Additional file 1 of Restrictive annuloplasty or replacement on reverse remodeling for nonischemic dilated cardiomyopathy

Supplementary Material 1: Patient flow-char

Development of PET Imaging to Visualize Activated Macrophages Accumulated in the Transplanted iPSc-Derived Cardiac Myocytes of Allogeneic Origin for Detecting the Immune Rejection of Allogeneic Cell Transplants in Mice

<div><p>Allogeneic transplantation (Tx) of induced pluripotent stem cells (iPSCs) is a promising tissue regeneration therapy. However, this inevitably induces macrophage-mediated immune response against the graft, limiting its therapeutic efficacy. Monitoring the magnitude of the immune response using imaging tools would be useful for prolonging graft survival and increasing the therapy longevity. Minimally invasive quantitative detection of activated macrophages by medical imaging technologies such as positron emission tomography (PET) imaging targets translocator protein (TSPO), which is highly expressed on mitochondrial membrane, especially in activated macrophage. <i>N</i>,<i>N</i>-diethyl-2-[4-(2-fluoroethoxy) phenyl]-5,7-dimethylpyrazolo[1,5-<i>a</i>]pyrimidine-3-acetamide (DPA-714) is known as a TSPO ligand used in clinical settings. We herein hypothesized that immune rejection of the transplanted iPSC-derived cardiomyocytes (iPSC-CMs) of allogeneic origin may be quantitated using ¹⁸F-DPA-714-PET imaging study. iPSC-CM cell-sheets of C57BL/6 mice origin were transplanted on the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (syngeneic Tx group), or Balb/c mice as an allogeneic model (allogeneic Tx group). ¹⁸F-DPA-714-PET was used to determine the uptake ratio, calculated as the maximum standardized uptake value in the anterior and septal wall of the LV. The uptake ratio was significantly higher in the allogeneic Tx group than in the syngeneic group or the sham group at days 7 and day 10 after the cell transplantation. In addition, the immunochemistry showed significant presence of CD68 and CD3-positive cells at day 7 and 10 in the transplanted graft of the allogeneic Tx group. The expression of TSPO, <i>CD68</i>, <i>IL-1</i> beta, and <i>MCP-1</i> was significantly higher in the allogeneic Tx group than in the syngeneic Tx and the sham groups at day 7. The ¹⁸F-DPA-714-PET imaging study enabled quantitative visualization of the macrophages-mediated immune rejection of the allogeneic iPSC-cardiac. This imaging tool may enable the understanding and monitoring host-immune response of the host, allogeneic cell transplantation therapy.</p></div

Protocol for the cardiomyogenic differentiation of murine iPSCs and cardiac cell-sheet generation, and transplantation of the cell-sheet into murine model.

<p>A, the protocol for cardiomyogenic differentiation and purification of murine iPSCs; B, C57BL/6 mice-derived iPSC-cardiac sheet was transplanted over the LV surface of the C57BL/6 mice or the Balb/c mice, as the syngeneic or the allogeneic cell transplantation models, respectively. Sham operation, thoracotomy and pericardiotomy, was performed in the Balb/c mice; C, murine iPSC-cardiac sheet transplantation to the surface of LV via left thoracotomy, dotted line indicates iPSC-cardiac cell sheet; D, the protocol for evaluation by bioluminescence imaging, ¹⁸F-DPA-714 PET/ CT imaging, autoradiography, or immunohistochemistry of the cardiac tissue for immunochemical histology; E, iPSC-CMs stained with anti-alpha-actinin antibody (Alexa Fluor 647), anti-troponin I (Alexa Fluor 488) and DAPI, were analyzed by confocal laser scanning microscopy; F, iPSC-CMs stained with Alexa Fluor 647-conjugated anti-troponin T antibody or the isotype control were analyzed by flow cytometry.</p

Accumulation of ¹⁸F-DPA-714 after the allogeneic iPSC-Cardiac sheet transplantation.

<p>A, ¹⁸F-DPA-714 PET/ CT imaging in the allogeneic Tx group, the syngeneic Tx group, and sham group at days 1, 7, and 10 after iPSC-cardiac sheet transplantation on the surface of LV; B, comparing uptake ratio among the allogeneic Tx group, the syngeneic Tx group, sham group and non-surgical control group at days 1, 7, and 10 after iPSC-cardiac sheet transplantation; * indicates <i>P</i> <0.05 N.S, not significant.</p

Bioluminescence imaging.

<p>A, serial evaluation of transplanted graft survival by bioluminescence imaging in the allogeneic and syngeneic Tx groups; B, quantitative comparison of bioluminescence between the allogeneic and syngeneic Tx groups; C, teratoma formation in the syngeneic Tx group at day 7, bar = 100μm; Tx indicates transplantation; iPSC, induced pluripotent stem cells; LV, left ventricle; * indicates P <0.01; N.S, not significant.</p

Infiltration of activated macrophages and T lymphocytes into the transplanted graft.

<p>A, infiltration of CD68 positive activated macrophages into the transplanted grafts in the allogeneic Tx group, the syngeneic Tx group, and sham group at days 1, 7, and 10. bar = 100μm; B, infiltration of CD3 positive T lymphocytes into the transplanted grafts in the allogeneic Tx group, the syngeneic Tx group, and sham group at days 1, 7, and 10. bar = 100μm; C, comparison of the infiltrating CD68 positive cell counts per HPF among the allogeneic Tx group, the syngeneic Tx group, and sham group at days 1, 7, and 10; D, comparison of the infiltrating CD3 positive cell counts per HPF among the allogeneic Tx group, the syngeneic Tx group, and sham group at days 1, 7, and 10 * indicates <i>P</i> <0.05; N.S, not significant.</p

Expression of TSPO and associated proteins after iPSC-cardiac sheet transplantation.

<p>A-E, serial changes in the expression of <i>TSPO</i>, <i>CD68</i>, <i>IL-1beta</i>, <i>MCP-1</i> and <i>IL-2</i> in the allogeneic Tx group, the syngeneic Tx group, and sham group at day 7 after transplantation; F-H, correlation between the uptake ratio and expression of <i>TSPO</i>, <i>CD68</i> and <i>MCP-1; TSPO</i> indicates translocator protein; <i>IL-1</i>, interleukin-1; iPSC, induced pluripotent stem cells; Tx, transplantation; LV, left ventricle; * indicates <i>P</i> <0.05; N.S, not significant.</p

¹⁸F-DPA-714 detectable by PET-CT imaging.

<p>A, left side: systemic distribution of ¹⁸F-DPA-714 injected into tail vein was assessed in the normal Balb/c mice by PET-CT imaging, right side: ¹⁸F-DPA-714 PET imaging of transverse plane at the level of each red dotted lines; B, detection by PET/CT imaging of physiologic accumulation of ¹⁸F-DPA-714 in the anterior (solid line) and septal (dotted line) wall in LV in the normal Balb/c mice; C, white solid arrow indicates the site of transplantation of allogeneic iPSC-CMs in the dorsal side of subcutaneous space in Balb/c mice; dotted arrow indicates non-transplanted site; D, comparison of the mean SUVmax obtained by ¹⁸F-DPA-714 PET imaging in the transplanted site and contralateral side of subcutaneous tissue; E, correlation between SUVmax in the heart obtained by ¹⁸F-DPA-714 PET imaging and radioactivity obtained by autoradiography; DPA714, * indicates P <0.01.</p

Image2_Safety confirmation of induced pluripotent stem cell-derived cardiomyocyte patch transplantation for ischemic cardiomyopathy: first three case reports.jpeg

IntroductionWith the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies.Study designThe inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response.ResultsIn the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation.ConclusionsOur case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.</p

Table1_Safety confirmation of induced pluripotent stem cell-derived cardiomyocyte patch transplantation for ischemic cardiomyopathy: first three case reports.docx

IntroductionWith the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies.Study designThe inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response.ResultsIn the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation.ConclusionsOur case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.</p