SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection. SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection

Nearly 20% of hospitalized patients diagnosed with severe SARS-CoV-2 infection are at risk for thromboembolic events. We developed a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells, pericytes, and smooth muscle cells to recapitulate the vascular pathology associated with SARS-CoV-2 exposure. Our results demonstrate that perivascular cells, particularly smooth muscle cells (SMCs), are a specifically susceptible vascular target for SARS-CoV-2 infection. Utilizing RNA sequencing, we characterized the transcriptomic changes accompanying SARS-CoV-2 infection of SMCs, and endothelial cells (ECs). We observed that infected human SMCs shift to a pro-inflammatory state and increase the expression of key mediators of the coagulation cascade. Further, we showed human ECs exposed to the secretome of infected SMCs produce hemostatic factors that can contribute to vascular dysfunction, despite not being susceptible to direct infection. The findings here recapitulate observations from patient sera in human COVID-19 patients and provide mechanistic insight into the unique vascular implications of SARS-CoV-2 infection at a cellular level. Overall design: Human endothelial cells (ECs), pericytes (PCs), and smooth muscle cells (SMCs) were differentiated from human pluripotent stem cells(hPSCs). hPSC-derived ECs and SMCs were treated with either live SARS-CoV-2, heat-inactivated (HI) SARS-CoV-2 or mock-infected (no virus). We compared the transcriptional changes induced by each of these conditions in both ECs and SMCs. We also compared the transcriptional changes induced by the treatment of ECs with media from SMCs that were infected with SARS-CoV-2 or HI SARS-CoV-2. For the media transfer experiments, ECs were exposed to media from SMCs that had been exposed to live or HI SARS-CoV-2, and then RNA was isolated from the ECs 48 hours after exposure.

近20%确诊为重症严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）感染的住院患者，存在血栓栓塞事件风险。本研究采用人诱导多能干细胞来源的内皮细胞、周细胞和平滑肌细胞，构建了新冠病毒感染模型，以复现新冠病毒暴露相关的血管病理改变。研究结果显示，血管周细胞，尤其是平滑肌细胞（smooth muscle cells, SMCs），是新冠病毒感染的特异性易感血管靶标。本研究通过RNA测序技术，解析了新冠病毒感染的SMCs及内皮细胞（endothelial cells, ECs）的转录组变化特征。研究发现，感染新冠病毒的人源SMCs会向促炎表型转化，并上调凝血级联反应关键介质的表达水平。此外，本研究证实，尽管人源ECs无法被新冠病毒直接感染，但暴露于感染SMCs的分泌组后，其可产生可诱发血管功能障碍的止血因子。本研究结果复现了新冠患者血清中的临床观测结果，并从细胞层面揭示了新冠病毒感染独特的血管病理机制。
实验整体设计：从人多能干细胞（human pluripotent stem cells, hPSCs）分化获得人源内皮细胞（ECs）、周细胞（pericytes, PCs）和平滑肌细胞（SMCs）。将hPSC来源的ECs与SMCs分别接种活新冠病毒、热灭活（heat-inactivated, HI）新冠病毒，或进行模拟感染（未添加病毒）处理。本研究对比了上述三种处理方式在ECs与SMCs中诱导的转录组变化。此外，本研究还对比了使用感染新冠病毒或热灭活新冠病毒的SMCs培养基处理ECs后，ECs的转录组变化差异。在培养基转移实验中，ECs先暴露于经活新冠病毒或热灭活新冠病毒处理的SMCs培养基，随后于暴露48小时后收集ECs并提取总RNA。