ADSC to Porcin MI model20171013



Cell Spray Transplantation of Adipose-Derived Mesenchymal Stem Cell Recovers Ischemic Cardiomyopathy in a Porcine Model

Daisuke Mori,1 MD, Shigeru Miyagawa, MD, PhD,1 Shin Yajima, MD,1 Shunsuke Saito, MD, PhD,1 Satsuki Fukushima, MD, PhD,1 Takayoshi Ueno, MD, PhD,1 Koichi Toda, MD,1 PhD, Kotoe Kawai,3,5 Hayato Kurata,3,5 Hiroyuki Nishida,3,5 Kayako Isohashi, MD,4 PhD, Jun Hatazawa,4 MD, PhD, and Yoshiki Sawa, MD, PhD1,2

1Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Japan

2Medical Center for Translational Research, Osaka University Hospital, Osaka, Japan

3Institute of Advanced Stem Cell Therapy, Osaka University, Osaka, Japan

4Department of Nuclear Medicine and Tracer Kinetics, Osaka University Graduate School of Medicine, Suita, Japan

5ROHTO Pharmaceutical Co.,Ltd

*Corresponding author

Yoshiki Sawa, MD, PhD

Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

E-mail: sawa-p@surg1.med.osaka-u.ac.jp

TEL: +81-6-6879-3154/FAX: +81-6-6879-3163

Author contributions

D.M. participated in research design performance of research, analyzed data, and wrote the article.

S.Y., K.K., H.K., and H.N. performed research and analyzed data.

K.I. and J.H. participated in performing PET.(helped with some experiments)

S.S., S.F., T.U., and K.T. reviewed all data and article.

S.M. and Y.S.participated in research design, writing of the article and reviewed all data and article.

All authors have met the following criteria: drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Abbreviations

ADSCs, adipose-derived mesenchymal stem cells; bFGF, basic fibroblast growth factor; BM-MSCs, bone marrow-derived mesenchymal stem cells; CFR, coronary flow reserve; EDPVR, end-diastolic pressure-volume relationship; ESPVR, end-systolic pressure-volume relationship; ICM, ischemic cardiomyopathy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HGF, human growth factor; LAD, left anterior descending coronary artery; LV, left ventricle; LVDd, left ventricular end-diastolic diameter; LVDs, left ventricular end-systolic diameters; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; LVEF, left ventricular ejection fraction; MBF, myocardial blood flow; MFR, myocardial flow reserve; MI, myocardial infarction; MRI, magnetic resonance imaging; NH3-PET, ammonia-positron emission tomography; PBS, phosphate-buffered saline; PAS, periodic acid-Schiff; PSLS, peak-systolic longitudinal strain; RT-PCR, reverse transcription-polymerase chain reaction; SDF-1, stromal cell-derived factor 1; VEGF, vascular endothelial growth factor

Disclosure

This study was supported by Rohto Pharmaceutical Co., Ltd. Dr. Sawa serves as an advisor for the sponsor. Dr. Sawa and Dr. Miyagawa received a speaking fee from the sponsor. Ms. Kawai, Mr. Kurata, and Mr. Nishida receive a salary from the sponsor where they are employees. The sponsor had no control over the interpretation, writing, or publication of this work. The terms of this arrangement have been reviewed and approved by Osaka University in accordance with its policy on objectivity in research.

Funding

This work was supported by the Department of Advanced Stem Cell Therapy (Rohto Pharmaceutical Co. Ltd.).

Abstract

Background: Allogeneic adipose-derived mesenchymal stem cells (ADSC) are promising cell sources for cell therapy to treat ischemic cardiomyopathy (ICM).

We hypothesized that ADSC transplantation via the new cell spray method may be a feasible, safe, and effective treatment for ICM. Methods: Human ADSCs were acquired from white adipose tissue. Porcine ICM models were established by constriction of the left anterior descending coronary artery. ADSCs were spread over the surface of the heart via cell spray in fibrinogen and thrombin solutions. The cardiac function was compared to that of the control group. Results: ADSCs were successfully transplanted forming a graft-like gel film covering the infarct myocardium. Premature ventricular contractions were rarely detected in the first 3 days after transplantation. Echocardiography and magnetic resonance imaging revealed improved cardiac performance of the ADSC group at 4 and 8 weeks after transplantation. Systolic and diastolic parameters were significantly greater in the ADSC group at 8 weeks after transplantation. Histological examination showed significantly attenuated left ventricular remodeling and a greater vascular density in the infarct border area in the ADSC group. Moreover, the coronary flow reserve was maintained, and expression levels of angiogenesis-related factors in the infarct border and remote areas were significantly increased. Conclusion: Spray method implantation of allogenic ADSCs can improve recovery of cardiac function in a porcine infarction model. This new allogenic cell delivery system may help to resolve current limitations of invasiveness and cost in stem cell therapy.

INTRODUCTION

The field of regenerative medicine is expanding at a rapid rate with a large number of clinical trials and studies being conducted worldwide, and the long-term effects of these treatments will soon come to light to usher in a new era of stem cell therapy.1 Currently, the main challenges of regenerative medicine include determination of the optimal cell type, timing of incorporation, and site of transplantation, especially in treatment for ischemic cardiomyopathy (ICM), which shows a highly variable pathophysiology between the acute phase and chronic phase after onset.2

Mesenchymal stem cells (MSCs) are considered to be suitable for clinical application, because they show good immunotolerance along with vascularization and anti-inflammatory effects, so that they can be effective at both the acute and chronic phases of ischemia. As an innovative treatment for heart failure, autologous cell transplantation has been introduced in clinical settings with ease because of the minimal ethical problems. However, this treatment strategy also has some drawbacks such as maintaining cell uniformity and high costs.3

Considering these problems, it is important to investigate suitable methods for introducing appropriate cells in clinical treatment, and to deliver the cells effectively to the myocardium in a less invasive manner while reducing the overall cost of the treatment.4 In this regard, introduction of immunotolerant MSCs with allogeneic or xenogeneic cell transplantation holds great promise for achieving cell uniformity at a low cost with clinical convenience. In particular, adipose-derived mesenchymal stem cells (ADSCs), which can be automatically isolated from the fat tissue and industrially cultured to generate a large number of cells, show cytokine paracrine ability with a relatively low immune response in vivo.5,6

With respect to the transplantation method, the use of epicardial transplantation such as the cell sheet method, intracoronary administration, intramyocardial injection, or intravenous injection have all been used in clinical applications to date.7–9 Although intracoronary administration and intramyocardial injection are less invasive methods, they are also associated with certain complications such as microembolization, leading to myocardial infarction, arrhythmia, and pulmonary infarction, which has raised some concern and uncertainty with regard to the use of this transplantation site and cell transplantation method. By contrast, the epicardial implantation method has been reported to show improved restoration of implanted cells compared with other methods, although this method is relatively more invasive.10 Thus, to promote the practical use of ADSC transplantation, it is essential to develop a less invasive method and to improve the accuracy of cell delivery so as to achieve maximal effectiveness for the treatment of ischemic heart disease; however, no such method has been established to date that can meet all of the requirements for clinical application in a variety of situations.

We recently developed “the cell spray method” as a new cell delivery system in which fibrinogen- and thrombin-containing cells are directly spread onto the epicardium of the infarct area. This allowed for tissues to be regenerated at the targeted site without a cell processing center. We hypothesized that implantation of ADSCs with the cell spray method might have a positive impact on the recovery of cardiac function. Therefore, in the present study, we evaluated the clinical feasibility of this method using a porcine infarction model, with the aim of translating this new cell delivery system to clinical application.

MATERIAL AND METHODS

Ethical Statement

All experimental procedures were approved by the Institutional Ethics Committee. Animal care was conducted humanely in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Animal Resources and published by the National Institutes of Health (Eighth Edition, revised 2011).

Human ADSC Preparation

Normal human ADSCs of fourth passage were purchased from Lonza Japan, Ltd. (Tokyo, Japan) and cultured at 37C and 5% CO2 for a few days. The ADSCs were then subcultured 4 times and cryopreserved. (SDC, Materials and Methods)

Flow Cytometry

The identity of ADSCs was confirmed by evaluation of various cell surface markers with flow cytometry. ADSCs were mechanically dissociated and resuspended in fluorescence-activated cell sorting (FACS) staining buffer (PBS supplemented with 5% fetal bovine serum). The antibodies used for FACS analysis were mouse anti-human CD11b, CD45, CD73, CD90, and the corresponding mouse IgG1 isotype conjugated to fluorescein isothiocyanate or phycoerythrin. The cells were stained for 30 min at room temperature, and then washed and examined using a BD FADSverse instrument (BD Biosciences, San Jose, CA, USA). The data were analyzed using the BD FACSDiva software (BD Biosciences).

ADSC Graft Solution and Spraying Procedure

The fibrin and thrombin solution for the cell spray was prepared using a Beriplast P Combi-Set Tissue adhesion kit (CLS Behring. Co. Ltd.). The 2 solutions were prepared, solution A and solution B. Solution A contained 80 mg/mL of fibrinogen, 60 IU of factor XIII, and 5000 KIE of bovine aprotinin in Buffer A, and solution B contained 300 IU/mL of thrombin in Buffer B. Each solution was then diluted with Hanks’ balanced salt solution. ADSCs (1 × 108/graft) were mixed with solution A just before spraying. The final concentrations of the fibrinogen and thrombin solution are provided in Table 1.

The solutions were spread over the infarct area from a distance of 5–10 cm by 0.05 Pa CO2 gas using a balloon-ring device which stabilize the beating heart and prevent the leakage of solution.

These 2 solutions are mixed in the air completely and takes few second until the graft has harden and adhered to the heart (Fig. 4A, 4B, and Supplemental video).

Enzyme-linked Immunosorbent Assay (ELISA)

Human ADSCs from passage 4 were seeded by the cell spray method on a 6-well plate and the small scale graft was cultured for 72 h. The culture supernatant was collected and the concentrations of various cytokines were assessed by ELISA as described previously.11

Generation of the Porcine ICM Model and ADSC Implantation

Myocardial infarction (MI) was induced in 12 female minipigs (Crown minipig; Japan Farm, Kagoshima, Japan) weighing 20–25 kg.12

Four weeks after MI induction, the minipigs were randomly divided into 2 treatment groups (n = 6 per group): ADSC transplantation or sham operation. In the ADSC transplant group, 1 × 108 ADSCs were sprayed over the infarct area of the myocardium, which was identified visually on the basis of surface scarring and abnormal wall motion. Echocardiography, cardiac magnetic resonance imaging (MRI), ammonia-positron emission tomography (NH3-PET), and cardiac catheterization were serially performed at pretransplantation (baseline), 4 weeks, and 8 weeks after ADSC transplantation. In the sham operation group, the chest was opened but no ADSC transplantation was performed.

At the endpoint of the study (8 weeks after cell transplantation), the animals were humanely sacrificed for histologic and biochemical analyses of the heart tissue. None of the animals received immunosuppression after transplantation (Fig. 1) (SDC, Materials and Methods).

Graft Survival

To detect the transplanted ADSCs in the heart tissue, the ADSCs were labeled prior to transplantation or stained with anti-human mitochondria antibody (Merck KGaA, Darmstadt, Germany) at 2 days, 4weeks and 8weeks after transplantation (SDC, Materials and Methods).

Echocardiography

The minipigs were anesthetized with inhalational anesthetic without intubation, and echocardiography was performed with the commercially available echocardiograph ProSound F75 Premier (Hitachi Ltd., Tokyo, Japan). An 8.0-MHz annular array transducer was used for cardiac evaluation. The minipigs were examined in a shallow left lateral decubitus position. The left ventricle (LV) end-diastolic and end-systolic diameters (LVDd and LVDs, respectively) were measured, while the LV end-diastolic and end-systolic volumes (LVEDV and LVESV, respectively) and LV ejection fraction (LVEF) were calculated from the the Teichholz formula: LVEF (%) = 100 × (LVEDV - LVESV)/(LVEDV).13

Cardiac Catheterization

At pretreatment and at the end of the study (8 weeks after treatment), the following indices were calculated: dP/dtmax, dP/dtmin, the time constant of isovolumic relaxation (τ), end-diastolic pressure-volume relationship (EDPVR), and end-systolic pressure-volume relationship (ESPVR). These parameters were myocardial-specific parameters that are independent of systemic condition such as blood pressure. (SDC, Materials and Methods)

MRI

The pigs were sedated by intramuscular injection of 10 mg/kg ketamine + 2 mg/kg xylazine and atropine 1A. After sedation, the pigs were shaved and a tracheal tube was inserted. The peripheral intravenous lines were inserted before placing the pig on the examination table in the MRI room. While in the supine position, the distribution pipe of the anesthesia machine was connected to the tracheal tube, and the electrocardiogram electrodes were attached to the chest. Isoflurane was used as the anesthetic, and the concentration was maintained at 1–2%. The coil was fit to cover the chest area, and the pig was moved into the tunnel of the MRI machine (Signa EXCITE XI Twin Speed 1.5T Ver.11.1, GE Healthcare).

Two types of imaging were performed: cine MRI, which shows the heart movement, and delayed enhanced MRI using a gadolinium-based contrast. (SDC, Materials and Methods)

13N-NH3 myocardial perfusion PET

13N-NH3-PET protocol

Six consecutive pigs subject to MI underwent rest and stress 13N-NH3 myocardial perfusion PET before and after treatment. PET was performed with the Headtome-V/SET 2400 W PET system (Shimadzu, Kyoto, Japan). (SDC, Materials and Methods)

Histological Analysis

The ADSCs and excised heart specimens were fixed with 10% buffered formalin and embedded in paraffin. The paraffin-embedded sections were stained with hematoxylin and eosin and visualized using standard light microscopy. Picosirius red or periodic acid-Schiff (PAS) staining was performed to assess the degree of interstitial fibrosis or cardiomyocyte hypertrophy, respectively.14,15 Endothelial cells were labeled with rat monoclonal anti-CD31 antibody (Abcam, Cambridge, UK; 1:50) and visualized by corresponding secondary antibodies (Alexafluor 488 or Alexafluor 555; Alexafluor 647 Molecular Probes, Eugene, OR, USA), and then counterstained with Hoechst 33342 (Dojindo, Kumamoto, Japan). Five different fields were randomly selected, and the number of stained vascular endothelial cells in each field was counted using a light microscope under high-power magnification (400×). The number of stained blood vessels from the 5 fields was averaged and the results are expressed as the vascular density (per square millimeter).

Reverse Transcription (RT)-PCR

One specimen was excised from each minipig heart to perform RT-PCR. Immediately after sacrifice, the samples were extracted and fixed with RNA stabilization reagent (RNAlater; QIAGEN, Inc., Tokyo, Japan). Total RNA was extracted from the cardiac tissue, reverse-transcribed using TaqMan reverse transcription reagent (Applied Biosystems, Stockholm, Sweden), and RT-PCR was performed with the ABI ViiA7 system (Thermo Fisher Scientific Inc.) using pig-specific primers for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and stromal cell-derived factor 1 (SDF-1). Complementary DNA samples were prepared and assayed in triplicate. The average copy number of gene transcripts was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample.16

Statistical Analysis

Values are given as the mean ± standard error of the mean. Student’s t test (two-tailed) was used to compare 2 groups of independent samples. One-way and two-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons were performed to assess within- and between-group differences following the treatments. Following ANOVA, between-group comparisons were made using a Student’s t-test (two-tailed). JMP 13 software (SAS Institute Inc., Cary, NC, USA) was used for all analyses, with P values < 0.05 deemed to indicate statistical significance.

RESULTS

Characteristics of Human ADSCs

Immunophenotyping of undifferentiated human ADSCs for various cell surface markers was carried out by flow cytometry, which revealed that the fluorescent intensity and distribution of the cells stained for CD11b and CD45 were not significantly different from those of the cells stained with isotope controls. These results indicated that the cells were devoid of any hematopoietic stem and/or progenitor cells. In contrast, human ADSCs displayed high expression of CD73, CD90, and CD105 surface antigens. The expression profiles of these surface molecules were consistent with previous reports (Fig. 2A).

Serum-free conditioned media from the ADSCs were screened for secreted factors using ELISA. The media contained high concentrations of various factors such as human growth factor (HGF), VEGF, and SDF-1, which have previously been reported as cardiac-protective and remodeling suppressive factors (Fig. 2B).

ADSC Properties in the Spraying Fluid

Using a balloon of 3 cm inside diameter for the prevention of fluid leakage, the solution of the same amount as in vivo experiment was sprayed on a petri dish and. formed a round-shaped ADSC graft. The graft strongly adhered onto the surface of the culture dish and the heart. Cross-sectional analysis of the small-scaled ADSC graft with hematoxylin-eosin staining revealed a regular structure approximately 10 mm in diameter and 500–600 μm thick (Fig. 3A). Cells in the graft were distributed uniformly and there was no apparent microscopic change in the shape (Fig. 3B and 3C).

Some of the cells in the graft became spindle-shaped and showed an actin-positive nanotube-like structure after 72 h of culture (Fig. 3D). To assess the cell survival and proliferation in the fibrin gel, immunohistochemical staining with Ki-67 was performed. Approximately 10–20% of the cells were Ki-67-positive after 24-h culture, indicating active proliferation (Fig. 3E). Moreover, ELISA showed that human VEGF, HGF, and SDF-1 were secreted from the ADSC graft into the culture supernatant over time (Fig. 3F).

Feasibility and Safety of ADSC Spray Transplantation for the Porcine ICM Model

Transplantation of ADSCs was successfully performed through median sternotomy under general anesthesia in 6 minipigs with LVEF values of 30–50% due to the induced chronic MI. There was no mortality related to the procedure or other factors before the planned euthanasia .

Cardiac Function after ADSC Transplantation

Serial standard transthoracic echocardiography was performed before and 4 weeks after transplantation or sham operation (Fig. 5A). The baseline LVDd, LVDs, and LVEF did not differ significantly between the 2 groups. The minipigs in the sham group exhibited a nonsignificant upward trend in LVDd and LVDs, and a downward trend in the LVEF 4 weeks after surgery. The LVEF was significantly greater in the transplant group than in the sham group 4 weeks after treatment (p < 0.05 for the global effect of time and group in the repeated ANOVA). The LVDs was significantly smaller in the transplant group than in the sham group at 4 weeks after the treatment (p < 0.05 for the global effect of time and group in the repeated ANOVA), whereas the LVDd and the LV anterior wall thickness did not differ significantly between the 2 groups (Fig. 5B) (Table S1 and S2).

Global and Regional Cardiac Strain, ESPVR, and EDPVR Evaluated with MRI and an LV Catheter

The LVEF calculated from all 4 views (4-chamber, 2-chamber, long-axis, and short axis at the papillary muscle level) of the cine MRI demonstrated clear improvement in the transplant group (Fig. 6A). Similarly, peak-systolic longitudinal strain (PSLS) was improved in the transplant group compared with that of the sham group. There was no individual decrease in the EF and PSLS at 4 weeks after treatment (Fig. 6B–F) (Table S3, S4 and S5).

In addition, invasive hemodynamic analysis showed that the ESPVR and EDPVR were well preserved after implantation of ADSCs compared to those of the sham group (Fig. 7A and B) (Table S6 and S7).

Global and Regional Changes in MBF and Coronary Flow Reserve (CFR)

Four weeks after MI induction, the global MBF at rest and during stress showed a substantial decrease in both groups, especially in the LAD territory (Fig. 8A), with no significant differences. Similarly, the global CFR did not differ between the 2 groups. However, at 4 weeks after treatment, the global CFR was substantially higher in the transplant group than in the sham group. The LAD area showed the most remarkable improvement with regard to the magnitude of change in the territorial CFR, as evidenced by a higher ratio of post to pretreatment CFR (p < 0.05 respectively; Fig. 8B).

Graft Survival

The stem cells labeled with MitoTracker was transplanted on the surface of the heart and we could detect the graft present in the heart macroscopically and microscopically 2 days after transplantation.

Immunohistochemistry using a human-specific anti-mitochondrial antibody demonstrated that 4 weeks after epicardial transplantation, implanted ADSCs were still present in the heart (Fig. 9). At 2 days after transplantation, real-time PCR detected 0.794 ng of human DNA among 20 ng of the genomic DNA extracted from heart tissues, whereas no human DNA was detected among the total DNA extracted from the heart tissues at 8 weeks after transplantation. (Table 2)

Pathological Hypertrophy, Fibrosis, and Vascular Density

The heart tissues excised 8 weeks after transplantation were assessed histologically. Representative whole heart specimens from both groups are shown in Fig. 10A, indicating that the thickness of the myocardium in the ADSC group was well-maintained, unlike that of the sham group. The extent of pathological cardiomyocyte hypertrophy, interstitial fibrosis, and vascular density was assessed at 8 weeks after treatment semi-quantitatively by PAS staining (Fig. 10D), Masson’s trichrome staining, and immunohistochemistry for anti-CD31 antibody, respectively (Fig. 10F). Masson's trichrome staining showed that the infarct area was significantly smaller in the ADSC transplant group than that in the sham group (p < 0.05; Fig. 10B). There was consistently significantly less accumulation of interstitial fibrosis in the remote area in the transplant group than in the sham group (p < 0.0001; Fig. 10C). In addition, the diameters of the cardiomyocytes in the peri-infarct area were significantly smaller in the transplant group than in the sham group (p < 0.05; Fig. 10E). Moreover, the capillary density in the border area was significantly greater in the ADSC group than that in the control group (p < 0.05; Fig. 10G).

Expression of VEGF, bFGF, and SDF-1 in the Heart

The expression levels of growth factors that are expressed in the myocardium and are potentially related to neovascularization were quantified by RT-PCR at 8 weeks after treatment. VEGF showed a tendency toward an increase in the transplant group, and the expression levels of SDF-1 and bFGF in the infarct border and in the remote area were significantly greater in the transplant group than in the sham group (p < 0.05, Fig. 11A–C).

DISCUSSION

In this study, we found that the transplantation of allogenic human ADSCs with our newly developed spray method was feasible, safe, and effective in a porcine MI model. After the treatment, cardiac function was significantly improved based on echocardiography and there was no mortality related to the procedure. Moreover, cardiac MRI and histological examination indicated that LV remodeling was attenuated and neovascularization in the infarct border area significantly increased after ADSC transplantation. Furthermore, NH3-PET revealed improvement in coronary blood flow in not only the infarct border area but also in areas remote to the infarction site.

Recently, basic research and clinical trials have shown that allogeneic or autologous ADSC transplantation by trans-endocardial injection, direct surgical injection, or intracoronary injection is minimally invasive and resulted in improvement in cardiac function.17–19 However, intra-myocardial injection via an epicardial or endocardial approach is also associated with the risk of inducing arrhythmia, and there is currently uncertainty related to transplantation procedures such as the blowing out of cells from the myocardium, which results in difficulty for delivering a sufficient number of cells to the heart.20–22 Compared with these methods, the cell sheet method has been reported to be safer with less arrhythmogenicity, and shows advantages with respect to graft retention; however, this method is also associated with disadvantages in graft preparation, as it requires a cell processing center, bringing about high costs and surgical invasiveness.23

On the other hand, the cell spray method may have potential to achieve transplantation through a relatively less invasive surgery with an endoscope, since only syringes are required for transplantation, and has an additional advantage in that the cells can be implanted immediately after dissolving the frozen cell stock (Fig. 12). Therefore, our results demonstrate the possibility of developing a new transplant technology toward realizing less invasiveness in clinical application that could be equivalent to endovascular catheter treatment.

In the cell spray method, fibrinogen functions as an extracellular matrix, which might provide a bed for the cell engraftment and further enhance cytokines production in the matrix containing ADSCs.24,25 We further speculated that the transplanted ADSCs would be under a hypoxic condition in the fibrin glue, which would enhance cytokine expression and angiogenesis in the damaged myocardium.26 Indeed, our ADSCs embedded in the fibrin gel secreted substantially more cytokines than simply cultured ADSC, which may support this hypothesis.

Overall, this study suggested that ADSCs transplantation by the cell spray method provides compatible functional recovery via angiogenesis to that of the previously reported autologous myoblast sheet method. Thus, the cell spray method may be superior with respect to the less invasiveness given that it can be performed through an endoscopic approach compared with the cell sheet method and is of lower cost because of the lack of need for a cell processing center.27

Although we can transplant a large number of uniform ADSCs with the spray method, there are still substantial concerns related to the immunogenicity of nonself cells and the cell fate after transplantation.28 Some clinical trials suggested that allogeneic cells may be superior to autologous cells for the treatment of ICM, which supports our proposed strategy.29

The potential for the induction of immunological tolerance of MSCs is well-known based on evidence of their lack of major histocompatibility complex class II, 1 of the co-stimulatory molecules. In addition, MSCs could suppress rejection by inducing polarization from naive T cells to regulatory T cells. In addition, bone marrow-derived MSCs (BM-MSCs) have been reported to engraft in the allogeneic host with immunotolerance and effectiveness through the possible mechanisms mentioned above.

By contrast, myoblasts have poor survival and effectiveness because of their high immunogenicity compared to MSCs.30 Therefore, it is important to choose an appropriate cell type from an immunological standpoint. From this perspective, allogenic MSCs may be a suitable candidate cell source for heart failure treatment.31

Although allogenic BM-MSCs show immunologic tolerance in allogenic cell transplantation, we have not yet examined the immunologic characteristic of allogeneic ADSCs. Thus, further study may be needed to elucidate the immunologic features in allogenic ADSCs; however, we speculate that ADSCs may have immunological tolerance because the implanted cells survived for at least 1 month without the administration of immunosuppressive drugs in the current study.32

Moreover, although the transplanted cells were not detected at 8 weeks after transplantation, the improvement in cardiac function was preserved. We also speculate that the implanted cells produced cytokines that enhanced angiogenesis and cell recruitment from bone marrow cells, and that the angiogenic action continued for up to 1 month after implantation. Thus, the mobilized MSCs introduced maturation of newly born vasculature in the damaged myocardium.33

In conclusion, we have demonstrated that implantation of ADSCs with a spray method has a positive impact on the recovery of cardiac function in a porcine infarction model. This new cell delivery system may be translated to a clinical scenario, thereby resolving the current limitations of invasiveness and high cost in stem cell therapy.

ACKNOWLEDGMENTS

The authors appreciate Hamaguchi Laboratory Animals, Ltd., and Takashi Nakazaki for their excellent technical assistance.

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Table 1

Final concentrations of the ADSC, fibrinogen, and thrombin solutions used to prepare the sprayed fluid.

Figure 1

Study protocol of the minipig experiment and the evaluation of cardiac function and histological analysis. UCG: Ultrasonocardiography, PET: Positron Emission Tomography, cMRI: Cardiovascular Magnetic Resonance Imaging

Figure 2

Characteristics of human adipose-derived mesenchymal stem cell (ADSC)

(A)Immunophenotypic characterization of ADCs by flow cytometry. ADSCs stained positive for CD73 and CD90, whereas they were negative for CD11b and CD45. ADSC: green, Isotype control: red

(B)In vitro screening for cytokines and growth factors. Several factors that may potentially be involved in neovascularization and cardiac repair were detected at relatively high concentrations in the medium.

Figure 3

(A)The sprayed and congealed ADSC solution in a 150-mm culture dish. The size of the ADSC graft was 3 cm in diameter.

(B)Hematoxylin and eosin (HE) staining of ADSC graft. The small-scaled ADSC graft was about 100–200 mm thick and 15mm in diameter.

(C)High-magnification image of the ADSC graft.

(D)Immunohistochemical staining of actin filament and nuclei. Arrowheads show the filopodium formation of ADSC.

Green indicates actin filament; blue, nuclei.

(E)anti-Ki 67 staining showed that some of the ADSCs actively grew in the fibrin graft. Arrows show Ki-67 positive cells.

(F)Cytokine concentrations in culture supernatant of ADSC-graft by ELISA analysis. Concentration of angiogenetic factors and SDF-1 became higher over time.

Figure 4

(A)Operative photograph. The balloon device was developed for preventing cells from spattering. The target lesion was infarct area, so balloon was attached surrounding that area.

(B)Feasibility and safety of sprayed ADSC graft for a porcine ICM model. The sprayed ADSC graft was strongly adhered on the anterior surface of the heart.

Figure 5

(A)Representative echocardiographic pictures of pre and postoperative LV wall motion.

(B)Echocardiographic evaluation.

The global cardiac function as assessed by the left ventricular ejection fraction (LVEF) was significantly better in the ADSC group. In ADSC group, wall thickening is preserved compared with baseline. The left ventricular end-diastolic diameter (LVDd) did not differ significantly between the ADSC and control groups. The left ventricular end-systolic diameter (LVDs) was significantly smaller in the ADSC group than in the control group.

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