Gene Expression
Nebulas
Gene Expression Nebulas概要: The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human fetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of fetal liver and maintain a unique transcriptional profile distinct from fetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogenicity within the EpCAM+ population of freshly isolated fetal and adult human liver reveals diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolated fetal HHyPs and confirmed their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.
项目整体设计: Primary samples from 5 2nd trimester human fetal livers and 3 uninjured adult human livers for single cell RNA sequencing by Smartseq2.
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| 生长方案: | - |
| 处理方案: | - |
| 提取方案: | Human fetal tissue was dissociated by Collagenase XI enzymatic dissociation for 25 minutes at 37 37℃ with agitation. Samples were stained with the following primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324208, APC, mouse; Biolegend), NCAM (362524, PE, mouse; Biolegend) all at 1:100 dilution and incubated for 30mins at 4 37℃. DAPI (D1306, ThermoFisher Scientific) at 1:1000 dilution was used for live/dead staining. Cells were sorted using a BD FACS Aria II instrument and deposited as single cells into 96-well plates, pre-loaded with lysis buffer (1% Triton X-100, 1mM dNTP, 1μM oligo-dT30, 1:1.2x106 ERCC ExFold RNA spike-in, Recombinant RNase Inhibitor (2313B, Takara Clontech). Single-cell sequencing was performed using SmartSeq2. |
| 建库方案: | RNA was converted into cDNA using SMARTScribe Reverse Transcriptase (639538, Takara Clontech) and amplified for 21 cycles (Kapa HiFi HotStart ReadyMix 2x, KK2602, KAPA Biosystems). Successful single cell libraries were identified by capillary gel electrophoresis (DNF-474-1000, High Sensitivity NGS Fragment Analysis Kit, AATI) and converted into sequencing libraries using a Nextera XT DNA Sample Preparation Kit (FC-131-1096, Illumina). Barcoded libraries were pooled and subjected to 75 base pair paired-end sequencing on a Illumina HiSeq 2500 instrument. |
| 分子类型: | poly(A)+ RNA |
| 库的片段类型: | PAIRED |
| 库的链类型: | -; Forward |
| 测序平台: | ILLUMINA |
| 测序仪型号: | Illumina HiSeq 2500 |
| 链特异性: | Unspecific; 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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