Gene Expression
Nebulas
Gene Expression NebulasSummary: By taking advantage of the foregut generation method, we initially differentiated iPSCs to foregut spheroids through definitive endoderm specification as described. The foregut spheroids were embedded in Matrigel and cultured with retinoic acid (RA). Following 4-day RA treatment, we switched into hepatocyte maturation media for the induction of the hepatocyte differentiation process to establish human liver organoids, hereafter defined as HLO, as early as day 20. To gain quantitative insights regarding the cellular composition in HLO, single-cell RNA sequencing was used to analyze their mRNA expression from 4,059 cells. t-distributed stochastic neighbor embedding analysis confirmed the five distinct major clusters among the cells in HLO, containing hepatocyte-, biliary cell-, hepatic stellate cell-, Kupffer cell-, biliary tree (or peribiliary gland) stem cell-like populations.
Overall Design: Examination of mRNA expression profile at single cell level in multicellular human liver organoids
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Growth Protocol: | iPSCs were differentiated into definitive endoderm. At day1, medium was changed to RPMI 1640 medium containing 100 ng/mL Activin A and 50 ng/mL bone morphogenetic protein 4, 100 ng/mL Activin A and 0.2% fetal calf serum at day 2, and 100 ng/mL Activin A and 2% FCS at day 3. On Day4-6, cells were cultured in Advanced DMEM/F12 with B27 and N2 containing 500 ng/ml fibroblast growth factor and 3 μM CHIR99021. Cells were maintained at 37 ℃ in 5% CO2 with 95% air and the medium was replaced every day. At day 6, spheroids and attached cells were gently pipetted to be delaminated from dishes. They were centrifuged at 800 rpm for 3 minutes, embedded in a 100% Matrigel drop on the dishes in Advanced DMEM/F12 with B27, N2 and 2 μM retinoic acid, and cultured for 4 days. After RA treatment, the media was switched to Hepatocyte Culture Medium with 10 ng/mL hepatocyte growth factor, 0.1 碌M Dexamethasone and 20 ng/mL Oncostatin M. Cultures for HLO induction were maintained at 37 ℃ in 5% CO2 with 95% air and the medium was replaced every 3 days. |
| Treatment Protocol: | - |
| Extract Protocol: | 17,500 cells were underwent the single cell RNA-seq |
| Library Construction Protocol: | Libraries were prepared by the Chromium Single Cell 3' Reagent version 2 kit and Chromium Controller (10X Genomics, CA, USA) as previously described at Zheng, G. X. Y. et al Nature communications 2017. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | 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 |
|---|