Kargaran Parisa K, Garmany Armin, Garmany Ramin, Stutzman Marissa J, Sadeghian Maryam, Ackerman Michael J, Perez-Terzic Carmen M, Terzic Andre, Behfar Atta
Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN, USA.
Center for Regenerative Biotherapeutics, Mayo Clinic, Rochester, MN, USA.
Sci Rep. 2025 Apr 24;15(1):14399. doi: 10.1038/s41598-025-97676-y.
Cardiac differentiation of human induced pluripotent stem cells is readily achievable, yet derivation of mature cardiomyocytes has been a recognized limitation. Here, a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5, GATA4, TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further, Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition, transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus, Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment.
人类诱导多能干细胞向心脏分化很容易实现，但成熟心肌细胞的诱导一直是一个公认的限制因素。在此，设计了一种中胚层启动方法，以促进在标准心脏分化条件下源自多能干细胞的心肌细胞后代的成熟。在谱系分化开始前转染中胚层转录因子短尾相关蛋白（Brachyury）后，通过对源自诱导多能干细胞的心肌细胞进行多参数评估，来评估成熟的功能和结构特征。用短尾相关蛋白转染导致更早诱导出心脏生成状态，其特征是心脏特异性转录因子NKX2.5、GATA4、TBX20的早期上调。短尾相关蛋白预处理后肌节成熟增强，表现为表达肌球蛋白轻链心室异构体的细胞比例增加以及肌节长度增加。通过线粒体大小增加和基础氧消耗率提高确定，中胚层启动的细胞对线粒体呼吸的依赖性增加。此外，短尾相关蛋白启动促进了钙处理的成熟，使转染细胞能够在更高的外部场刺激速率下维持钙瞬变形态，并增强了钙释放和摄取动力学。此外，转染细胞表现出更成熟的动作电位形态，去极化和复极化动力学增加。用短尾相关蛋白转染的衍生细胞对阿霉素的毒性反应增加，这通过钙瞬变形态的受损来确定。因此，此处的短尾相关蛋白预处理实现了一种简化策略，以促进人类多能干细胞衍生心肌细胞的成熟，建立了一个可推广的平台以供应用。
