Gene Expression
Nebulas
Gene Expression NebulasSummary: Differentiation of human pluripotent stem cells toward definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA-sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. We further show that differentiation-arrested phenotype is inversely correlated with zinc concentration in the differentiation media. This study improves our understanding of in-vitro DE differentiation and provides actionable options to improve DE differentiation efficiency.
Overall Design: RNA-sequencing of 329 single cells collected at four time points during a 4-day DE differentiation to identify mechanisms leading to cellular heterogeneity during differentiation
| Strategy: |
|
| Species: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Development Stage: |
|
| Growth Protocol: | hESCs are routinely maintained in E8 medium (Stem Cell Technologies). For differentiation, Advanced RPMI 1640 (Gibco) was supplemented with l-glutamine and antibiotics, with Activin A (R & D Systems) and CHIR99021 (Tocris) added to specified concentrations and time. |
| Treatment Protocol: | - |
| Extract Protocol: | Single-cell whole transcriptome amplification (WTA) was performed using the Fluidigm C1 Single-Cell Auto Prep System (C1 System) as per the manufacturer recommendations (full details available at Fluidigm.com). Briefly, cells from each time point were single cell collected with Accutase and were resuspended at a concentration of 250 cell per L of complete media, mixed 7:3 with C1 suspension reagent (Fluidigm), and loaded onto C1 Single-Cell Auto Prep chips (C1 chips; Fluidigm). After loading, each of the cell isolation chambers on the C1 chip was optically inspected for the presence of a cell. Subsequently, these cells were lysed and SMART-Seq (Ramskold et al., 2012) whole transcriptome amplified (WTA) products were prepared with the C1 System using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) and protocols provided by Fluidigm. |
| Library Construction Protocol: | - |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Unspecific |
| 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 |
|---|