Intracellular delivery of nitric oxide enhances the therapeutic efficacy of mesenchymal stem cells for myocardial infarction

Cell therapy by autologous mesenchymal stem cells (MSCs) is a clinically acceptable strategy for treating various diseases. Unfortunately, the therapeutic efficacy is largely affected by the low quality of MSCs collected from patients. Herein, we showed that the gene expression of MSCs from patients with diabetes was differentially regulated compared to that of MSCs from healthy controls. Then, MSCs were genetically engineered to catalyse an NO prodrug to release NO intracellularly. Compared to extracellular NO conversion, intracellular NO delivery effectively prolonged survival and enhanced the paracrine function of MSCs, as demonstrated by in vitro and in vivo assays. The enhanced therapeutic efficacy of engineered MSCs combined with intracellular NO delivery was further confirmed in mouse and rat models of myocardial infarction, and a clinically relevant cell administration paradigm through secondary thoracotomy has been attempted.

Methods
RNA sequencing was performed by the BGI (Shenzhen, China). Briefly, RNA from the adipose-derived mesenchymal stem cells (ADMSCs) of healthy people and patients with diabetes was extracted using TRIzol reagent (Yeasen, China). RNA samples were sequenced on the BGISEQ platform. The raw data containing low-quality reads, adaptor sequences, and high levels of N bases were filtered before analysis. Then, the clean reads were mapped to the reference genome using HISAT, and Bowtie2 was used to align the clean reads to the reference genes. The reference genome source is NCBI, and the reference genome version is GCF_000001405.39_GRCh38.p13. The expression levels of genes were quantified to identify differentially expressed genes by RSEM. The data uploaded here has not undergone any processing.

自体间充质干细胞（autologous mesenchymal stem cells, MSCs）疗法是临床认可的多种疾病治疗策略。然而，从患者体内采集的间充质干细胞质量欠佳，极大地制约了其治疗效果。本研究发现，糖尿病患者来源的间充质干细胞基因表达调控模式与健康对照者来源的间充质干细胞存在显著差异。随后，本研究通过基因工程改造间充质干细胞，使其能够催化一氧化氮（nitric oxide, NO）前体药物在细胞内释放一氧化氮。与细胞外一氧化氮转化方式相比，细胞内一氧化氮递送策略可有效延长间充质干细胞的存活时长并增强其旁分泌功能，该结论已通过体外及体内实验得到证实。在心肌梗死小鼠与大鼠模型中，基因工程改造联合细胞内一氧化氮递送的间充质干细胞疗法的治疗效果得到进一步验证；同时，本研究已尝试采用二次开胸的临床相关细胞给药范式进行细胞递送。

方法
转录组测序由中国深圳华大基因（BGI）完成。具体流程如下：使用TRIzol试剂（中国翌圣生物）提取健康人群与糖尿病患者脂肪来源间充质干细胞（adipose-derived mesenchymal stem cells, ADMSCs）的总RNA，随后在BGISEQ测序平台完成RNA测序。分析前需对原始数据进行过滤，去除其中的低质量读段、接头序列以及高N碱基占比的读段。将过滤得到的清洁读段（clean reads）通过HISAT比对至参考基因组，同时使用Bowtie2将清洁读段比对至参考基因集。本研究使用的参考基因组来源于NCBI，版本号为GCF_000001405.39_GRCh38.p13。通过RSEM对基因表达水平进行定量分析，以筛选差异表达基因。本次上传的数据集未经过任何预处理。