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. 2024 Oct 23;27(11):111234.
doi: 10.1016/j.isci.2024.111234. eCollection 2024 Nov 15.

Metabolic changes of human induced pluripotent stem cell-derived cardiomyocytes and teratomas after transplantation

Yusuke Soma  1   2 , Shugo Tohyama  1   2 , Akiko Kubo  3 , Tomoteru Yamasaki  4 , Noriko Kabasawa  1   2   5 , Kotaro Haga  1 , Hidenori Tani  1   2   6 , Yuika Morita-Umei  1   7 , Tomohiko C Umei  1   2 , Otoya Sekine  2 , Masashi Nakamura  2 , Taijun Moriwaki  1   2 , Sho Tanosaki  2 , Shota Someya  2 , Yujiro Kawai  8 , Masatoshi Ohno  1   8 , Yoshikazu Kishino  2 , Hideaki Kanazawa  2 , Jun Fujita  2   9 , Ming-Rong Zhang  4 , Makoto Suematsu  3   10   11 , Keiichi Fukuda  2   5 , Masaki Ieda  2
Affiliations

Metabolic changes of human induced pluripotent stem cell-derived cardiomyocytes and teratomas after transplantation

Yusuke Soma et al. iScience. .

Abstract

Cardiac regenerative therapy using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has been applied in clinical settings. Herein, we aimed to investigate the in vivo metabolic profiles of hiPSC-CM grafts. RNA sequencing and imaging mass spectrometry were performed in the present study, which revealed that hiPSC-CM grafts matured metabolically over time after transplantation. Glycolysis, which was active in the hiPSC-CM grafts immediately after transplantation, shifted to fatty acid oxidation. Additionally, we examined the metabolic profile of teratomas that may form when non-CMs, including undifferentiated human induced pluripotent stem cells (hiPSCs), remain in transplanted cells. The upregulated gene expression of amino acid transporters and the high accumulation of amino acids, such as methionine and aromatic amino acids, were observed in the teratomas. We show that subcutaneous teratomas derived from undifferentiated hiPSCs can be detected in vivo using positron emission tomography with [18F]fluorophenylalanine ([18F]fPhe). These results provided insights into the clinical application of cardiac regenerative therapy.

Keywords: cardiovascular medicine; cell biology.

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Conflict of interest statement

K.F. is the co-founder and CEO of Heartseed Inc. S.Tohyama is an advisor from Heartseed Inc. S. Tohyama, H.K., J.F., and K.F. own equity in Heartseed Inc.

Figures

None
Graphical abstract
Figure 1
Figure 1
Maturation of hiPSC-CMs transplanted into the myocardium of NOG mice (A) Scheme of the experiment. (B) Graft staining for sarcomere structure (α-actinin, red). Scale bar, (upper) 100 μm; (middle) 50 μm; and (lower) 10 μm. (C) Quantification of sarcomere length (n = 20 sarcomeres at each time point. Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.0005, ∗∗∗∗p < 0.0001. Error bars represent SEM). (D) Graft staining for the mature ventricular phenotype (MLC2v, red) or the immature ventricular/atrial/nodal cardiomyocyte phenotype (MLC2a, green). (E) The proportion of MLC2a- and MLC2v-positive CMs. (F) Graft staining for Ki67 (green) and human-specific cTnI (red). hs: human-specific. (G) The proportion of Ki67-positive cells in the graft.
Figure 2
Figure 2
Functional and metabolic maturation of hiPSC-CM grafts evaluated using RNA sequencing and IMS (A) Scheme of the experiment. (B) Heatmap showing RNA sequencing of hiPSC-CM grafts (2 weeks, n = 1; 4 weeks, n = 2; and 12 weeks, n = 3). (C) Principal component analysis of the early-stage group (2 weeks and 4 weeks) and the late-stage group (12 weeks). (D) Gene ontology analysis of the genes that were upregulated in the late-stage group compared with those in the early-stage group. (E) Gene ontology analysis of the genes that were downregulated in the late-stage group compared with those in the early-stage group. (F) Heatmap showing characteristic gene expression of hiPSC-CM grafts. (G) Representative data of the accumulation of hexose diphosphate in hiPSC-CM grafts and host myocardium measured by IMS. Scale bar, 1 mm. (H) The ratio of glycolytic metabolites between hiPSC-CM grafts and host myocardium measured via IMS at 2, 4, and 12 weeks (n = 5, respectively. Dunn’s multiple comparisons test. ∗p < 0.05. Error bars represent SEM). G3P/DHAP, glyceraldehyde-3-phosphate/dihydroxyacetone phosphate; 2PG/3PG, 2-phosphoglycerate/3-phosphoglycerate; PEP, phosphoenolpyruvic acid.
Figure 3
Figure 3
Metabolic differences between hiPSC-CM and -T grafts (A) Scheme of the experiment. (B) Upper: heatmap showing the results of RNA sequencing on hiPSC-CM and hiPSC-T grafts. Lower: heatmap showing the gene expression of amino acid transporters. (C) Principal-component analysis of hiPSC-CM (red) and -T (blue) grafts at 2, 4, and 12 weeks. (D) Gene set enrichment analysis demonstrating differences in gene expression between hiPSC-CM and -T grafts.
Figure 4
Figure 4
Metabolism of hiPSC-Ts evaluated via IMS and PET imaging (A) The ratio of accumulation of glycolytic metabolites between hiPSC-Ts and the host myocardium measured by IMS (n = 5, respectively. Dunn’s multiple comparisons test. ∗p < 0.05. Error bars represent SEM). G3P/DHAP: glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. 2PG/3PG, 2-phosphoglycerate/3-phosphoglycerate; PEP, phosphoenolpyruvic acid. (B) The ratio of accumulation of amino acids between hiPSC-Ts and the host myocardium measured by IMS (n = 5, respectively. Dunn’s multiple comparisons test. ∗p < 0.05. Error bars represent SEM). (C) Representative data showing the accumulation of amino acids in hiPSC-Ts and the host myocardium measured by IMS. (D) Scheme of the PET imaging experiment using [18F]fPhe. (E) Representative PET image of the subcutaneous hiPSC-T by [18F]fPhe 9 weeks after transplantation (40–60 min, integrated images). (F) Time-activity curve expressing the radioactivity of [18F]fPhe in each organ. (G) Autoradiography of the heart and subcutaneous hiPSC-T graft. (H) Time-activity curve expressing the mean values of the radioactivity of [18F]fPhe in hiPSC-Ts and in skeletal muscles and the tumor/muscle ratio (n = 6. Error bars represent SEM). (I) The mean values of the radioactivity of [18F]fPhe in hiPSC-Ts and skeletal muscles around 60 min (n = 6. One-sample Wilcoxon signed rank test. ∗p < 0.05. Error bars represent SEM).
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