Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: Identification of iAT1 cells in FD‐AOs. A, A schematic representation of the analysis of scRNA‐seq in FD‐AOs. FD‐EpCAM + cells (P2) were collected from two independent FD‐AOs using FACS, followed by single‐cell isolation using a Fluidigm C1 system and RNA sequencing. Bioinformatics data were processed using Seurat (version 3.1.2) including FD‐SFTPC + cells (P2) (GSE90813). B, Flow cytometric analyses of FD‐AOs (P2) with or without treatment with DMSO from day 2 to 14 before being subjected to scRNA‐seq. C, UMAP plots displaying cell clusters of FD‐EpCAM + cells (P2) in FD‐AOs. We defined five major types of FD‐EpCAM + cells: iAT1 cells, FD‐iAT2 cells, mitotic cells, ASCL1 + PNECs, and NKX2‐1 + cells. D, UMAP plots showing the expression of the selected marker genes; AT1 cells ( AGER , CAV1 , and PDPN ), AT2 cells ( SFTPB and SFTPC ), mitotic cells ( TOP2A ), ASCL1 + PNECs ( ASCL1 ), NKX2‐1 + cells ( NKX2‐1 ), and alveolar epithelial cells ( CPM ). E, Violin plots showing the gene expression distributions of the selected marker genes across cell types. F,G, Immunostaining of AT1 markers (HT1‐56, PDPN, and AGER) and SFTPC‐GFP in FD‐AOs (P3). Scale bars = 10 μm. DMSO, dimethyl sulfoxide; FACS, fluorescence‐activated cell sorting; FD‐AOs, fibroblast‐dependent alveolar organoids; scRNA‐seq, single‐cell RNA sequencing; UMAP, uniform manifold approximation and projection

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Single-cell Isolation, RNA Sequencing, Expressing, Marker, Gene Expression, Immunostaining, Fluorescence, FACS

Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: iAT1 cells are present in hiPSC‐derived EpCAM + cell population in FD‐AOs but poorly detected in FF‐AOs. A, A schematic representation of the analysis of scRNA‐seq in FD‐AOs and FF‐AOs. FD‐EpCAM + cells (P0) and FF‐EpCAM + cells (P0) were isolated using FACS. Next, scRNA‐seq was performed and sequencing data, including that of FD‐EpCAM + cells (P2), FD‐EpCAM + /DMSO cells (P2), and FD‐SFTPC + cells (P2), were analyzed. B, Flow cytometric analyses of FD‐EPCAM + cells (P0) and FF‐EpCAM + cells (P0) used for scRNA‐seq. C, UMAP plots displaying cell clusters in FD‐AOs and FF‐AOs. We defined eight major types of hiPSC‐derived EpCAM + cells: iAT1 cells, FD‐iAT2 cells, mitotic cells, ASCL1 + PNECs, ID1 + cells, NKX2‐1 + cells, gastric‐like cells, and FF‐EpCAM + cells. D, Violin plots showing the gene expression distributions of each representative marker gene. E, Immunostaining of AT1 markers (HT1‐56, PDPN, and/or AGER) and SFTPC‐GFP in the FD‐AO (P3) and FF‐AO (P0). Scale bars = 10 μm. DMSO, dimethyl sulfoxide; FACS, fluorescence‐activated cell sorting; FD‐AOs, fibroblast‐dependent alveolar organoids; FF‐AOs, fibroblast‐free alveolar organoids; hiPSC, human induced pluripotent stem cell; scRNA‐seq, single‐cell RNA sequencing; UMAP, uniform manifold approximation and projection

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Derivative Assay, Isolation, Sequencing, Gene Expression, Marker, Immunostaining, Fluorescence, FACS, RNA Sequencing

Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: The transcriptomic profiles of iAT1 cells were similar to those of primary AT1 cells. A,B, Combined UMAP plots of single cells from FD‐EpCAM + cells (P0 and P2), FD‐EpCAM + /DMSO cells (P2), FD‐SFTPC + cells (P2), and FF‐EpCAM + cells (P0) and adult donor lung cells extracted from the database (GSE122960) described in . Adult donor lung cells are shown in gray, FD‐EpCAM + cells (P0 and P2), FD‐EpCAM + /DMSO cells (P2), and FD‐SFTPC + cells (P2) in red, and FF‐EpCAM + cells (P0) in blue (A). UMAP plots revealed 13 clusters in adult donor lung cells and 8 clusters in FD‐EpCAM + and FF‐EpCAM + cells (B). C, Violin plots showing the gene expression distributions of the representative markers indicated in Figure . AT1, alveolar type I

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Gene Expression

Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: The transcriptomes of iAT1 cells were more similar to those of primary AT1 cells than those of 2D‐AT1‐like cells. A, GSEA using the top 100 gene sets of primary AT1 cells or AT1 cells on human postnatal day 1 from the LungGENS database. Significantly up‐ or downregulated genes ( P < .05) ranked by the log difference in average gene expression between FD‐iAT2 and iAT1 cells, or primary 2D‐AT1‐like and AT2 cells (GSE38569) were used for the analysis. B, UMAP plots of 2D‐iAT1 cells added to those of Figure . FD‐SFTPC + cells (P2) were differentiated in 2D cultures for 7 days and subjected to scRNA‐seq. Next, scRNA‐seq was performed and sequencing data, including that of FD‐EpCAM + cells (P0 and P2), FD‐EpCAM + /DMSO cells (P2), FD‐SFTPC + cells (P2), and FF‐EpCAM + cells (P0), were analyzed. C, Violin plots showing the gene expression distributions of the representative lineage markers with COL4A1 , TP63 , KRT5 , KRT17 , and SFN for characterizing 2D‐iAT1 cells. AT1, alveolar type I; GSEA, gene set enrichment analysis; scRNA‐seq, single‐cell RNA sequencing

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Gene Expression, Sequencing, RNA Sequencing

Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: The transcriptomes of HFLF and hiPSC‐derived EpCAM + cells in FD‐AOs. A, A schematic diagram of scRNA‐seq analysis of FD‐AOs. B, UMAP plots displaying cell clusters in FD‐AOs. Six major types of FD‐EpCAM + cells (FD‐iAT2 cells, mitotic cells, ASCL1 + PNECs, INSM1 + cells, NKX2‐1 + cells, and hepatic‐like cells) and three types of fibroblasts (PDGFRA + , WNT5A + /PDGFRA + , and RARRES1 + fibroblasts) were identified. C, Violin plots indicating the gene expression distributions of each fibroblast cluster marker genes. D, Pathway enrichment analysis of PDGFRA + , WNT5A + /PDGFRA + , and RARRES1 + fibroblasts. Significantly upregulated genes ( P < .05) among PDGFRA + , WNT5A + /PDGFRA + , or RARRES1 + fibroblasts compared with either or both of the other cell clusters were used for analysis. The top 100 differential pathways with a P < .05 are shown. E, GSEA using the gene set of β‐catenin degradation by the destruction complex. Significantly up‐ or downregulated genes ( P < .05) ranked by the log difference in average gene expression between iAT1 cells and FD‐iAT2 cells were used for analysis. FD‐AOs, fibroblast‐dependent alveolar organoids; GSEA, gene set enrichment analysis; HFLF, human fetal lung fibroblasts; hiPSC, human induced pluripotent stem cell; scRNA‐seq, single‐cell RNA sequencing; UMAP, uniform manifold approximation and projection

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Derivative Assay, Gene Expression, Marker, RNA Sequencing

Journal: Stem Cells (Dayton, Ohio)

Article Title: Directed induction of alveolar type I cells derived from pluripotent stem cells via Wnt signaling inhibition

doi: 10.1002/stem.3302

Figure Lengend Snippet: XAV‐939 promotes iAT1 cell differentiation in FD‐AOs. A, FD‐AOs (P2) were treated with 10 μM XAV‐939 or DMSO (vehicle control) from day 2 to 14, and then the FD‐EpCAM + cells collected using FACS were subjected to scRNA‐seq. B, GSEA using the gene set of β‐catenin degradation by the destruction complex. Significantly up‐ or downregulated genes ( P < .05) ranked by the log difference in average gene expression between DMSO‐ and XAV‐939‐treated FD‐EpCAM + cells (P2) were used for the analysis. C, UMAP analysis showing cell clusters in XAV‐939‐treated FD‐AOs. XAV‐939‐treated FD‐EpCAM cells (P2) were included among iAT1 cells. D,E, UMAP and violin plots showing the expression of the representative lung epithelial cell marker genes; AT1 cells ( AGER , CAV1 , and PDPN ), AT2 cells ( SFTPB and SFTPC ), mitotic cells ( TOP2A ), ASCL1 + PNECs ( ASCL1 ), ID1 + cells ( ID1 ), NKX2‐1 + cells ( NKX2‐1 ), basal cells ( TP63 ), subpopulation of AT1 cells ( IGFBP2 ), and alveolar epithelial cells ( CPM ). DMSO, dimethyl sulfoxide; FACS, fluorescence‐activated cell sorting; FD‐AOs, fibroblast‐dependent alveolar organoids; GSEA, gene set enrichment analysis; scRNA‐seq, single‐cell RNA sequencing; UMAP, uniform manifold approximation and projection

Article Snippet: For the Chromium Single Cell Gene Expression Solution Platform (10x Genomics), single cells derived from FD‐AO (P0) were processed using the 3' Library and Gel Bead Kit following the manufacturer's user guide (v2, rev B).

Techniques: Cell Differentiation, Control, Gene Expression, Expressing, Marker, Fluorescence, FACS, RNA Sequencing