From the Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta (D.T., M.E.B., M.E.D.).
Division of Pediatric Cardiology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA (J.T.M., C.X., M.E.D.).
Circ Res. 2019 Feb 15;124(4):526-538. doi: 10.1161/CIRCRESAHA.118.313845.
RATIONALE: Congenital heart disease can lead to life-threatening right ventricular (RV) heart failure. Results from clinical trials support expanding cardiac progenitor cell (CPC) based therapies. However, our recent data show that CPCs lose function as they age, starting as early as 1 year. OBJECTIVE: To determine whether the aggregation of child (1-5-year-old) CPCs into scaffold-free spheres can improve differentiation by enhancing Notch signaling, a known regulator of CPC fate. We hypothesized that aggregated (3-dimensional [3D]) CPCs will repair RV heart failure better than monolayer (2-dimensional [2D]) CPCs. METHODS AND RESULTS: Spheres were produced with 1500 CPCs each using a microwell array. CPC aggregation significantly increased gene expression of Notch1 compared with 2D CPCs, accompanied by significant upregulation of cardiogenic transcription factors (GATA4, HAND1, MEF2C, NKX2.5, and TBX5) and endothelial markers (CD31, FLK1, FLT1, VWF). Blocking Notch receptor activation with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) diminished these effects. To evaluate the therapeutic improvements of CPC aggregation, RV heart failure was induced in athymic rats by pulmonary artery banding, and cells were implanted into the RV free wall. Echocardiographic measurements 28 days postimplantation showed significantly improved RV function with 3D compared with 2D CPCs. Tracking implanted CPCs via DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)-labeling showed improved retention of 3D CPCs. Transducing 3D CPCs with Notch1-shRNA (short hairpin RNA) did not reduce retention, but significantly reduced RV functional improvements. Histological analyses showed 3D treatment reduced RV fibrosis and increased angiogenesis. Although 3D CPCs formed CD31 vessel-like cells in vivo, these effects are more likely because of improved 3D CPC exosome function compared with 2D CPC exosomes. CONCLUSIONS: Spherical aggregation improves child CPC function in a Notch-dependent manner. The strong reparative ability of CPC spheres warrants further investigation as a treatment for pediatric heart failure, especially in older children where reparative ability may be reduced.
背景：先天性心脏病可导致危及生命的右心室（RV）心力衰竭。临床试验结果支持扩大基于心脏祖细胞（CPC）的治疗方法。然而，我们最近的数据表明，CPC 随着年龄的增长而丧失功能，早在 1 岁时就开始出现这种情况。 目的：确定将儿童（1-5 岁）CPC 聚集到无支架球体中是否可以通过增强 Notch 信号转导来改善分化，Notch 信号转导是 CPC 命运的已知调节剂。我们假设聚集的（3 维[3D]）CPC 比单层（2 维[2D]）CPC 更能修复 RV 心力衰竭。 方法和结果：使用微井阵列，每个球体产生 1500 个 CPC。与 2D CPC 相比，CPC 聚集显着增加了 Notch1 的基因表达，同时伴随着心脏发生转录因子（GATA4、HAND1、MEF2C、NKX2.5 和 TBX5）和内皮标志物（CD31、FLK1、FLT1、VWF）的显着上调。用γ-分泌酶抑制剂 DAPT（N-[N-（3,5-二氟苯乙酰基）-L-丙氨酰]-S-苯基甘氨酸叔丁酯）阻断 Notch 受体激活会减弱这些作用。为了评估 CPC 聚集的治疗改善效果，通过肺动脉缩窄在无胸腺大鼠中诱导 RV 心力衰竭，并将细胞植入 RV 游离壁。植入后 28 天的超声心动图测量显示，与 2D CPC 相比，3D CPC 显着改善了 RV 功能。通过 DiR（1,1'-二辛基-3,3,3',3'-四甲基吲哚碳酰亚胺碘化物）标记追踪植入的 CPC 显示，3D CPC 的保留得到改善。用 Notch1-shRNA（短发夹 RNA）转导 3D CPC 不会降低保留率，但显着降低了 RV 功能改善。组织学分析显示，3D 治疗减少了 RV 纤维化并增加了血管生成。尽管 3D CPC 在体内形成 CD31 管状细胞，但这些作用更可能是由于与 2D CPC 外泌体相比，3D CPC 外泌体的功能得到了改善。 结论：球体聚集以 Notch 依赖性方式改善儿童 CPC 的功能。CPC 球体的强大修复能力值得进一步研究，作为小儿心力衰竭的治疗方法，特别是在修复能力可能降低的大龄儿童中。
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