Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation
- Stanford Cardiovascular Institute, Stanford University, United States
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, United States
- Department of Pharmacy, Sungkyunkwan University, United States
- Division of Pediatric Cardiology, Department of Pediatrics, Stanford University, United States
- School of Medicine, University of California, San Diego, United States
- Department of Biomedicine, Aarhus University, Denmark
- Department of Pediatrics, Stanford University, United States
- Division of Cardiovascular of Medicine, Department of Medicine, Stanford University, United States
Figures
Figure 1 with 1 supplement
Integration of T-box transcription factor (TBX5)/myosin light chain-2 (MYL2) lineage tracing reporter system into human-induced pluripotent stem cells.
(A) Schematic of lineage tracing strategy for identifying left ventricular cardiomyocytes in vitro. (B) CRISPR/Cas9 gene targeting strategy of genetic constructs for TBX5 lineage tracing and MYL2 …
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Figure 1—source data 1
Raw and uncropped DNA electrophoresis image data for genotyping PCR for genetic constructs presented in Figure 1C.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig1-data1-v2.zip
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Figure 1—source data 2
Single guide RNA sequences used for gene targeting.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig1-data2-v2.xlsx
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Figure 1—source data 3
Genotyping primer sequences for identification of genetic constructs.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
Flow cytometry analysis of TurboGFP expression in genome-edited human-induced pluripotent stem cells (hiPSCs) after 30 days of pluripotency culture.
Genome edited SCVI-111 and WTC were cultured in pluripotency media for 30 days. Flow cytometry plots are shown for day 0 of culture and day 30 of culture.
Figure 2 with 1 supplement
T-box transcription factor (TBX5)-lineage tracing reveals a predominance of lineage positive cardiomyocytes through the course of human-induced pluripotent stem cell (hiPSC) cardiac differentiation.
(A) Schematic of analysis approach of reporter system expression through the course of cardiac differentiation. (B) Representative flow cytometry plots for the expression of TurboGFP and cardiac …
Figure 2—figure supplement 1
FACS sorted TurboGFP positive cells enrich for T-box transcription factor (TBX5) xxpression.
(A) Representative FACS plot of sort GFP+ and GFP− day 15 WTC reporter line cardiac differentiations. (B) RT-qPCR for TBX5 and TurboGFP expression of GFP+ and GFP− sorted day 15 WTC cardiac …
Figure 3
Immunofluorescence imaging of day 30 reporter cardiomyocytes and RT-qPCR profiling of first heart field (FHF) and second heart field (SHF) markers across differentiation.
(A) Immunofluorescence images of TurboGFP, TdTomato, and TNNT2 expression at day 30 of cardiac differentiation for WTC and SCVI-111 reporter lines. (B) RT-qPCR profiling of FHF markers HAND1 and …
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Figure 3—source data 1
qPCR human primer sequences.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig3-data1-v2.xlsx
Figure 4 with 7 supplements
single-cell RNA sequencing (scRNA-seq) profiling and trajectory inference reveals emergence of myocardial and epicardial lineages during human-induced pluripotent stem cell (hiPSC) cardiac differentiation.
(A) Diagram of scRNA-seq multiplexing for profiling of 12 timepoints across two lines during hiPSC cardiac differentiation. (B) Left, UMAP plot with identification of 13 cell populations over the …
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Figure 4—source data 1
GEO Accession Numbers for Datasets.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig4-data1-v2.xlsx
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Figure 4—source data 2
Differentially Expressed Genes Identified For Annotated Cell Types.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig4-data2-v2.xlsx
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Figure 4—source data 3
Genes Correlated with Differentiation Trajectories Identified During hiPSC Cardiac Differentiation.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig4-data3-v2.xlsx
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Figure 4—source data 4
Description of scRNA-seq Run and hashtag oligo sequences used for sample multiplexing.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig4-data4-v2.xlsx
Figure 4—figure supplement 1
Quality control and hashtag oligo labeling of samples for scRNA-seq sample Galdos_Seq_Run1.
(A) Cutoff values (dotted red lines) were calculated for quality control filtering of four metrics evaluated. (B) Ridge plots demonstrating highly specific labeling of chemically modified oligos for …
Figure 4—figure supplement 2
Quality control and hashtag oligo labeling of samples for scRNA-seq sample Galdos_Seq_Run2.
(A) Cutoff values (dotted red lines) were calculated for quality control filtering of four metrics evaluated. (B) Ridge plots demonstrating highly specific labeling of chemically modified oligos for …
Figure 4—figure supplement 3
Quality control and hashtag oligo labeling of samples for scRNA-seq sample Galdos_Seq_Run3.
(A) Cutoff values (dotted red lines) were calculated for quality control filtering of four metrics evaluated. (B) Ridge plots demonstrating highly specific labeling of chemically modified oligos for …
Figure 4—figure supplement 4
Unsupervised clustering and marker expression of combined human-induced pluripotent stem cell (hiPSC) single-cell RNA sequencing (scRNA-seq) data from WTC and SCVI-111 lines.
(A) UMAP embedding for combined scRNA-seq data from WTC and SCVI-111 lines, along with unsupervised clustered identified. (B) Stacked violin plots for distinct cell markers enriched across all …
Figure 4—figure supplement 5
Trajectory inference analysis of human-induced pluripotent stem cell (hiPSC) cardiac differentiation across WTC and SCVI-111 Lines.
(A) UMAP embedding reproduced from Figure 4A demonstrates annotation of 12 distinct cell types. (B) Compositional analysis showing the proportion of each cell type identified across all timepoints …
Figure 4—figure supplement 6
Feature plots of selected first heart field (FHF), second heart field (SHF), endoderm, and cardiomyocyte markers.
(A) UMAP of human-induced pluripotent stem cell (hiPSC) cardiac differentiation showing cell type annotation, (B) cell line of origin, and (C) timepoint during differentiation. (D) Feature plots …
Figure 4—figure supplement 7
Comparison of expression of atrial and ventricular cardiomyocyte markers in human-induced pluripotent stem cell (hiPSC)-CM single-cell RNA sequencing (scRNA-seq) time course.
(A) UMAP of hiPSC cardiac differentiation showing cell type annotation, (B) cell line of origin, and (C) timepoint during differentiation. (D) Feature plots showing gene expression of atrial markers …
Figure 5
Comparison of heart field development between murine and human-induced pluripotent stem cell (hiPSC) cardiac differentiation reveals first heart field (FHF) identity of hiPSC cardiac lineages.
(A) Top, UMAP plot showing clustering of murine cell types encompassing early mesodermal progenitors, FHF/second heart field (SHF) progenitors, LV/RV/OFT cardiomyocytes, and epicardial cells. …
Figure 6
Comparison of 2D and 3D cardiac differentiation uncovers potential of organoid system for second heart field (SHF) generation.
(A) Schematic of comparison strategy of single-cell RNA sequencing (scRNA-seq) data from 2D human-induced pluripotent stem cell (hiPSC) cardiac differentiation generated using our 2D protocol and a …
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Figure 6—source data 1
Differential gene expression analysis between putative OFT and LV cardiomyocyte clusters in joint 2D and 3D hiPSC-CM Data.
Pct1 and Pct2 represent the percentage of cells in cluster 1 and all other that express a particular gene.
- https://cdn.elifesciences.org/articles/80075/elife-80075-fig6-data1-v2.xlsx
Videos
Video 1
Contractility of SCVI-111 reporter line cardiomyocytes at day 15 of differentiation.
Video 2
