Matured hiPSC-derived cardiomyocytes possess dematuration plasticity

Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used to identify potential factors capable of inducing endogenous cardiomyocyte proliferation to regenerate the injured heart. L-type calcium channel blockers have previously been identified as a class of factors capable of inducing matured hiPSC-CMs to proliferate. However, the mechanism by which L-type calcium channel blockers promote hiPSC-CM proliferation remains unclear. Here we provide evidence that matured hiPSC-CMs possess plasticity to undergo dematuration in response to certain pharmacological compounds. Consistent with primary cardiomyocyte maturation during perinatal development, we found that centrosome disassembly occurs in hiPSC-CMs during plate-based, temporal, maturation. A small molecule screen identified Nitrendipine, an L-type calcium channel blocker, and 1-NA-PP1, a Src kinase inhibitor, as factors capable of inducing centrosome reassembly in a subpopulation of hiPSC-CMs. Furthermore, centrosome-positive hiPSC-CMs were more likely to exhibit cell cycle activity than centrosome-negative hiPSC-CMs. In contrast, neither Nitrendipine or 1-NA-PP1 induced centrosome reassembly, or cell cycle activity, in neonatal rat ventricular myocytes (NRVMs). Differential bulk transcriptome analysis indicated that matured hiPSC-CMs, but not NRVMs, treated with Nitrendipine or 1-NA-PP1 undergo dematuration. ScRNA transcriptome analysis supported that matured hiPSC-CMs treated with either Nitrendipine or 1-NA-PP1 undergo dematuration. Collectively, our results indicate that matured hiPSC-CMs, but not primary NRVMs, possess plasticity to undergo dematuration in response to certain pharmacological compounds such as L-type calcium channel blockers and Src-kinase inhibitors. This study shows that once mature, hiPSC-CMs may not maintain their maturity under experimental conditions and thus may have implications for experimental systems where the state of hiPSC-CM maturation is relevant. Bulk RNA prep and sequencing P6-NRVMs and CDI-CMs were treated with Nitrendipine or 1-NA-PP1 for 2 days prior to RNA extraction. In addition, both P6-NRVMs and CDI-CMs during treatment with Nitrendipine or 1-NA-PP1 were cultured in the presence of araC. AraC was used to reduce overgrowth of contaminating cardiac fibroblasts (which generally amount to ~3% of cultured cells in primary cardiomyocyte isolation procedure). RNA was extracted from sub-confluent 10-cm plates using Qiagen RNeasy mini kit. RNA quality (RNA integrity number, RIN) and quantity was measured in a Bioanalyzer 2100 (Agilent) using the Agilent RNA 6000 Nano Kit (part number 5067-1511). The sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760S, New England Biolabs), starting with 200 ng of total RNA. In brief, mRNA was isolated and fragmented using the NEBNext poly(A) mRNA magnetic isolation module (E7490S, New England Biolabs), with the first and second strands of cDNA synthesized and purified using AmPure XP SPRI beads (A63880, Beckman Coulter). Then adaptor ligation and size selection were performed according to the manufacturer’s protocol. Next, the adaptor ligated cDNA was PCR enriched to incorporate an Illumina compatible index sequence (NEBNext Oligos for Illumina, Dual Index Primers Set1 (E7600S, New England Biolabs). All libraries were purified using AmPure XP SPRI beads, and the size distribution of the libraries was analyzed at the Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (part number 5067-4626). Subsequently, the quantification of the libraries was performed with the Qubit® 2.0 Fluorometer (ThermoFisher Scientific) and QubitTM dsDNA HS Assay Kits (Q32851, Invitrogen). Finally, all 19 libraries were pooled and diluted to 3.5 nM for sequencing on one lane of a Hiseq 3000 sequencer (Illumina), using a single read 50 bp and dual indexed sequencing strategy. *************************************************************** Submitter states that missing raw files are due to file loss. ***************************************************************

人类诱导多能干细胞衍生心肌细胞（Human induced Plurotent Stem Cell-derived cardiomyocytes，简称hiPSC-CMs）正日益被应用于筛选可诱导内源性心肌细胞增殖、实现受损心脏再生的潜在调控因子。既往研究已将L型钙通道阻滞剂（L-type calcium channel blockers）鉴定为一类可诱导成熟hiPSC-CMs发生增殖的因子，但其促进hiPSC-CMs增殖的具体分子机制仍未阐明。本研究证实，成熟hiPSC-CMs可响应部分药理学化合物发生去成熟化过程。与围产期发育过程中原代心肌细胞的成熟进程一致，我们发现基于平板培养的时序性成熟过程中，hiPSC-CMs会发生中心体解离。通过小分子筛选，我们鉴定出尼群地平（Nitrendipine，一种L型钙通道阻滞剂）与1-NA-PP1（一种Src激酶抑制剂）可在hiPSC-CMs的亚群中诱导中心体重组装。进一步研究发现，中心体阳性的hiPSC-CMs较中心体阴性细胞更易表现出细胞周期活性。与之相反，尼群地平与1-NA-PP1均无法在新生大鼠心室肌细胞（Neonatal rat ventricular myocytes，简称NRVMs）中诱导中心体重组装或细胞周期活性。批量转录组分析结果显示，经尼群地平或1-NA-PP1处理的成熟hiPSC-CMs（而非NRVMs）发生了去成熟化过程；单细胞RNA转录组分析亦证实，经尼群地平或1-NA-PP1处理的成熟hiPSC-CMs可发生去成熟化。综上，本研究结果表明，成熟hiPSC-CMs（而非原代NRVMs）具备响应部分药理学化合物（如L型钙通道阻滞剂与Src激酶抑制剂）发生去成熟化的可塑性。本研究显示，在实验条件下，成熟后的hiPSC-CMs并非维持其成熟状态，这一发现对以hiPSC-CMs成熟状态为关键变量的实验体系具有重要参考意义。

批量RNA制备与测序
实验前，将P6-NRVMs与CDI-CMs用尼群地平或1-NA-PP1处理2天，随后提取RNA。此外，处理过程中的P6-NRVMs与CDI-CMs均在阿糖胞苷（AraC）存在的条件下培养，阿糖胞苷用于减少污染性心脏成纤维细胞的过度增殖（原代心肌细胞分离过程中，污染性成纤维细胞约占培养细胞的3%）。
从汇合度低于饱和的10cm培养皿中提取RNA，使用Qiagen RNeasy迷你试剂盒（Qiagen RNeasy mini kit）。RNA的质量（RNA完整性数，RIN）与浓度通过Agilent Bioanalyzer 2100生物分析仪（Bioanalyzer 2100），搭配Agilent RNA 6000 Nano试剂盒（Agilent RNA 6000 Nano Kit，货号5067-1511）进行检测。
测序文库以200ng总RNA为起始材料，使用NEBNext Ultra II 定向RNA文库制备试剂盒（NEBNext Ultra II Directional RNA Library Prep Kit for Illumina，货号E7760S，New England Biolabs）构建。具体步骤如下：首先通过NEBNext poly(A) mRNA磁珠分离模块（NEBNext poly(A) mRNA magnetic isolation module，货号E7490S，New England Biolabs）分离并片段化mRNA，随后使用AmPure XP SPRI磁珠（AmPure XP SPRI beads，货号A63880，Beckman Coulter）合成并纯化cDNA的第一链与第二链。接着按照制造商的说明书进行接头连接与片段大小筛选。之后，将连接有接头的cDNA进行PCR富集，以引入适配Illumina平台的索引序列（使用NEBNext Oligos for Illumina双索引引物套装1，NEBNext Oligos for Illumina, Dual Index Primers Set1，货号E7600S，New England Biolabs）。所有文库均使用AmPure XP SPRI磁珠纯化，并通过Agilent Bioanalyzer 2100生物分析仪搭配Agilent High Sensitivity DNA试剂盒（Agilent High Sensitivity DNA Kit，货号5067-4626）分析文库的片段大小分布。随后，使用Qubit® 2.0荧光计（Qubit® 2.0 Fluorometer，ThermoFisher Scientific）与QubitTM dsDNA HS检测试剂盒（QubitTM dsDNA HS Assay Kits，货号Q32851，Invitrogen）对文库进行定量。最后，将全部19个文库混合并稀释至3.5 nM，使用Illumina Hiseq 3000测序仪进行单端50bp双索引测序，测序运行在单个测序泳道中完成。

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