Table_4_Single-Cell RNA-seq Identifies Cell Subsets in Human Placenta That Highly Expresses Factors Driving Pathogenesis of SARS-CoV-2.xls

Infection by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) results in the novel coronavirus disease COVID-19, which has posed a serious threat globally. Infection of SARS-CoV-2 during pregnancy is associated with complications such as preterm labor and premature rupture of membranes, and a proportion of neonates born to infected mothers are also positive for the virus. During pregnancy, the placental barrier protects the fetus from pathogens and ensures healthy development. To predict if the placenta is permissive to SARS-CoV-2, we utilized publicly available single-cell RNA-seq data to identify if the placental cells express the necessary factors required for infection. SARS-CoV-2 binding receptor ACE2 and the S protein priming protease TMPRSS2 are co-expressed by a subset of syncytiotrophoblasts (STB) in the first trimester and extravillous trophoblasts (EVT) in the second trimester human placenta. In addition, the non-canonical receptor BSG/CD147 and other proteases (CTSL, CTSB, and FURIN) are detected in most of the placental cells. Other coronavirus family receptors (ANPEP and DPP4) were also expressed in the first and second trimester placental cells. Additionally, the term placenta of multiple species including humans expressed ACE2, DPP4, and ANPEP along with the viral S protein proteases. The ACE2- and TMPRSS2-positive (ACE2 + TMPRSS2 +) placental subsets expressed mRNA for proteins involved in viral budding and replication. These cells also had the mRNA for proteins that physically interact with SARS-CoV-2 in host cells. Further, we discovered unique signatures of genes in ACE2 + TMPRSS2 + STBs and EVTs. The ACE2 + TMPRSS2 + STBs are highly differentiated cells and express genes involving mitochondrial metabolism and glucose transport. The second trimester ACE2 + TMPRSS2 + EVTs are enriched for markers of endovascular trophoblasts. Both these subtypes abundantly expressed genes in the Toll-like receptor pathway. The second trimester EVTs are also enriched for components of the JAK-STAT pathway that drives inflammation. We carried out a systematic review and identified that in 12% of pregnant women with COVID-19, the placenta was infected with SARS-CoV-2, and the virus was detected in STBs. To conclude, herein we have uncovered the cellular targets for SARS-CoV-2 entry and have shown that these cells can potentially drive viremia in the developing human placenta. Our results provide a basic framework toward understanding the paraphernalia involved in SARS-CoV-2 infections in pregnancy.

严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染可引发新型冠状病毒病（COVID-19），该疾病已对全球构成严重威胁。妊娠期感染SARS-CoV-2与早产、胎膜早破等并发症相关，且部分感染母亲所产新生儿的病毒检测结果也呈阳性。妊娠期，胎盘屏障可保护胎儿免受病原体侵害并保障其健康发育。为预测胎盘是否对SARS-CoV-2易感，我们利用公开的单细胞RNA测序（single-cell RNA-seq）数据，探究胎盘细胞是否表达病毒感染所需的必要功能因子。SARS-CoV-2的结合受体血管紧张素转换酶2（ACE2）以及S蛋白切割蛋白酶跨膜丝氨酸蛋白酶2（TMPRSS2），在人类胎盘早期合体滋养层细胞（syncytiotrophoblasts, STB）亚群与中期绒毛外滋养层细胞（extravillous trophoblasts, EVT）中共表达。此外，非经典受体BSG/CD147及其他蛋白酶（组织蛋白酶L、组织蛋白酶B与弗林蛋白酶）在多数胎盘细胞中均有检出。冠状病毒家族的其他受体氨肽酶N（ANPEP）与二肽基肽酶4（DPP4），在早、中期胎盘细胞中也有表达。包括人类在内的多个物种的足月胎盘，均表达ACE2、DPP4与ANPEP以及病毒S蛋白切割相关蛋白酶。共表达ACE2与TMPRSS2（ACE2+TMPRSS2+）的胎盘细胞亚群，可表达病毒出芽与复制相关蛋白的mRNA。这类细胞同时还表达宿主细胞中与SARS-CoV-2发生物理相互作用的蛋白对应的mRNA。进一步研究发现，ACE2+TMPRSS2+的STB与EVT中存在独特的基因表达特征。ACE2+TMPRSS2+ STB为高度分化的细胞，其表达与线粒体代谢及葡萄糖转运相关的基因。中期妊娠的ACE2+TMPRSS2+ EVT则富集血管内滋养层细胞标志物。这两种细胞亚型均大量表达Toll样受体通路相关基因。中期EVT还富集了驱动炎症反应的JAK-STAT通路相关组分。我们开展了一项系统综述，结果显示12%的COVID-19孕妇的胎盘感染了SARS-CoV-2，且病毒可在STB中被检测到。综上，本研究明确了SARS-CoV-2入侵胎盘的细胞靶点，并证实这些细胞可在发育中的人类胎盘中潜在引发病毒血症。本研究结果为理解妊娠期SARS-CoV-2感染相关机制提供了基础框架。