significant reduction in cross-sectional area and significant increase in cardiac myocytes nuclei per mm2 as compared with vehicle-treated animals. Significantly reduced myocyte apoptosis for 3 months may give insight into how there is more muscle mass, reduced scar, and more myocytes per mm2 as compared with vehicle-treated IR/MI hearts.

Figure 7.
Figure 7.

Cortical bone stem cells (CBSCs) reduce scar size, inhibit pathological hypertrophic remodeling, preserve myocyte nuclear density, and reduce cardiac myocyte death. Gross anatomy morphometric analysis for scar size, myocyte cross-sectional area (CSA), myocyte nuclear density, and TUNEL (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling) staining was performed 3 mo post-IR/myocardial infarction (MI) or in noninfarcted controls. A, 2-fold reduction in scar size with CBSC treatment. B and C, Representative gross anatomy cross-sections of mid-myocardium±CBSCs. Scale bar=10 mm. D, Average myocyte CSA in the border zone (BDZ) and (E) myocyte CSA distribution within group and representative 40× confocal images of wheat germ agglutinin (WGA) staining. F, Average myocyte CSA in the remote zone (RZ) and (G) myocyte CSA distribution within group and representative 40× confocal images of WGA staining. H, Average myocyte nuclei per mm2 and (I) distribution of nuclear density per group within viable tissue (BDZ and RZ combine). J, The mean number of TUNEL+ nonmyocytes per mm2 and (K) the mean number of TUNEL+ myocytes per mm2 of cardiac tissue is reduced in the CBSC group within viable tissue (BDZ and RZ combine). Representative confocal images of TUNEL+ myocytes (arrowheads) and nonmyocytes (arrows) in vehicle- (VEH) and CBSC-treated animals at 3 mo post-MI. Scale bar=50 μm. N=5 in each group. *P value ≤0.05, **P value ≤0.005, ****P value ≤0.0001 vs VEH-treated group, and ####P value ≤0.0001 vs CBSC group.

CBSCs Induce Myocyte Proliferation With Little Recruitment of Ckit+ Cells

EdU was infused into 3-month vehicle- and CBSC-treated animals (n=5, each) for the first 7 days after MI to identify cells that incorporate EdU into their DNA during this time period (Figure 8); EdU was also infused in noninfarcted controls (n=3) to observed baseline incorporation. We imaged and quantified the number of EdU+ cells at 3 months post-MI±CBSCs and in noninfarcted controls. Confocal images from the IZ, BDZ, RZ, and septal BDZ of all animals were examined (Figure 8A1 through 8A4 [vehicle] and Figure 8B1 through 8B4 [CBSC]) and EdU+ cells were identified and quantified.34 We calculated the total number of nuclei (DAPI+ [all cells]), EdU+/DAPI+ cells (total EdU+ cells), actin+/DAPI+ (total myocytes), and EdU+/actin+/DAPI+ cells (EdU+ myocytes; Figure 8; Online Figure VIIIA through VIIID) in each region. No GFP+ cells were found in any 3-month post-MI hearts, suggesting that none of the injected CBSCs were still present at this time point. The percentage of EdU+ nuclei (Figure 8A1 through 8A4 and 8B1 through 8B4 [arrowheads]) was not different in CBSC- versus vehicle-treated heart in any region (Figure 8C1 through 8C4). There was a significant increase in the total EdU+ cells in both treatment arms when compared with noninfarcted controls in the IZ and BDZs. However, EdU+ cardiac myocytes were easily identified in every heart with CBSC treatment (Figure 8B1 through 8B4 [arrows]; Online Figure IX). None of these myocytes were GFP+, so they were not derived from injected CBSCs. The percentage of EdU+ myocytes out of total myocytes was significantly greater in CBSC hearts in all regions adjoining the MI, and the majority of the EdU+ myocytes were at or near the infarct BDZ (Figure 8B1 through 8B4 [arrows] and Figure 8D1 through 8D4). In noninfarcted controls, the percentage of EdU+ myocytes was significantly reduced in the IZ (ie, anterior wall) and both BDZs (ie, lateral wall and septal wall). The percentage of EdU+ myocytes was not significantly increased in the areas of the heart that are remote from the infarct (Figure 8D3).

Figure 8.
Figure 8.

Cortical bone stem cell (CBSC) treatment increases EdU+ myocytes in the border zones. EdU+ cells were imaged and quantified at 3 mo post-myocardial infarction (MI) in (A) vehicle- (VEH) treated heart, (B) CBSC-treated heart, or noninfarcted controls. A1–A4, Representative confocal images (20×) from the 4 distinct regions of the myocardium post-IR/MI+VEH. B1–B4, Representative confocal images (20×) from the 4 distinct regions of the myocardium post-IR/MI+CBSCs. 40× representative confocal images illustrates EdU+ myocytes and nonmyocytes from hearts±CBSCs. EdU (green), α-actin (red), and DAPI (blue). Arrows=myocytes, arrowheads=nonmyocytes. 20× scale bars=200 μm, 40× scale bars=50 μm, and zoom/channel breakdown scale bars=25 μm. C1–C4, Quantification of total EdU+ cells as a percentage of total DAPI+ in each region. D1–D4, Quantification of total EdU+actin+DAPI+ cells as a percentage of total actin+DAPI+ (myocyte) in each region. N=5 in VEH and CBSC group. N=3 in noninfarcted controls. *P value ≤0.05, **P value ≤0.005 vs VEH, #P value ≤0.05, ##P value ≤0.005 vs CBSC. IZ indicates infarct zone; and RZ, remote zone.

To explore whether recruitment of endogenous stem cell populations to the areas of exogenous stem cell injections occurred, we examined the number of ckit+ stem cells near injection sites in all animals at 3 months post-MI. Only a few ckit+ cells were found (<0.01% of total nuclei in both groups). Representative images of ckit+ cells found near the injection sites are in the Online Data Supplement (Online Figure X).

CBSC Treatment Induces Vessel Formation

To investigate the formation of new vessel, we quantified the number of VWF+ and VWF+/smooth muscle actin+ vessel per mm2. A significant increase in VWF+ was found (versus vehicle) in CBSC-treated hearts, and dual positive vessels were observed in the IZ (Online Figure XIA and XIE.) However, in most other regions of the heart, there was no significant difference between groups (Online Figure XIB, XID, XIF, and XIH). Interestingly, there was a significant increase in the density of VWF+ vessels in the RZ of vehicle-treated animals versus CBSCs (Online Figure XIC). Significant increases in vessel density in CBSC-treated animals may be involved in the beneficial functional effects of CBSC treatment observed at 3 months post-MI.

Discussion

The present study was designed to test the safety and efficacy of transendocardial administration of CBSCs in a randomized, blinded, placebo-controlled preclinical swine model of I/R-induced MI. There were several major new findings. The studies performed showed that transendocardial injection of allogeneic CBSC is safe both acutely and chronically, giving no evidence of ectopic tissue formation or lethal arrhythmogenesis. CBSC retention within injection sites was shown at 72 hours post-IR/MI, and these CBSCs were EdU+, consistent with the idea that they were proliferative after injection. CBSCs had no effect on IR/MI-induced myocyte necrosis or on initial infarct size. However, CBSC treatment reduced reactive fibrosis (scar size) at 3 months post-IR/MI. Progressive structural pathological remodeling (ie, hypertrophy of viable tissue, wall thinning within the scar, and dilation of the LV chamber) was also reduced with allogeneic CBSC treatment. There was preservation of LV systolic and diastolic function and a greater cardiac functional reserve in the CBSC-treated animals. A small but significantly greater number of EdU+ cardiac myocytes were found in CBSC-treated hearts 3 months post-IR/MI. These studies strongly support the idea that delivery of CBSCs to the MI BDZ in the immediate post-IR/MI heart causes beneficial changes in post-MI remodeling that reduce adverse structural and functional derangements (Online Figure XII).

CBSCs Are Not Cardioprotective but Preserve Intrinsic Myocardial Performance

The present experiments showed that IR/MI induced similar amounts of cardiac injury in CBSC- and vehicle-treated animals. All IR/MI animals had similar increases in circulating troponin levels (Figure 2B and 2C). In addition, the amount of necrotic tissue identified with triphenyltetrazolium chloride staining at 3 days post-MI was identical in both treatment groups. These results strongly support the idea that CBSCs do not provide cardioprotection, to reduce the initial infarct size, at least with the approaches used in these experiments. However, we did not measure the potential effects of CBSCs on the area at risk, using Evan blue staining. These data would give insight to amount of tissue that could be lost to infarct expansion over time or rescued by CBSC therapy. Regardless, these results are not surprising because our IR/MI model causes necrotic cell death within the infarct core and an infarct BDZ of uncoupled cardiac tissue, as we have shown previously.30 Our results do suggest that CBSCs reduce infarct expansion, which would likely result from progressive death of those myocytes at the MI BDZ that have uncoupled from the parent myocardium and have survived the necrotic cell death caused by IR/MI. TUNEL staining revealed a reduction in nonmyocyte death 3 days post-MI and a trend toward a reduction in TUNEL+ myocyte in the CBSC-treated group. Others have shown acute cardioprotective features of exosomes from cardiosphere-derived stem cells under similar experimental conditions.39 One possible explanation for these differences is that our IR/MI model causes a large necrotic infarct core, which develops within minutes on reperfusion, whereas the damage in the previous report seems to produce an infarct with intact, non-necrotic myocytes with apoptotic myocyte death in the infarct core and BDZs.39

CBSC injections did not improve the echocardiographic-determined cardiac volumes (end-diastolic volume, end-systolic volume, and LVEF) or structural dimensions at 72 hours, providing additional evidence that these cells did not provide immediate cardiac protection. However, myocardial performance, measured by LV longitudinal and radial strain, was better preserved in CBSC-treated animals. Speckle-tracking echocardiographic strain analysis is known to be more sensitive and identifies global and regional abnormalities in cardiac muscle contraction earlier than classical ECHO-derived volumetric measurements (ie, LVEF).40,41 The basis for these CBSC-mediated improvements in global strain in the absence of a cardioprotective effect are unclear at present but might be best explained by a wall stabilizing effect of injected CBSCs.29

CBSCs Survive and Expand During the First Few Days Post-IR/MI

GFP+ CBSCs injected into the IR/MI BDZ were found in every animal at 3 days post-IR/MI. EdU was infused for 3 days, and most CBSCs were also EdU+, showing that a portion of the injected cells survive and proliferate during this period. We also found a large percentage of the nuclei in the injured regions of the heart were EdU+ (≈20%), and the vast majority of these cells were nonmyocytes. The number of EdU+ nonmyocytes in the damaged regions of the heart was increased in CBSC-treated animals. The identity of these proliferative cells was determined to primarily consist of VWF+ cells, smooth muscle actin+ cells, and CD45+ cells confirming that post-IR/MI, infiltrating cells are primarily from the circulation and myofibroblasts.42,43 The potential links between increases in proliferative nonmyocytes in CBSC-treated animals and the subsequent reduction in pathological post-MI remodeling needs to be defined.

CBSC Therapy Reduces post-MI Structural and Functional Remodeling

The 3-month post-IR/MI studies explored CBSC effects on scar size, cardiac hypertrophy, progressive LV-chamber remodeling, loss of contractility, and functional reserve. Similar to our previous results in mouse MI models,29 CBSC treatment resulted in a reduction in scar size (Figure 7A through 7C). It is not entirely clear how CBSCs cause a reduced scar burden, but modulation of the post IR/MI inflammatory response is likely to be involved44 and represents a topic for future studies and a target for future translational experiments. An inhibition or reduction in low levels of myocyte apoptosis (Figure 7) for long periods of time post-MI may also be a contributing factor to the increased myocyte mass and reduced scar burden in CBSC-treated animals.

Ventricular chamber dilation is a hallmark feature of pathological post-IR/MI remodeling.45 The present study showed that although CBSCs did not prevent necrotic cell death in the immediate post-MI time frame, they significantly slowed the rate of and reduced the amount of ventricular remodeling as independently observed by ECHO and invasive hemodynamics studies.

Depression of cardiac systolic function with reduced contractile reserve is a classical feature of human HF.46 The amount/extent of dysfunction is proportionate to the burden of the HF syndrome. The swine model of IR/MI captures early features of MI-induced HF. ECHO measurements showed progressive cardiac chamber dilation that was associated with progressive depression in cardiac systolic function (Figures 5 and 6 and Online Table IV). Our results also show a significant shift in end-diastolic and end-systolic pressure–volume relationships measured with invasive hemodynamic techniques (Figure 6A through 6C). Changes in the pressure–volume loops of 3-month post-MI animals document cardiac chamber dilation and reduced basal cardiac function (Figure 6A through 6F). Cardiac contractile reserve, as measured by a dobutamine response, was also significantly reduced in vehicle- versus CBSC-treated MI animals. Collectively, our studies show that CBSC treatment reduced post-MI structural remodeling and preserved post-MI cardiac systolic function and inotropic reserve (Figure 6G through 6I).

Our studies did not find evidence of allogeneic CBSC persistence or differentiation into new cardiac tissue (blood vessels or myocytes) at 3 months post-MI. These results suggest that paracrine factors released from CBSCs during the first few days/weeks after IR/MI are responsible for reduced post-MI remodeling and improved cardiac pump function.28,29 CBSCs were alive and proliferative in vivo at 72 hours post-MI. Collectively, our results suggest that CBSCs release factors that modify post-IR/MI wound healing to reduce scar size and possibly limit myocyte death in the MI BDZ in the weeks after MI, when infarcts size can expand.29,47

CBSC Therapy Increases Muscle Mass in the Post-MI Heart

The HW and HW/body weight ratio of vehicle- and CBSC-treated animals were not different 3 months after MI (Online Table II), but scar mass was significantly smaller in CBSC-treated hearts, so muscle mass was greater in these hearts. Myocyte size was significantly smaller in CBSC- versus vehicle-treated hearts with greater myocyte nuclear density per mm2 within viable cardiac tissue (eg, BDZ and RZ). In addition, there was significantly less cardiac myocyte apoptosis in CBSC- versus vehicle-treated hearts in the viable regions (eg, BDZ and RZ; Figure 7). These results, with simple, reliable techniques, strongly support the idea that there are more myocytes in the ventricles of CBSC-treated as compared with vehicle-treated hearts. There are many possible reasons for the greater number of myocytes found in CBSC-treated hearts at 3 months post-MI: (1) progressive and chronic expansion of myocyte death in vehicle-treated animals would leave these hearts with progressively fewer myocytes, whereas (2) CBSCs might prevent the death of myocytes in the infarct BDZs, preserving myocyte number and leaving these hearts with more myocytes than the vehicle-treated counterparts, and (3) CBSC treatment could induce new myocyte formation either through stimulation of endogenous myocyte division or recruitment of endogenous stem cells which then differentiate into new myocytes.48 We are confident that there are more cardiac myocytes in the ventricles of CBSC-treated hearts (versus vehicle-treated hearts) 3 months post-MI. Both reduced myocyte death and enhanced myocyte regeneration were found at specific time periods. However, we cannot predict the overall contribution of these two processes to the greater myocyte number in CBSC-treated hearts because we did not measure them continuously, and we do not know the temporal nature of the two processes.

Our results suggest that CBSC treatment induces an increase in the number of newly formed myocytes within the first week post-MI, particularly in the BDZs, and that these myocytes are derived from the host myocardium, not from recruitment of endogenous stem cell populations (Figure 8; Online Figures IX and X). Previous studies of post-MI cell therapy have reported a range of new myocyte formation from 1%12 to ≥50%,49,50 and the source of these new myocytes has been widely debated.12,49–52 The current consensus in the field is that the normal adult heart has a limited ability to form new myocytes but that after injury, there is a small but significant increase in new myocyte formation, with the new myocytes being derived primarily from preexisting myocytes.53–55 Our data are consistent with these ideas, and our previous in vitro study28 suggests that the paracrine factors released from CBSCs could significantly enhance new myocyte formation. However, the number of EdU+ myocytes identified is rather small (≈1.5% of the total myocytes counted) and EdU could be incorporated into cardiac myocytes that are not dividing.56 Also, the number of EdU+ myocytes observed (≈1.5% in the IZ and BDZs) in CBSC-treated hearts does not seem to be sufficient to explain the improved cardiac function. However, EdU labeling was only performed in the first week post-MI, and new myocyte formation that occurred after the first week post-MI would not be measured with our techniques. Multiple labeling periods could be used in future studies to address this issue. What is clear is that any newly formed myocytes were not derived from the injected CBSCs.

Study Limitations

This study looked at a single stem cell type, delivered using 1 technique (direct intramuscular delivery) and at 1 dose and 1 time point (immediate post-MI) in a small cohort of normal female pigs. The study showed significant CBSC benefits under the conditions tested. However, we recognize that our study was performed with a small sample size, so we are cautious with our conclusions. Future studies to translate these cells into a useful therapeutic would need to evaluate dose- and time-dependent effects and could include evaluations with magnetic resonance imaging with larger cohorts of animals. Mechanistic insights are almost always more limited in large animal studies. Investigating the area at risk with Evan blue staining would have helped confirm the potential for infarct expansion and any effects CBSC treatment may have had on the short- and long-term scar formation. Also, performing a study looking at EdU incorporation at different time points within the post-MI remodeling phase could help us understand the temporal nature of endogenous myocyte proliferation. However, we have used several independent, simple, and reliable methods to show that CBSCs improve post-MI remodeling and do so through paracrine effect(s) leading to preserved cardiac structure and function.

Conclusions

Our experiments show that CBSCs delivered by transendocardial injection into the infarct BDZ after IR/MI: (1) cause no adverse effects, (2) survive and expand for at least 72 hours in the infarct BDZ, (3) increase the number of proliferative cells in the injured heart after MI, (4) limit the progression of post-MI structural remodeling, and (5) preserve cardiac pump function and contractile reserve. This is done through (6) inhibition of myocytes apoptosis chronically in the heart and (7) an increase in the number of EdU+ cardiac myocytes. This leads to an increase in the total number of myocytes within the CBSC-treated hearts to be greater than in vehicle-treated hearts. These findings support the idea that CBSC therapy improves post-MI wound healing to reduce scar size, inhibit slow progressive myocyte death, increase myocyte number, and preserve cardiac function, limiting the progression toward HF with reduced ejection fraction.

Sources of Funding

This work was supported by the National Institute of Health, National Heart, Blood, and Lung Institute, Washington DC–Project Program grants 5P01HL108806-04 (S.R. Houser) and 1R56HL137850-01 (S. Mohsin) The work is also supported by American Heart Association–Predoctoral Fellowship (14PRE20450006; T.E. Sharp) and Scientist Development Grant (S. Mohsin).

Disclosures

S.R. Houser is a named inventor on intellectual property filings that are related to the subject of this paper. In addition, S.R. Houser is a co-founder, scientific advisor, and holds equity in Myocard Therapeutics, LLC, a biotech start-up which will license S.R. Houser’s cortical bone cell technology from Temple University for commercial development and clinical trials. Myocard Therapeutics, LLC has not funded any aspect of this research.

Footnotes

  • In August 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.65 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.311174/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    BDZ
    border zone
    CBSCs
    cortical bone stem cells
    HF
    heart failure
    HW
    heart weight
    IZ
    infarct zone
    LVEF
    left ventricular ejection fraction
    RZ
    remote zone
    VWF
    Von Willebrand Factor+

  • Received April 13, 2017.
  • Revision received August 30, 2017.
  • Accepted September 14, 2017.

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Novelty and Significance

What Is Known?

  • Ischemia–reperfusion myocardial infarction (MI) leads to scar formation within the infarct zone, pathological remodeling of remaining viable myocardium, and left ventricular chamber dilation leading to cardiac dysfunction, which is hallmarked by a reduced left ventricular ejection fraction.

  • Cortical bone stem cells (CBSCs) are unique from mesenchymal and cardiac-derived stem cells and may be superior in their ability to repair the heart post-MI.

What New Information Does This Article Contribute?

  • This is the first translational large animal study performed using CBSCs.

  • Allogeneic transendocardial delivery of CBSCs acutely post-MI is safe and efficacious, permitting cell retention for at least 3 days.

  • CBSC therapy alters the progression of pathological remodeling by increasing the number of proliferative cells at 3 days, reducing scar size through a reduction in myocyte apoptosis and an increase in endogenous myocytes proliferation at 3 months post-MI. This inhibition of pathological remodeling leads to preservation of cardiac structure and functional reserve.

Mesenchymal and cardiac stem cells have been studied during the last several decades as therapeutic strategies in the post-MI and heart failure settings. Although proven to be safe in patients, these cell types have shown modest improvements in functional and survival outcomes leading to the need for the investigation of novel cell and cell-based therapies. We identified a novel stem cell population derived from the CBSCs. Previously, we performed in vitro and in vivo studies comparing CBSC with more well-established cell therapeutics (mesenchymal stem cells and cardiac-derived stem cells). This present study is the first preclinical large animal study to investigate the safety, efficacy, and ultimately, the translational application of this cell type. We observed a change in the pathological remodeling that is classically seen post-MI. There was reduced scar size, less reactive myocyte growth within viable tissue, reduced myocyte cell death, an increase in the number of myocytes, and increase in the number of myocytes that had proliferated with CBSC versus vehicle treatment. This alteration of classical pathological remodeling lead to preserved cardiac structure and function over time. Moreover, a hallmark of the progression toward heart failure is a lack on systolic functional reserve, which was preserved with CBSC treatment when compared with vehicle treatment. These findings together demonstrate the safety and efficacy of CBSCs as a novel stem cell therapy in a preclinical large animal model and will be the foundation of future studies investigating dosing, time of delivery, and comparison with standard of care.

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  • Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial InfarctionNovelty and Significance
    Thomas E. Sharp, Giana J. Schena, Alexander R. Hobby, Timothy Starosta, Remus M. Berretta, Markus Wallner, Giulia Borghetti, Polina Gross, Daohai Yu, Jaslyn Johnson, Eric Feldsott, Danielle M. Trappanese, Amir Toib, Joseph E. Rabinowitz, Jon C. George, Hajime Kubo, Sadia Mohsin and Steven R. Houser
    Circulation Research. 2017;121:1263-1278, originally published September 14, 2017
    https://doi.org/10.1161/CIRCRESAHA.117.311174
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