Gene Expression
Nebulas
Gene Expression Nebulas概要: Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells and lung organoids. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression in both human cardiac and lung epithelial cells and protected hESC-derived lung organoids against SARS-CoV-2 infection. To build an in vitro model of viral infection in human lung tissue, we set out to generate lung organoids from hESC using a slightly modified combination of previously established differentiation methods (Carvalho et al., 2019; Jacob et al., 2017; Miller et al., 2019). We performed single cell RNA sequencing of fully differentiated organoids to unbiasedly characterize the cell types present and validate them as a model of SARS-CoV-2 infection and antiandrogenic drug treatment
项目整体设计: Cells during HLO differentiation were harvested on Day 0 (hPSCs), 5, 9, 15, 25, 35, and 50, in duplicates, and used for RNAseq library preparation
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| 生长方案: | Human lung organoids (HLO) were derived from hPSCs as previously described (Carvalho et al., 2019; Jacob et al., 2017; Miller et al., 2019), with minor modifications. Briefly, H9 hPSCs were maintained in mTeSR Plus media and were seeded on Day 0 onto matrigel (Corning) coated surface. On Days 1-4 cells were treated with 100 ng/mL Activin A and increasing concentration of defined FBS (dFBS, HyClone) in RPMI 1640 medium (Thermo), to induce definitive endoderm formation (Day 1: 3 µM CHIR99021 and no dFBS; Day 2: 0.2% dFBS; Days 3-4: 2% dFBS). On Day 4, cells were >95% double positive for CXCR4/CD184 and c-Kit/CD117 as determined by flow cytometry. On Days 5-9 cells were treated with anterior foregut induction medium, containing 10 µM SB431542, 100 nM LDN193189, 2 µM CHIR99021, 1 µM SAG, 500 ng/mL FGF4 in Foregut Basal Medium (Advanced DMEM, 1x B27, 1x N2, 10 mM HEPES, 1x Glutagro (all Thermo), 50 µg/mL ascorbic acid (Sigma), 0.4 µM monothioglycerol (Sigma)). On Day 9, anterior foregut spheroids were harvested by gentle pipetting and transferred into an ultra-low attachment plate, in Lung Organoid Medium I (3 µM CHIR99021, 10 ng/mL BMP4, 10 ng/mL FGF7, 10 ng/mL FGF10, 50 nM all-trans retinoic acid, in Foregut Basal Medium). The medium was changed every two days until Day 15. On Day 15, the medium was changed to Lung Organoid Medium II (3 µM CHIR99021, 10 ng/mL FGF7, 10 ng/mL FGF10, in Foregut Basal Medium). On Day 25 the organoids were embedded in matrigel, in transwell inserts, and grown in Lung Organoid Medium III (3 µM CHIR99021, 10 ng/mL FGF7, 10 ng/mL FGF10, 50 nM dexamethasone (Sigma), 100 µM 8-bromo-cAMP (Sigma), 100 µM IBMX (Sigma), in Foregut Basal Medium). The medium was changed every 3 days. The CHIR99021 was withdrawn on Days 35-42. All media components were from Stem Cell Technologies unless noted otherwise. |
| 处理方案: | - |
| 提取方案: | RNA was extracted using Quick-RNA 96 Kit (Zymo) with on-column DNaseI treatment. |
| 建库方案: | QuantSeq FWD Kit (with UMI module, Lexogen) was used for RNAseq library preparation. |
| 分子类型: | Total RNA |
| 库的片段类型: | SINGLE |
| 库的链类型: | Reverse |
| 测序平台: | ILLUMINA |
| 测序仪型号: | Illumina HiSeq 4000 |
| 链特异性: | Specific |
| 数据来源 | GEN样本编号 | GEN数据集编号 | 系列编号 | 项目编号 | 样本编号 | 样本名称 | 生物样本编号 | 样本访问号 | 实验访问号 | 释放时间 | 提交时间 | 最后更新时间 | 物种 | 种族 | 族裔 | 年龄 | 年龄单位 | 性别 | 来源名称 | 组织 | 细胞类型 | 细胞亚型 | 细胞系 | 疾病 | 疾病状态 | 发育阶段 | 突变/变异 | 表型 | Condition Detail | 生长方案 | 处理方案 | 提取方案 | 建库方案 | 分子类型 | 库的片段类型 | 链特异性 | 库的链类型 | 加标（Spike-In） | 测序方法 | 测序平台 | 测序仪型号 | 细胞数 | 测序片段数 | 碱基数 | 平均测序片段长度_1 | 平均测序片段长度_2 | 唯一比对率 | 多重比对率 | 覆盖度 |
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