Gene Expression
Nebulas
Gene Expression NebulasSummary: 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.
Overall Design: 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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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | 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. |
| Library Construction Protocol: | 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. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | -; Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific; Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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