利用人诱导多能干细胞构建的心脏类器官在心脏疾病建模及药物评价中的应用价值

doi:10.12122/j.issn.1673-4254.2025.11.17

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (11): 2444-2455.doi: 10.12122/j.issn.1673-4254.2025.11.17

利用人诱导多能干细胞构建的心脏类器官在心脏疾病建模及药物评价中的应用价值

龚雪¹(), 樊雍扬²(), 罗开元², 燕翼², 李忠豪²()

^1.中国人民解放军南部战区总医院，广东广州 510010
^2.广州医科大学附属第三医院心血管内科//广东省产科重大疾病重点实验室//广东省妇产疾病临床医学研究中心，广东广州 510150

收稿日期:2025-04-03 出版日期:2025-11-20 发布日期:2025-11-28
通讯作者: 李忠豪 E-mail:503253172@qq.com;760445401@qq.com;525838244@qq.com
作者简介:龚雪，本科，主管护师，E-mail: 503253172@qq.com
樊雍扬，在读硕士研究生，E-mail: 760445401@qq.com
基金资助:
国家自然科学基金(82570445);广东省科技厅国际合作专项(2022A0505050079);广东省基础与应用基础研究基金面上项目(2024A1515012365);广州市科技局临床研究专项(202201020189)

Construction of cardiac organoids derived from human induced pluripotent stem cells for cardiac disease modeling and drug evaluation

Xue GONG¹(), Yongyang FAN²(), Kaiyuan LUO², Yi YAN², Zhonghao LI²()

^1.General Hospital of Southern Theater Command of PLA, Guangzhou 510010, China
^2.Department of Cardiology, Third Affiliated Hospital of Guangzhou Medical University//Guangdong Provincial Key Laboratory of Major Obstetric Diseases//Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, Guangzhou 510150, China

Received:2025-04-03 Online:2025-11-20 Published:2025-11-28
Contact: Zhonghao LI E-mail:503253172@qq.com;760445401@qq.com;525838244@qq.com
Supported by:
National Natural Science Foundation of China(82570445)

摘要/Abstract

摘要：

目的探讨人诱导多能干细胞衍生的心脏类器官在心脏疾病模型和药物评价中的潜在应用。方法通过调控Wnt信号通路构建由人诱导多能干细胞自组装衍生的心脏类器官，采用流式细胞术检测心脏类器官中心肌细胞比例，RT-qPCR检测干细胞标志基因（OCT4、Nanog、SOX2）和心肌细胞标志基因（TNNT2、NKX2.5、RYR2、KCNJ2）的表达，免疫荧光染色检测TNNT2、CD31、Vimentin的蛋白表达，钙瞬态检测心脏类器官跳动幅度；建立体外心肌损伤模型和缺血再灌注模型，Masson染色检测损伤水平，ELISA检测心肌肌钙蛋白T（cTnT）释放水平；利用TUNEL染色和钙瞬态检测分别评价化疗药物多柔比星和曲妥珠单抗对心脏类器官的毒副作用。结果心脏类器官在培养第8天开始跳动，心肌细胞在心脏类器官中占比为32.4%，心脏类器官可见心肌标志物TNNT2、NKX2.5、RYR2和KCNJ2大量表达（P<0.001）。不同批次分化的心脏类器官在形态大小、跳动频率、心肌细胞比例、和心肌收缩力方面均无显著差异。心脏类器官可维持体外培养≥50 d。液氮造成心脏类器官心肌损伤、 TNNT2和MYH7基因表达下降（P<0.05）、cTnT分泌增加（P<0.01）、跳动幅度下降（P<0.01）和峰值时间增加（P<0.01），而卡托普利处理可减轻损伤发生。低氧/复氧诱导心脏类器官缺血再灌注损伤，心肌纤维化和凋亡增多（P<0.001）。多柔比星处理心脏类器官24 h后，细胞死亡明显增加（P<0.05）、心脏类器官的跳动频率和细胞活力下降（P<0.05），且具有剂量依懒性。曲妥珠单抗引起心脏类器官收缩力和钙处理能力下降（P<0.05）。结论成功构建心脏类器官，并可用于心脏疾病建模及药物评价。

关键词: 人诱导多能干细胞, 心脏类器官, 疾病建模, 药物评价

Abstract:

Objective To explore the potential applications of human induced pluripotent stem cell-derived cardiac organoids in constructing cardiac injury models and drug evaluation. Methods Cardiac organoids derived from the self-assembled human induced pluripotent stem cells were constructed by regulating the Wnt signaling pathway. Flow cytometry was used to detect the proportion of cardiomyocytes in the cardiac organoids, and RT-qPCR was employed to detect the mRNA expressions. Immunofluorescence staining was used to detect the protein expressions of TNNT2, CD31, and vimentin. The beating amplitude of the cardiac organoids was determined with calcium transient. In vitro myocardial injury models and ischemia-reperfusion models were established, and the cell injuries were examined using Masson staining. TUNEL staining and calcium transient detection were used to evaluate the adverse effects of doxorubicin and trastuzumab in the cardiac organoids. Results The cardiac organoids began to beat on the 8th day of culture and consisted of 32.4% cardiomyocytes with high expressions of the myocardial markers TNNT2, NKX2.5, RYR2 and KCNJ2. No significant differences in morphological size, beating frequency, proportion of cardiomyocytes, or myocardial contractility were observed in the cardiac organoids differentiated from different batches. These cardiac organoids could be maintained in in vitro culture conditions for at least 50 days. Captopril treatment could obviously alleviate liquid nitrogen-induced myocardial injury in the cardiac organoids. Hypoxia/reoxygenation induced ischemia-reperfusion injury and promoted myocardial fibrosis and apoptosis in the cardiac organoids. Treatment with doxorubicin for 24 h resulted in significantly increased cell death and reduced beating frequency and cell viability in the cardiac organoids in a dose-dependent manner. Trastuzumab significantly impaired the contractile and calcium handling abilities of the cardiac organoids. Conclusion Cardiac organoids derived from human induced pluripotent stem cells have been successfully constructed and can be used for cardiac disease modeling and drug evaluation.

Key words: human induced pluripotent stem cells, cardiac organoids, disease modeling, drug evaluation

龚雪, 樊雍扬, 罗开元, 燕翼, 李忠豪. 利用人诱导多能干细胞构建的心脏类器官在心脏疾病建模及药物评价中的应用价值[J]. 南方医科大学学报, 2025, 45(11): 2444-2455.

Xue GONG, Yongyang FAN, Kaiyuan LUO, Yi YAN, Zhonghao LI. Construction of cardiac organoids derived from human induced pluripotent stem cells for cardiac disease modeling and drug evaluation[J]. Journal of Southern Medical University, 2025, 45(11): 2444-2455.

图/表 8

表1 RT-qPCR引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Forward Primer (5'→3')	Reverse Primer (5'→3')
OCT4	CTCTGAGGTGTGGGGGATTC	TCAGGCTGAGAGGTCCCAAG
Nanog	TTTGTGGGCCTGAAGAAAACT	AGGGCTGTCCTGAATAAGCAG
SOX2	GCCGAGTGGAAACTTTTGTCG	GGCAGCGTGTACTTATCCTTCT
TNNT2	GGAGGAGTCCAAACCAAAGCC	TCAAAGTCCACTCTCTCTCCATC
NKX2.5	CCAAGTGTGCGTCTGCCTTT	CGCACAGCTCTTTCTTTTCGG
RYR2	GGCAGCCCAAGGGTATCTC	ACACAGCGCCACCTTCATAAT
KCNJ2	GTGCGAACCAACCGCTACA	CCAGCGAATGTCCACACAC
MYH7	GGCAAGACAGTGACCGTGAAG	CGTAGCGATCCTTGAGGTTGTA
GAPDH	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG

图1 心脏类器官的构建

Fig.1 Construction of the Cardiac Organoids derived from human induced pluripotent stem cells. A: Schematic diagram depicting the protocol for constructing cardiac organoids derived from human induced pluripotent stem cells. B: Brightfield images of the developing cardiac organoids. C, D: Proportion of cardiomyocytes and endothelial cells in cardiac organoids determined by flow cytometry. E: Frozen sections of the developing cardiac organoids. F: Diameter of the developing cardiac organoids (n=10). G: The mRNA expressions in cardiac organoids determined by RT-qPCR. ***P<0.001, ****P<0.0001 vs D0.

图2 心脏类器官的鉴定

Fig.2 Characterization of the constructed cardiac organoids. A: Immunofluorescent staining of frozen sections of the cardiac organoids. B: Whole-mount staining of the cardiac organoids. C, D: Calcium transient assay of the cardiac organoids (n=3).

图3 不同批次的心脏类器官一致性检测

Fig.3 Consistency tests of the cardiac organoids from different batches. A: Brightfield images of the developing cardiac organoids from different batches. B: Beating frequency of the cardiac organoids from different batches (n=8). C: Proportion of cardiomyocytes in the cardiac organoids from different batches determined by flow cytometry. D: Measurement of the contractile ability of cardiac organoids using Image J.

图4 心脏类器官长期培养

Fig.4 Long-term culture of the cardiac organoids. A: Brightfield images of the developing cardiac organoids. B: Measurement of contractile ability of the cardiac organoids using Image J. C: Immunofluorescent staining of frozen sections of the cardiac organoids.

图5 心脏类器官建模

Fig.5 Disease modeling of cardiac organoids. A: Brightfield images of the cardiac organoids with different treatments. B: Masson's staining of the cardiac organoids. C: Calcium transient assay of the cardiac organoids. D: Amplitude of Ca2+ transient and the time to peak of Ca2+ transient (n=5). E: mRNA expressions of MYH7 and TNNT2 with or without cryoinjury and captopril treatment. F: Evaluation of cTnT levels in the culture medium by ELISA (n=6). G: Masson's staining of the cardiac organoids. H: TUNEL staining of the organoid sections after H/R treatment. I: Statistical analysis of apoptosis. *P<0.05, **P<0.01, ***P<0.001 vs control group; #P<0.05 vs cryoinjury group.

图6 多柔比星对心脏类器官的影响

Fig.6 Effect of doxorubicin on the cardiac organoids. A: Brightfield images of the cardiac organoids treated with doxorubicin (scale bar=200 μm). B: Dose-dependent effects of doxorubicin on beating frequency of the cardiac organoids (n=8). C: Dose-dependent effects of doxorubicin on cell vitality in the cardiac organoids by CCK8 assay. D: Statistical analysis of cell apoptosis. E: TUNEL staining of the organoid sections after exposure to doxorubicin for 24 h at the indicated concentrations. F: Measurement of contractile ability of the cardiac organoids using Image J. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control group (0).

图7 曲妥珠单抗引起心脏类器官功能异常

Fig.7 Trastuzumab causes dysfunction of the cardiac organoids. A: Calcium transient assay of the cardiac organoids with Trastuzumab treatment. B: Amplitude of Ca2+ transient and the time to peak of Ca2+ transient (*P<0.05, **P<0.01 vs control group, n=5). C: Measurement of contractile ability of the cardiac organoids with Trastuzumab treatment using Image J.

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利用人诱导多能干细胞构建的心脏类器官在心脏疾病建模及药物评价中的应用价值

Construction of cardiac organoids derived from human induced pluripotent stem cells for cardiac disease modeling and drug evaluation

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