Journal: iScience

Article Title: Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

doi: 10.1016/j.isci.2021.103149

Figure Lengend Snippet: Identification of significantly enriched drivers and cell types (A) Growth kinetics of round 1 teratoma formation for injections with driver library transduced hESCs vs control WT hESCs. (B) UMAP visualization of cell types from round 1 teratomas formed by driver library transduced hESCs. (C) Growth kinetics of round 2 tumors formed from re-injected cells from round 1 teratomas formed by driver library transduced hESCs vs WT hESCs. Control measurements are from a common set of tumors grown from the parent WT hESC line, which were used as growth controls for all experiments in this study. (D) UMAP visualization of cell types from round 2 tumors formed by re-injected cells from round 1 teratomas of driver library transduced hESCs. (E) Relative fraction of each cell type in round 1 teratomas formed from library transduced hESCs and WT hESCs, and log fold change with associated -log(p-value) of each cell type for driver library teratomas vs WT teratomas. (F) Relative fraction of each cell type in round 2 tumors formed from library transduced hESCs and WT hESCs, and log fold change with associated -log(p-value) of each cell type for driver library tumors vs WT tumors. (G) Relative fraction of top enriched drivers prior to injection and in each round of tumor formation. (H) H&E stained sections of round 2 tumors formed from WT and driver library transduced hESCs. WT tumors display mature cell types from all three germ layers, such as cartilage (top right), muscle (bottom right) and dermis-like epithelium (bottom left). Driver library tumors display disorganized and more homogeneous composition along with markers of transformation such as nuclear pleomorphism (top right), areas of high mitotic rates (bottom right) and areas of necrosis (bottom left). Scale bars for full sections are 5 mm, scale bars for magnified images are 50 μm.

Article Snippet: H1 male hESC cell line was maintained under feeder-free conditions in mTeSR1 medium (Stem Cell Technologies) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific).

Techniques: Control, Injection, Staining, Transformation Assay

Journal: iScience

Article Title: Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

doi: 10.1016/j.isci.2021.103149

Figure Lengend Snippet: Identification of significantly enriched drivers and cell types for tumors formed with driver libraries without c-MYC and myr-AKT1 (A) Growth kinetics of round 1 teratoma formation for injections with driver library transduced hESCs vs WT hESCs. (B) UMAP visualization of cell types from round 1 teratomas formed by driver library transduced hESCs. (C) Growth kinetics of round 2 tumors formed from re-injected cells from round 1 teratomas formed by driver library transduced hESCs vs WT hESCs. Control measurements are from the common set of tumors grown from the parent WT hESC line, which were used as growth controls for all experiments in this study. (D) UMAP visualization of cell types from round 2 tumors formed by re-injected cells from round 1 teratomas of driver library transduced hESCs. (E) Relative fraction of each cell type in round 1 teratomas formed from library transduced hESCs and WT hESCs, and log fold change with associated -log(p-value) of each cell type for driver library teratomas vs WT teratomas. (F) Relative fraction of each cell type in round 2 tumors formed from library transduced hESCs and WT hESCs, and log fold change with associated -log(p-value) of each cell type for driver library tumors vs WT tumors. (G) Relative fraction of top enriched drivers prior to injection and in each round of tumor formation. (H) H&E stained sections of round 1 and round 2 tumors formed from hESCs transduced with driver libraries without c-MYC or myr-AKT1 . Round 1 tumors contain neuroectodermal and epithelial cell types as the majority of cells, while round 2 tumors contain mesenchymal cell types as the majority of cells. Scale bars for full sections are 5 mm, scale bars for magnified images are 100 μm.

Article Snippet: H1 male hESC cell line was maintained under feeder-free conditions in mTeSR1 medium (Stem Cell Technologies) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific).

Techniques: Injection, Control, Staining, Transduction

Journal: iScience

Article Title: Charting oncogenicity of genes and variants across lineages via multiplexed screens in teratomas

doi: 10.1016/j.isci.2021.103149

Figure Lengend Snippet: Validation of tumor formation and proliferative advantages of significantly enriched drivers from library screens (A) Growth kinetics of round 1 and round 2 tumors formed from a mixture of hESCs transduced with driver hits (c-MYC, myr-AKT1, c-MYC + myr-AKT1 or MEK1 (S218D, S222D) and hESCs transduced with a negative control (mCherry). Control measurements are from the common set of tumors grown from the parent WT hESC line, which were used as growth controls for all experiments in this study. (B) Fraction of reads detecting either the driver or negative control barcodes at each stage: pre-injection, round 1 tumor formation and round 2 tumor formation. Barcodes were amplified from genomic DNA. (C) Gene expression of lineage-specific markers for round 2 tumors driven by individual hits. Expression values are normalized and log transformed. (D) H&E stain of round 2 tumor driven by myr-AKT1 showing regions of poor differentiation (top right) and necrosis (bottom right) interspersed with regions of organized tissue (bottom left). (E) H&E stain of round 2 tumor driven by c-MYC showing regions of poor differentiation (top and bottom right) and regions of necrosis (bottom left). (F) H&E stain of round 2 tumor driven by c-MYC + myr-AKT1 showing regions of poor differentiation (top and bottom right) and regions of necrosis (bottom left). (G) H&E stain of round 2 tumor driven by MEK1 (S218D, S222D) showing primarily regions of mature mesenchymal fibroblast-like tissue (top and bottom right) and cartilage-like (bottom left). (D–G) Scale bars for full sections are 5 mm, scale bars for magnified images are 50 μm. (H) Immunofluorescence micrographs of DAPI and Ki-67 stained sections. Scale bars are 75 μm.

Article Snippet: H1 male hESC cell line was maintained under feeder-free conditions in mTeSR1 medium (Stem Cell Technologies) supplemented with 1% antibiotic-antimycotic (Thermo Fisher Scientific).

Techniques: Biomarker Discovery, Transduction, Negative Control, Control, Injection, Amplification, Gene Expression, Expressing, Transformation Assay, Staining, Immunofluorescence