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. 2015 Feb 11;8(5):425–431. doi: 10.1111/cts.12251

Autologous c‐Kit+ Mesenchymal Stem Cell Injections Provide Superior Therapeutic Benefit as Compared to c‐Kit+ Cardiac‐Derived Stem Cells in a Feline Model of Isoproterenol‐Induced Cardiomyopathy

Sharven Taghavi 1,2, Thomas E Sharp III 2, Jason M Duran 2, Catherine A Makarewich 2, Remus M Berretta 2, Tim Starosta 2, Hajime Kubo 2, Mary Barbe 3, Steven R Houser 2,✉
PMCID: PMC5351102  PMID: 25684108

Abstract

Background

Cardiac‐ (CSC) and mesenchymal‐derived (MSC) CD117+ isolated stem cells improve cardiac function after injury. However, no study has compared the therapeutic benefit of these cells when used autologously.

Methods

MSCs and CSCs were isolated on day 0. Cardiomyopathy was induced (day 28) by infusion of L‐isoproterenol (1,100 ug/kg/hour) from Alzet minipumps for 10 days. Bromodeoxyuridine (BrdU) was infused via minipumps (50 mg/mL) to identify proliferative cells during the injury phase. Following injury (day 38), autologous CSC (n = 7) and MSC (n = 4) were delivered by intracoronary injection. These animals were compared to those receiving sham injections by echocardiography, invasive hemodynamics, and immunohistochemistry.

Results

Fractional shortening improved with CSC (26.9 ± 1.1% vs. 16.1 ± 0.2%, p = 0.01) and MSC (25.1 ± 0.2% vs. 12.1 ± 0.5%, p = 0.01) as compared to shams. MSC were superior to CSC in improving left ventricle end‐diastolic (LVED) volume (37.7 ± 3.1% vs. 19.9 ± 9.4%, p = 0.03) and ejection fraction (27.7 ± 0.1% vs. 19.9 ± 0.4%, p = 0.02). LVED pressure was less in MSC (6.3 ± 1.3 mmHg) as compared to CSC (9.3 ± 0.7 mmHg) and sham (13.3 ± 0.7); p = 0.01. LV BrdU+ myocytes were higher in MSC (0.17 ± 0.03%) than CSC (0.09 ± 0.01%) and sham (0.06 ± 01%); p < 0.001.

Conclusions

Both CD117+ isolated CSC and MSC therapy improve cardiac function and attenuate pathological remodeling. However, MSC appear to confer additional benefit.

Keywords: heart failure, cardiomyopathy, cardiovascular diseases

Introduction

The mammalian heart contains a resident population of stem cells with the capacity for regeneration.1, 2, 3, 4, 5 This regeneration can occur during the normal aging process or in response to pathological stress, such as myocardial infarction.3, 6, 7, 8 These cardiac‐derived progenitor cells have been isolated and shown to have the capability of regenerating damaged myocardium in animal and human studies.9, 10, 11, 12 In addition, stem cells derived from bone marrow have shown similar regenerative capacity when used therapeutically.13, 14, 15, 16, 17, 18, 19, 20 One subpopulation of cells known to have cardiac regenerative capacity are those containing the cell marker c‐Kit. These stem cells are multipotent with capability of differentiating into myocytes, endothelial cells, and smooth muscle cells.5, 21, 22, 23, 24

The successful use of stem cells to repair damaged myocardium in animal studies has led to their use in human trials and substantial progress has been made with two adult stem cell sources—those from the heart itself and those from bone marrow.25, 26, 27, 28, 29, 30 While most clinical trials have focused on the use of mesenchymal stem cells (MSC), derived from bone marrow,31, 32, 33, 34 cardiac‐derived stem cells (CSC) have also been used with observed improvement in cardiac function.35 While both stem cell lineages are being studied, there is no clear consensus on which source of cells has the greatest capacity for repairing damaged myocardium. One study demonstrated that human fetal CSCs have greater capacity for regeneration than MSCs when injected into mice.36 However, no study to date has done a direct comparison of cardiac‐derived and bone marrow‐derived CD117+ isolated stem cells, when administered in autologous fashion.

The present study compared the ability of CD117+ isolated MSC and CSC to repair damaged myocardium in an established model of isoproterenol cardiomyopathy.3 CD117+ isolated CSCs were isolated from right atrial biopsy and culturing of this cardiac tissue and CD117+ isolated MSCs were isolated from bone marrow aspirates using established techniques.6, 24 Injury was induced by infusion of isoproterenol11 for 10 days, which causes diffuse myocardial injury. We infused 5‐bromodeoxyuridine (BrdU) on days 3 to 10, to identify proliferative cells. On day 10, the ISO injured animals were given intracoronary infusion of 1 × 106 MSC or CSC. Cardiac function was compared by echocardiogram37 and invasive hemodynamics 28 days after stem cell injection. Our results indicate that both MSCs and CSCs improve cardiac function, however, MSCs provide additional therapeutic benefit.

Methods

Isolation of c‐Kit+ isolated CSCs from the feline heart

Right atrial tissue was obtained by right‐sided thoracotomy on day 0. The atrial tissue was finely minced and cultured for 7–10 days on a fibronectin coated dish with cardiac stem cell expansion medium (DMEM‐ F12 supplemented with 10% FBS (Life Technologies, Norwalk, CT, USA), 0.2% insulin‐transferin‐selenium (Lonza, Walkersville, MD, USA), 1% penicillin‐streptomycin‐glutamine (Invitrogen, Norwalk, CT, USA), 1,000 U/mL LIF (Millipore, Darmstadt, Germany), 20 ng/mL bFGF (Peprotech, Rocky Hill, NJ, USA), and 40 ng/mL EGF (Sigma‐Aldrich, Allentown, PA, USA). The cells that grow from the tissue chunks were then dissociated and filtered through a 100 μm nylon mesh, and cultured on a poly‐D‐lysine coated dish for 7–10 days. These clusters were collected and c‐kit+ isolated cells were further isolated by incubating the suspenion with magnetic microbeads conjugated with human c‐kit antibody (Miltenyi Biotec Inc., San Diego CA, USA). The magnetic bead bound cells were collected using the OctoMACS magnetic separation unit (Miltenyi Biotec Inc.). The cells were then cultured on a tissue culture treated dish. CSCs were infected with a lentivirus carrying GFP (Qiagen Sciences, Germantown, MD, USA) using Lenti‐X Accelerator (Clontech, Mountain View, CA, USA).

Isolation of mesenchymal stem cells

Bone marrow aspiration of the humerus was carried out using an 18‐gauge aspiration catheter on day 0. Mononuclear cells were isolated from the aspirate (1–3 mL) using Ficoll‐paque (Stem Cell Technologies, Vancouver, BC, Canada). To isolate c‐kit+ bone marrow stem cells (BMSCs), the MNCs were incubated with magnetic microbeads conjugated with human c‐kit antibody (Miltenyi Biotec Inc.). The magnetic beads bound mononuclear cells were collected using the OctoMACS magnetic separation unit (Miltenyi Biotec Inc.). The cells were then cultured on a tissue culture dish with cardiac stem cell expansion medium. MSCs that were adhesive to the culture dish were retained for expansion and infected with a lentivirus carrying GFP (Qiagen Sciences) using Lenti‐X Accelerator (Clontech).

Isoproterenol (catecholamine)‐induced cardiomyopathy

Isoproterenol injury was carried out in adult, male felines approximately 5–6 months old as demonstrated in established techniques3 on day 28. Animals were divided into 4 groups: those receiving atrial biopsy and CSC injection (n = 7), those receiving atrial biopsy and sham injection (n = 5), those receiving bone marrow aspiration and MSC injection (n = 4), and those receiving bone marrow aspiration and sham injections (n = 6). Atrial surgery and bone marrow aspiration was carried out 4 weeks prior to isoproterenol injury. Isoproterenol was infused for 10 days, followed by removal of ISO pumps and stem cell injection on day 38, followed by sacrifice on day 66. BrdU pumps were inserted on day 31 and removed with ISO pumps on day 38. BrdU incorporates and identified DNA synthesis. Echocardiography was carried out at baseline on day 28, on day 38, and prior to sacrifice on day 66. Invasive hemodynamics were carried out prior to euthanization.

Intracoronary stem cell injection

A 4‐French through lumen balloon catheter (Edwards Lifesciences) was inserted into the right carotid artery and threaded into the aortic root under fluoroscopy. Aortic occlusion was carried out with inflation of the balloon. Aortic occlusion and coronary injection was verified using contrast medium and fluoroscopy. Approximately 1 × 106 MSCs or CSCs in 1 mL of phosphate buffered saline (PBS) were injected. Sham animals received a 1 mL injection of PBS.

Other techniques

All other techniques used in this study have been described in previous studies.3 Ejection fraction (EF) was measured at baseline, after 10 days of ISO injury, and at sacrifice.

Invasive hemodynamics

Invasive hemodynamic were carried out at sacrifice using a Scisense pressure system. The femoral artery was cannulated with a 4‐French fluid filled pressure transducing catheter. The catheter was threaded into the left ventricle under fluoroscopic guidance and pressure measurements were recorded for 10‐second intervals. End‐diastolic pressure, tau index, maximum dp/dt, and contractility index were calculated using the Scisense SP200 software (Transonic Systems, Inc., Ithaca, NY, USA).

Results

Improvement in cardiac function

Isoproterenol injury is known to decrease cardiac function. It reliably decreases EF and lowers fractional shortening. In addition, it results in worsened diastolic dysfunction as seen by a lower transmitral E/A ratio.3 CSCs and MSCs are known to improve cardiac function in the damaged heart.35, 38, 39, 40 This study tested the ability of these stem cells to improve cardiac function in ISO‐induced cardiomyopathy in a large animal.

Echo was carried out at baseline, after 10 days of ISO injury, and 4 weeks after stem cell or sham injection. Results of echo findings are shown in Figure 1. A comparison of EF is shown in Figure 1 A. Animals receiving MSCs had a superior improvement in EF as compared to those receiving CSCs (27.7 ± 0.1% vs. 19.9 ± 0.4%, p = 0.021). A comparison of fractional shortening is shown in Figure 1 B. Both the MSC‐treated (25.1 ± 0.2% vs. 12.1 ± 0.5%, p = 0.006) and CSC‐treated group (26.9 ± 1.1% vs. 16.1 ± 0.2%, p = 0.014) showed improvement in fractional shortening as compared to their control groups. In addition, both the MSC (26.8 ± 7.1% vs. 9.6 ± 0.6%, p = 0.009) and CSC groups (15.9 ± 0.8% vs. 6.9 ± 0.5%, p = 0.001) showed improvement in diastolic dysfunction as measured by E/A ratio as compared to control (Figure 1 C).

Figure 1.

Figure 1

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A comparison of (A) ejection fraction, (B) fractional shortening, (C) transmitral E/A ratio, (D) left ventricle end‐diastolic volume, (E) left ventricle end‐systolic volume, and (F) diastolic internal diameter is shown at baseline, after isoproterenol injury, and at sacrifice.

Invasive hemodynamics were used to measure cardiac function as shown in Figure 2. Maximum dp/dt is shown in Figure 2 A. Both MSC (2,098.3 ± 96.8 vs. 1,297.8 ± 48.4 mmHg/sec, p = 0.001) and CSC (1,659.7 ± 126.7 vs. 1,150.2 ± 56.6 mmHg/sec, p = 0.007) treated animals had higher maximum dp/dt as compared to control. However, the MSC‐treated animals had significantly higher dp/dt than CSC‐treated (2,098.3 ± 96.8 vs. 1,659.7 ± 126.7 mmHg/sec, p = 0.037). Contractility index is shown in Figure 2 B. MSC‐treated animals displayed better contractility over sham injected (41.6 ± 3.3 vs. 26.6 ± 1.6, p = 0.005) and CSC treatment resulted in better contractility over sham (40.5 ± 4.4 vs. 25.8 ± 2.0, p = 0.023).

Figure 2.

Figure 2

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A comparison of (A) maximum dp/dt, (B) contractility index, (C) left ventricle end‐diastolic pressure, and (D) relaxation index (tau) as measured by invasive hemodynamics is shown.

Attenuation of pathological remodeling

MSC treatment results in superior reversal of cardiac dilatation in isoproterenol cardiomyopathy as seen in Figure 1. Decrease in left ventricular end‐diastolic volume (37.7 ± 3.1% vs. 19.9 ± 9.4%, p = 0.029) and end‐systolic volume (58.8 ± 10.5 vs. 39.9 ± 5.8%, p = 0.014) was greater in the MSC‐treated as compared to the CSC‐treated as shown in Figures 1 D and 1 E. There was no significant difference in decline in end‐diastolic diameter as seen in Figure 1 F.

Hemodynamic measures of cardiac dilatation are shown in Figure 2. Figure 2 C demonstrates that MSC treatment results in lower end‐diastolic pressure than control (6.3 ± 1.3 vs. 13.3 ± 0.7 mmHg, p = 0.008) and CSC treatment (6.3 ± 1.3 vs. 9.3 ± 0.7 mmHg, p = 0.047). The relaxation index tau is shown in Figure 2 D. Animals treated with MSC (10.8 ± 1.1 vs. 17.0 ± 0.6, p = 0.002) and CSC (13.1 ± 1.4 vs. 19.2 ± 0.6, p = 0.010) had a lower relaxation index than sham. There was no significant difference between MSC‐ and CSC‐treated in the relaxation index.

Reduction of cardiac hypertrophy

Measures of left ventricle chamber wall thickness are shown in Figure 3. Reversal of cardiac hypertrophy as measured by wall thickness was greater in the MSC‐treated animals. As seen in Figure 3 A, animals receiving MSC had greater reversal of diastolic anterior wall thickening than sham (31.2 ± 1.3 vs. +40.1 ± 25.3%, p = 0.021) and those receiving CSC (31.2 ± 1.3 vs. 13.9 ± 3.3%, p = 0.008). In addition, MSC‐treated animals had greater improvement in diastolic posterior wall thickening as compared to sham‐ (35.9 ± 1.2 vs. +59.0 ± 0.8%, p < 0.001) and CSC‐treated (35.9 ± 1.2 vs. 2.2 ± 2.0%, p = 0.031) as shown in Figure 3 B. Animals receiving MSC demonstrated decreased heart weight/tibia length ratio as compared to CSC (1.359 ± 0.121 vs. 1.708 ± 0.232, p = 0.021) as seen in Figure 3 C.

Figure 3.

Figure 3

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A comparison of (A) diastolic anterior wall thickness, (B) diastolic posterior wall thickness, and (C) heart weight to tibia length ratio is shown.

Decrease in replacement fibrosis

A comparison of collagen deposition in representative animals shown in Figure 4. Animals treated with MSC (13.1 ± 5.6 vs. 29.4 ± 2.4%, p < 0.001) and CSC (20.1 ± 6.6 vs. 29.4 ± 2.4%, p = 0.002) had lower percentage of collagen deposition than MSC sham animals as shown in Figure 4 A. However, animals treated with MSC had less replacement fibrosis than those treated with CSC (13.1 ± 5.6 vs. 20.1 ± 6.6%, p = 0.004). Representative images are shown in Figure 4. An MSC sham animal in Figure 4 B is compared to a CSC treated in 4C and an MSC treated in Figure 4 D.

Figure 4.

Figure 4

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A comparison of percentage of left ventricle collagen deposition is shown in (A). A representative image of (B) MSC sham animals, (C) CSC treated animals, and (D) MSC treated animals is shown.

Increase in proliferative cells

Left atrial and ventricle counts for BrdU positive myocytes and nuclei are shown in Figure 5. These BrdU positive cells could have the potential to form myocytes. Left atrial BrdU positive cells were greatest in MSC treated as seen in Figure 5 A. MSC‐treated animals had significantly higher BrdU positive myocytes as compared to MSC sham animals (1.16 ± 0.27 vs. 0.42 ± 0.12%, p = 0.045) and CSC treated (1.16 ± 0.27 vs. 0.96 ± 0.23%, p = 0.049). CSC‐treated animals had a significantly greater number of left atrial BrdU positive myoctes as compared to MSC sham (0.96 ± 0.23 vs. 0.42 ± 0.12%, p = 0.043). Number of total left atrial BrdU positive nuclei was not significantly different as seen in Figure 5 B.

Figure 5.

Figure 5

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A comparison of (A) left atrium BrdU+ myocytes, (B) left atrium BrdU+ nuclei, (C) left ventricle BrdU+ myocytes, and (D) left ventricle BrdU+ nuclei is shown.

Total left ventricular BrdU positive myocytes and BrdU positive nuclei are shown for representative animals in Figures 5 C and 5 D. Left ventricle BrdU positive myocytes were greatest in MSC treated (0.13 ± 0.06%), followed by the CSC treated (0.10 ± 0.04%), then the MSC sham group (0.04 ± 0.02%); p < 0.001. Number of total left ventricle BrdU positive nuclei was not significantly different as seen in Figure 5 D. A left atrial BrdU positive myocyte is shown in Figure 6. GFP+ myocytes could not be located in MSC‐ or CSC‐treated animals via immunohistochemistry.

Figure 6.

Figure 6

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Stains for (B) α‐actin, (C) BrdU, (D) DAPI, and (A) a merged image of all stains is shown.

Discussion

Cardiac disease remains a leading cause of morbidity and mortality worldwide.41 The heart was once thought to be a terminally differentiated organ without capacity for regeneration.1, 2, 42 However, studies have shown that the adult heart contains resident progenitor cells with the capacity for myocyte regeneration.5, 7, 43 This has opened up the door for using these progenitor cells for therapeutic benefit. Multiple sources of stem cells have been demonstrated to have capability to improve cardiac function in the face of physiological stress.13, 14, 31, 35 While most clinical trials have focused on stem cells derived from bone marrow,25, 31, 34 the optimal source of stem cells has yet to be determined.

Our previous study has shown that ISO infusion causes reliable cardiac injury. This feline cardiomyopathy model results in depressed systolic and diastolic function with profound dilatation of atria and ventricles. In addition, there is significant myocyte hypertrophy and replacement fibrosis. Myocytes in this model behave like myocytes in end‐stage human heart failure.3 The purpose of this study was to perform a direct comparison of the therapeutic benefit of CD117+ isolated MSCs and CSCs when used in autologous fashion.

CD117+ isolated MSCs are superior to CD117+ isolated CSCs in improving cardiac function

Our experiments show that both sources of stem cells give therapeutic benefit, however, MSCs provide additional advantage. Treatment with MSCs resulted in improved contractility and better systolic and diastolic function. In addition, MSCs were better at attenuating the pathological changes of heart failure. It reversed ventricular dilatation and decreased LV end diastolic pressure to a greater extent than CSCs. While a direct comparison of autologous MSC and CSC has not been carried out to this point, there is evidence that MSCs can act synergistically with CSCs,44 although stem cell delivery in this study was not done autologously. Previous work has shown that in this model of isoproterenol cardiomyopathy, there are resident progenitor cells in the myocardium that regenerate myocardium. Treatment with autologous MSCs and the presence of these resident CSCs in the myocardium may allow for this synergistic effect, which results in better cardiac function than treatment with CSCs alone. Future studies are needed to examine this treatment strategy.

Previous comparisons of MSCs and CSCs have been limited to in vitro studies or nonautologous animal studies. One such study compared the cardiac differentiation ability of MSCs and CSCs in monoculture and with neonatal rat cardiac myocytes.45 In this study, CSCs showed greater potential for differentiating into cardiac myocytes.45 While CSCs may have greater ability to commit to the cardiac lineage and transdifferentiate into cardiac myocytes,45 this in vitro study cannot account for the ability of MSCs to improve cardiac function through a paracrine effect. MSCs are known to secrete paracrine factors that contribute to endogenous cardiomyogenesis and angiogenesis.46, 47, 48, 49, 50 MSCs secrete a number of cytokines and growth factors that can inhibit fibrosis and apoptosis and enhance the ability of tissue‐specific stem cells to differentiate.51, 52, 53, 54 Given the inability to find stem cells that transdifferentiated into myocytes, evidence from our study indicates that it is through this paracrine effect that MSCs provide superior therapeutic benefit.

Another study has compared human CSCs and MSCs in a murine model of myocardial infarction. While this study showed CSCs to be superior to MSCs in improving cardiac function, cell treatments were not autologous and were carried out in a small animal model.36 Our study is the first to use large mammals and with autologous stem cell treatments.

MSCs and CSCs cells have a paracrine effect

In the present study, there was no evidence of CSC or MSC transdifferentiation into myocytes. However, we demonstrated a significant increase in BrdU‐positive myocytes in both stem cell treated groups, which may represent proliferating cells. These findings suggest that the improvement in cardiac function seen with stem cell therapy occurs predominately through a paracrine effect. This has been supported in previous studies.55, 56, 57 Previous studies demonstrate that endogenous cardiac repair occurs after removal of ISO.3 Therefore the injected stem cells likely secrete paracrine factors early in the recovery phase and die off prior to sacrifice at 28 days post‐ISO. These paracrine factors result in improved cardiac function and decreased pathological remodeling. This paracrine effect allows for MSCs to augment cardiac repair to a greater extent than CSCs.

Increase in proliferative cells for both sources but greater in MSC

Our previous work has shown that ISO injury leads to proliferation of a pool of cells with cardiogenic potential as demonstrated with BrdU+ labeled myocytes. These cells are transiently proliferative and form new myocytes upon removal of ISO.3 Treatment with both MSCs and CSCs resulted in an increased number of these BrdU+ cells as compared to control. Animals treated with MSCs may have a greater number of proliferating cells than those treated with CSCs. While direct engraftment and differentiation may play a role in mediating cardiac repair, data from this study indicate that the enhanced recovery seen in MSC‐treated animals is more likely through secreted soluble paracrine factors. Previous studies have shown that MSCs engraft when injected directly into the myocardium, however, improvement in cardiac function is predominately through the stimulation of resident progenitor cells in the myocardium.57

The atria regenerates more than the ventricle

Our previous study has demonstrated that ISO injury results in proliferation of cells in the atria and ventricle and there appears to be more robust regeneration in the atria.3 In this study, treatment with CSC and MSCs resulted in increased proliferation in both the atria and ventricle. Even after treatment with MSC and CSCs, proliferation of progenitor cells is greater in the atria than the ventricle. Further studies are needed to compare the proliferative capacity of atrial and ventricular tissue.

Limitations

This study was performed with an ISO injury model rather than a myocardial infarction model. This was done for several reasons. Significant infarcts are not easily obtained in felines due to their substantial coronary collateral circulation. In addition, ISO injury provides a reliable and reproducible injury. This injury is diffuse and the injury phase can be rapidly terminated by removal of the ISO pumps. This allows for there to be a defined injury phase followed by rapid removal of the noxious stimulus. In addition, we did not perform flow cytometry (FACS) analysis of the stem cells that were administered. Commercially available CD117 antibodies that work routinely in rodent and human cells do not work well in feline cells. In addition, it has become increasingly clear that CD117 expression is reduced both with time and with passage in culture,58 making FACS analysis of our stem cells problematic. For these reasons we did not perform FACS analysis.

We must be cautious in saying BrdU is used to identify new myocytes because it could be incorporated into myocyte DNA during repair or in myocytes with DNA synthesis without cytokinesis. However, previous data have shown that this is unlikely and that BrdU+ myocytes likely represent proliferating cells.3

Conclusion

In summary, our data show that both MSCs and CSCs enhance repair of the ISO injured heart. MSCs appear to be superior to CSCs in providing therapeutic benefit. These progenitor cells may work through a paracrine mechanism. Future studies should focus on MSCs as they appear to be the optimal source of stem cells for treatment of cardiac disease.

Disclosures

The authors have no disclosures to report.

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