Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, β-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, β2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/β, PCG 1α/β, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

中文翻译：

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β-肾上腺素能刺激和旋转悬浮培养可增强人脂肪形成的间充质干细胞向高传导性心脏祖细胞的转化。

使用人脂肪形成的间充质干细胞（hAdMSCs）治疗心脏病的临床试验表明，心脏功能有所改善，并且被证明是安全的。但是，hAdMSC不能有效地转化为心肌细胞（CMs）或脉管系统。因此，在将来的研究中，将hAdMSCs重编程为肌细胞祖细胞可能会更好。为了将hAdMSCs重编程为导电性心脏祖细胞，我们率先采用了三步重编程策略，该策略使用了经验证的MESP1 / ETS2转录因子，在三维（3D）心球体中诱导的β-肾上腺素和低氧信号传导。在阶段1中，在hAdMSCs转化为心脏祖细胞的过程中，ETS2和MESP1激活了NNKX2.5，TBX5，MEF2C，dHAND和GATA4。接下来，在阶段2 β2AR激活将心脏祖细胞重新定位在新生的传导性心肌细胞中，并出现了RYR2，CAV2.1，CAV3.1，NAV1.5，SERCA2和CX45基因转录本，并显示了动作电位。在第3阶段中，由Synthecon®，Inc.旋转生物反应器中形成的3D心球体促进的导电诱导了缺氧基因的出现：HIF-1α/β，PCG1α/β和NOS2，与之强烈激活了成年收缩基因，包括MLC2v，TNNT2和TNNI3，离子通道基因，以及超极化激活和环状核苷酸门控通道（HCN1-4）的出现。缺氧后，传导速度加倍至〜200 mm / s，而3D细胞簇解离至〜400 mm / s后又再次加倍。通过对比，在整个心脏中，正常的心室心肌细胞内的正常传导速度为0.5到1 m / s。肾上腺素刺激贴片中的第3阶段心脏细胞导致电波振幅增加，表明传导性心脏细胞。我们将hAdMSCs转化为高传导性心脏祖细胞的有效方案证明了将3期细胞用于组织工程应用中进行心脏修复的潜在用途。