Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Krakow, Poland.
Doctoral School of Exact and Natural Sciences, Jagiellonian University, Kraków, Poland.
Stem Cell Res Ther. 2024 Oct 29;15(1):382. doi: 10.1186/s13287-024-03956-1.
Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects not only the joints but also has significant cardiovascular (CV) manifestations. The mechanistic interplay between RA and cardiovascular complications is not yet well understood due to the lack of relevant in vitro models. In this study, we established RA cardiac microtisses (cMTs) from iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs) to investigate whether this fully human 3D multicellular system could serve as a platform to elucidate the connection between RA and CV disorders.
PBMC and FLS from healthy and RA donors were reprogrammed to hiPSCs with Sendai vectors. hiPSCs pluripotency was assessed by IF, FACS, spontaneous embryoid bodies formation and teratoma assay. hiPSCs were differentiated to cardiac derivatives such as CMs, ECs and CFs, followed by cell markers characterizations (IF, FACS, qRT-PCR) and functional assessments. 3D cMTs were generated by aggregation of 70% CMs, 15% ECs and 15% CFs. After 21 days in culture, structural and metabolic properties of 3D cMTs were examined by IF, qRT-PCR and Seahorse bioanalyzer.
hiPSCs demonstrated typical colony-like morphology, normal karyotype, presence of pluripotency markers, and ability to differentiate into cells originating from all three germ layers. hiPSC-CMs showed spontaneous beating and expression of cardiac markers (cTnT, MYL7, NKX2.5, MYH7). hiPSC-ECs formed sprouting spheres and tubes and expressed CD31 and CD144. hiPSC-CFs presented spindle-shaped morphology and expression of vimentin, collagen 1 and DDR2. Self-aggregation of CMs/ECs/CFs allowed development of contracting 3D cMTs, demonstrating spherical organization of the cells, which partially resembled the cardiac muscle, both in structure and function. IF analysis confirmed the expression of cTnT, CD31, CD144 and DDR2 in generated 3D cMTs. RA cMTs exhibited significantly greater formation of capillary-like structures, mimicking enhanced vascularization-key RA feature-compared to control cMTs. Seahorse examination of cMTs revealed changes in mitochondrial and glycolytic rates in the presence of metabolic substrates and inhibitors.
The cMTs model may represent an advanced human stem cell-based platform for modeling CV complications in RA. The highly developed capillary-like structures observed within RA cMTs highlight a critical feature of inflammation-induced CV dysfunction in chronic inflammatory diseases.
类风湿关节炎（RA）是一种慢性炎症性疾病，不仅影响关节，而且还具有显著的心血管（CV）表现。由于缺乏相关的体外模型，RA 和心血管并发症之间的机制相互作用尚不清楚。在这项研究中，我们从诱导多能干细胞（iPSC）衍生的心肌细胞（CMs）、内皮细胞（ECs）和心脏成纤维细胞（CFs）中建立了 RA 心脏微组织（cMTs），以研究这个完全基于人的 3D 多细胞系统是否可作为阐明 RA 与 CV 疾病之间联系的平台。
使用 Sendai 载体将来自健康和 RA 供体的 PBMC 和 FLS 重编程为 hiPSCs。通过免疫荧光、流式细胞术、自发胚状体形成和畸胎瘤测定评估 hiPSCs 的多能性。hiPSCs 分化为心脏衍生物，如 CMs、ECs 和 CFs，随后进行细胞标志物特征（免疫荧光、流式细胞术、qRT-PCR）和功能评估。通过 70%CMs、15%ECs 和 15%CFs 的聚集生成 3D cMTs。培养 21 天后，通过免疫荧光、qRT-PCR 和 Seahorse 生物分析仪检查 3D cMTs 的结构和代谢特性。
hiPSCs 表现出典型的菌落样形态、正常核型、多能性标志物的存在以及分化为来自三个胚层的细胞的能力。hiPSC-CMs 表现出自发搏动和心脏标志物（cTnT、MYL7、NKX2.5、MYH7）的表达。hiPSC-ECs 形成发芽球体和管，并表达 CD31 和 CD144。hiPSC-CFs 呈纺锤形形态，表达波形蛋白、胶原蛋白 1 和 DDR2。CMs/ECs/CFs 的自聚集允许收缩 3D cMTs 的发育，显示出细胞的球形组织，在结构和功能上都部分类似于心肌。免疫荧光分析证实了生成的 3D cMTs 中 cTnT、CD31、CD144 和 DDR2 的表达。与对照 cMTs 相比，RA cMTs 中形成的毛细血管样结构明显更多，模拟了增强的血管生成-RA 的关键特征。在存在代谢底物和抑制剂的情况下，对 cMTs 的 Seahorse 检查显示线粒体和糖酵解率的变化。
cMTs 模型可能代表用于模拟 RA 中 CV 并发症的先进的基于人类干细胞的平台。在 RA cMTs 中观察到高度发达的毛细血管样结构突出了慢性炎症性疾病中炎症诱导的 CV 功能障碍的一个关键特征。
