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. 2021 Feb 19;6(3):239-254.
doi: 10.1016/j.jacbts.2020.11.017. eCollection 2021 Mar.

Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals

Shinji Kawaguchi  1 , Yusuke Soma  2 , Kazuaki Nakajima  2 , Hideaki Kanazawa  2 , Shugo Tohyama  2   3 , Ryota Tabei  2 , Akinori Hirano  1 , Noriko Handa  4 , Yoshitake Yamada  5 , Shigeo Okuda  5 , Shuji Hishikawa  4 , Takumi Teratani  4 , Satoshi Kunita  4 , Yoshikazu Kishino  2 , Marina Okada  2 , Sho Tanosaki  2 , Shota Someya  2 , Yuika Morita  2 , Hidenori Tani  2 , Yujiro Kawai  1 , Masataka Yamazaki  1 , Akira Ito  6 , Rei Shibata  7 , Toyoaki Murohara  8 , Yasuhiko Tabata  9 , Eiji Kobayashi  3   4 , Hideyuki Shimizu  1 , Keiichi Fukuda  2 , Jun Fujita  2   10
Affiliations

Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals

Shinji Kawaguchi et al. JACC Basic Transl Sci. .

Abstract

The severe shortage of donor hearts hampered the cardiac transplantation to patients with advanced heart failure. Therefore, cardiac regenerative therapies are eagerly awaited as a substitution. Human induced pluripotent stem cells (hiPSCs) are realistic cell source for regenerative cardiomyocytes. The hiPSC-derived cardiomyocytes are highly expected to help the recovery of heart. Avoidance of teratoma formation and large-scale culture of cardiomyocytes are definitely necessary for clinical setting. The combination of pure cardiac spheroids and gelatin hydrogel succeeded to recover reduced ejection fraction. The feasible transplantation strategy including transplantation device for regenerative cardiomyocytes are established in this study.

Keywords: CM, cardiomyocyte; CMR, cardiac magnetic resonance; CS, cardiac spheroid; ECG, electrocardiogram; EF, ejection fraction; FAC, fractional area change; GH, gelatin hydrogel; HF, heart failure; LV, left ventricular; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; VEGF, vascular endothelial growth factor; cardiac spheroids; cardiomyocyte; cell transplantation; dp/dtmax, maximum rate of left ventricular pressure rise; hPSC, human pluripotent stem cell; heart failure; hiPSC, human induced pluripotent stem cell; human iPS cells; sCM, single cardiomyocyte.

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Conflict of interest statement

This work was supported by the Highway Program for Realization of Regenerative Medicine (17bm054006h0007 [to Dr. Fukuda]) and the Research Project for Practical Applications of Regenerative Medicine (17bk010462h0001 [to Dr. Fukuda]) from the Japan Agency for Medical Research and Development, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (nos. 15K09098 [to Dr. Kanazawa], 16K09507 [to Dr. Fujita], 19H03660 [to Dr. Fujita], 17H05067 [to Dr. Tohyama], 18K15903 [to Dr. Nakajima]). Drs. Kanazawa, Tohyama, Fukuda, and Fujita have patents related to this work. Drs. Tohyama, Shimizu, Kanazawa, Fukuda, and Fujita own equity in Heartseed, Inc. Dr. Tohyama is an advisor of Heartseed, Inc. Dr. Fukuda is a co-founder and CEO of Heartseed, Inc.; and receives a salary from Heartseed, Inc. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures

None
Graphical abstract
Figure 1
Figure 1
hIPSC-Derived CSs (A) Immunostained images of differentiated human induced pluripotent stem sell (hiPSC)–derived cardiomyocytes (CMs) before metabolic selection. (B) Immunostained images of differentiated hiPSC-derived CMs after metabolic selection. Note that most of the cells are α-actinin–positive CMs. Scale bars represent 200 μm in upper panels and 100 μm in lower panels. (C) hiPSC-derived CMs are positive for MLC2a (myosin light chain 2a), indicating immature CMs, at day 21 (upper panel). Most of purified hiPSC-derived CMs become positive for MLC2v, indicating ventricular CMs, at 50 days (lower panel). Scale bars represent 100 μm. (D) Differentiation efficiency is ∼80%. (E) After purification, the proportion of cardiac troponin T–positive CMs is >98%. (F) Cardiac spheroids (CSs) are generated from hiPSC-derived CMs in microwell plates. (G) Systolic and diastolic movements of beating CMs are analyzed by Cell Motion Imaging System SI8000. Stimulation of β-adrenoreceptor significantly increased the beats rate of hiPSC-derived CSs (p < 0.001) (n = 5). ∗∗ p < 0.01.
Figure 2
Figure 2
hiPSC-Derived CSs Significantly Improved Cardiac Function in Immunocompromised Rats With Heart Failure (A) Transplantation protocol for immunocompromised rats is shown. (B) Representative short-axis and M-mode images of hearts in the control (gelatin hydrogel [GH]), single CM (sCM), and CS groups. (C) Cardiac ejection fraction (EF) significantly improved in the CS group (p < 0.001). Post hoc analysis also showed the significant improvement in the CS group at 2 months (CS vs. GH; p = 0.022 CS vs. sCM; p = 0.002). (D) Cardiac fractional area change (FAC) significantly improved in the CS group (p = 0.005). Post hoc analysis showed that the CS group significantly improved at 2 months in comparison with the sCM group, and tended to improve in comparison with the GH group (CS vs. GH: p = 0.073; CS vs. sCM: p = 0.029). (E) Hemodynamic data showed that the +dp/dtmax significantly improved in the CS group (p = 0.003). Post hoc analysis showed that the CS group significantly improved +dp/dtmax at 2 months (CS vs. GH: p = 0.003; CS vs. sCM: p = 0.009). (F) Diastolic function (–dp/dtmax) tended to improve in the CS group (p = 0.129). ∗p < 0.05; ∗∗p < 0.01. M = month; W = week.
Figure 3
Figure 3
hiPSC-Derived CSs Strongly Engrafted in Immunocompromised Rats With Heart Failure (A) sCMs engrafted rarely in a recipient’s heart. (B) Large amount of transplanted CMs, which is positive for both human nuclear antigen and cardiac troponin I, strongly engrafted in recipients’ hearts in the CS group. (C) Cardiac troponin I–positive CMs are also positive for α-actinin. (D) Connexin43 was expressed in transplanted cardiomyocytes. MLC2v was more dominant than MLC2a in hiPSC-derived CMs. Tom20 staining indicated that the transplanted CMs had immature mitochondria in comparison with those of adult CMs. Scale bars represent 100 μm in A and B and 50 μm in C and D. DAPI = 4′,6-diamidino-2-phenylindole; other abbreviations as in Figures 1 and 2.
Figure 4
Figure 4
hiPSC-Derived CSs Were Transduced by a Lentiviral Vector Encoding Venus and Luciferase (A) Venus was expressed in hiPSC-derived CSs by transduction of a lentiviral vector encoding Venus and luciferase. (B) Luciferin-added CSs express bioluminescence signal. The representative bioluminescence signal in the XSCID rat's heart. (C) The bioluminescence signal decreased 1 month after cell transplantation; however, significant signals remained at 2 months (n = 3). Abbreviations as in Figure 1.
Figure 5
Figure 5
hiPSC-Derived CSs Significantly Improved Swine Cardiac Function in Swine Heart Failure (A) Transplantation protocol. (B) An injection device has 6 needles with a blind tip and side holes. (C) CSs are transplanted by the injection device to anterior wall, which is stabilized by a stabilizer (Videos 1 and 2). (D) Representative images of systolic and diastolic phase of hearts in the GH and CS groups (Videos 3 and 4). (E) EF significantly improved in the CS group (p = 0.001). Post hoc analysis showed that the CS group significantly improved at 2 months in comparison with the GH group, and tended to improve in comparison with sham group (CS vs. GH: p = 0.002; CS vs. sham: p = 0.074). (F) Left ventricular end-systolic volume significantly improved in CS group (p = 0.023). Post hoc analysis showed that the CS group significantly improved at 2 months in comparison with the GH group, and tended to improve in comparison with the sham group (CS vs. GH: p = 0.037; CS vs. sham: p = 0.262). (G) Left ventricular end-diastolic volume remained unaltered between the control and CS groups (p = 0.192). ∗p < 0.05. ∗∗p < 0.01. MRI = magnetic resonance imaging; other abbreviations as in Figures 1, 2 and 3.
Figure 6
Figure 6
Morphometric Analysis of Infarction Area in Swine Hearts (A) Representative late gadolinium enhancement images of cardiac magnetic resonance in the control and CS groups. (B) Infarcted area was assessed by late gadolinium enhancement. The proportion of infarcted area remarkably decreased in the CS group (p < 0.001). Post hoc analysis showed that the CS group significantly decreased the proportion of infarcted area at 2 months in comparison with the GH and sham groups (CS vs. GH: p < 0.001; CS vs. sham: p < 0.001). (C) Representative short-axis figures of TTC (2,3,5-triphenyl tetrazolium chloride)-stained hearts. (D) Infarcted area was assessed by TTC staining. These data confirmed that infarcted area in CS group decreased significantly (infarct size: p = 0.006; rate: p = 0.001). Post hoc analysis showed that the CS group significantly improved at 2 months in comparison with the GH and sham groups (for infarct size, CS vs. GH: p = 0.016; CS vs. sham: p = 0.013; for rate, CS vs. GH: p = 0.006; CS vs. sham: p = 0.002) (E) Representative α-actinin staining of cardiac tissues in remote area. Scale bars represent 100 μm. (F) The average size of CMs in the CS group is not different from that of the control and sham groups (p = 0.769). ∗p < 0.05. ∗∗p < 0.01. Abbreviations as in Figures 1 and 2.
Figure 7
Figure 7
Human CSs Strongly Induced Angiogenesis in Swine Hearts (A, B) Representative figures of von Willebrand Factor–stained endothelial cells and troponin I–stained CMs at border zone in control and CS groups. Scale bars represent 100 μm. (C, D) Representative images of endothelial cells and CMs at the remote zone in the control and CS groups. (E) Angiogenesis was significantly promoted at border zone in the CS group (p < 0.001). ∗∗p < 0.01. (F) Angiogenic and inflammatory cytokines were measured by multiplex cytokine assay. Vascular endothelial growth factor (VEGF) was remarkably released from CSs. Only a small amount of cytokines were released from hiPSC-derived CSs except VEGF. (G) Transplanted hiPSC-derived CMs expressed VEGF in vivo. Vascular cells also expressed VEGF (arrows). The scale bar represents 100 μm. EGF = epidermal growth factor; FGF = fibroblast growth factor; G-CSF = granulocyte colony-stimulating factor; PDGF = platelet-derived growth factor-AB/BB; IL = interleukin; TNF = tumor necrosis factor; other abbreviations as in Figures 1, 2 and 3.
Figure 8
Figure 8
Ventricular Arrhythmia Was Induced in the hiPSC-Derived CS Group in the Swine Heart Failure Model (A, B) No control swine showed either tachycardia or ventricular arrhythmia. (C, D) hiPSC-derived CS-transplanted swine increased their heart rate and the number of wide QRS complex for 20 days after cell transplantation. (E) Ventricular tachycardia emerged in correlation with the emergence of sinus or paroxysmal supraventricular tachycardia (PSVT), but no fatal arrhythmia happened. T/P = transplantation; other abbreviations as in Figures 1 and 2.
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