Journal: Transplantation

Article Title: Extracorporeal Photopheresis Enhances the Frequency and Function of Highly Suppressive FoxP3 + Treg Subsets in Heart Transplanted Individuals

doi: 10.1097/TP.0000000000005201

Figure Lengend Snippet: Absolute numbers and percentages of circulating immune cells in HTx participants before (T0) and after 3 (T1), 6 (T2), and 12 (T3) mo of ECP treatment

Article Snippet: Specifically, PBMCs were stained with a mix of the following monoclonal antibodies: APC-H7 anti-human CD4 (BD Pharmingen, clone RPA-T4), FITC-anti-human CD45RA (Miltenyi Biotec, clone REA562), BB700 anti-human CCR7 (BD Horizon, clone 3D12), PECy7 anti-human CD25 (BD Pharmingen, clone M-A251), BV421 anti-human PD-1 (BD Horizon, clone EH12-1), antigen-presenting cell anti-human CD152/CTLA-4 (BD Pharmingen, clone BN13), PE anti-human FoxP3-All (BD Pharmingen, clone 259D/C7), or PE anti-human FoxP3-E2 (ThermoFisher Scientific, clone 150D/E4).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Deriving Human Intestinal Organoids with Functional Tissue-Resident Macrophages All From Pluripotent Stem Cells

doi: 10.1016/j.jcmgh.2024.101444

Figure Lengend Snippet: Direct differentiation of monocytes/macrophages from hPSCs. ( A, B ) Schematic and representative images of the directed differentiation of hPSCs into HPCs followed by subsequent differentiation into monocytes/macrophages (mono/macro) using M-CSF and GM-CSF. ( C ) Representative flow cytometric analysis of hPSC-derived myeloid cells stained with viability 7AAD, panhematopoietic CD45, mono/macro CD14 and CD11c, and macrophage CD163. No stain controls show gating strategy. ( D, E ) Schematic and gross images of Day 21 mono/macro embedded in Matrigel and cultured in HIO media with and without supplemented M-CSF and GM-CSF. ( F ) Representative flow cytometry of Day 35 mono/macro with and without supplemented M-CSF and GM-CSF. Adv. DMEM, Advanced DMEM; CSF, colony-stimulating factor; EGF, epidermal growth factor; L-Glut, L-glutamine; mono/macro, monocyte and macrophage; P/S, penicillin /streptomycin.

Article Snippet: Anti-human CD45 FITC , Tonbo Biosciences (35-9459-T025).

Techniques: Derivative Assay, Staining, Cell Culture, Flow Cytometry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Deriving Human Intestinal Organoids with Functional Tissue-Resident Macrophages All From Pluripotent Stem Cells

doi: 10.1016/j.jcmgh.2024.101444

Figure Lengend Snippet: Generation of HIOs containing hPSC-derived macrophages. ( A ) Schematic of protocol for mixing Day 28 hPSC-derived macrophages (Macro) with Day 21 HIOs during followed by 2 weeks of coculture in Matrigel and standard HIO media (no M-CSF). ( B ) Representative whole-mount immunofluorescence of Day 35 HIOs and HIO + Macro stained for macrophages CD163 ( red ), endothelial CD34 ( green ), and epithelial CDH1 ( white ). Macro were added to HIOs either by mixing within Matrigel during plating ( center ) or in media/supernatant overlaying the Matrigel ( right ). Higher magnification image shown of boxed area. Scale bars: 200 μm for low and 50 μm for high magnifications. ( C ) Relative expression of mono/macro markers CD163 , CD14 , ITGAX/CD11c and cytokines IL6 , IL10 , and TNFα in Day 35 HIO and HIO + Macro. ( D ) Representative flow cytometric analysis and gating of viable (7AAD-), mono/macro (CD45+ CD14+), macro (CD163+ CD11c+) cells in Day 35 HIOs and HIO + Macro. ( E ) Quantification of percent of viable CD14+ CD45+ gated cells in Day 35 HIO and HIO + Macro. Statistical significance was set at ∗∗ P < .01 and ∗∗∗ P < .001, according to t test (n = 4). ( F ) UMAP visualization of scRNA-sequencing of Day 35 HIOs and HIO + Macro from 2 independent differentiations (N = 2). ( G ) Seurat clustering UMAP colored by annotated major cell populations. ( H ) Cell proportions of major cell types present in HIO and HIO + Macro compared with published proportions in 2 human gastrointestinal tract scRNA-seq atlases. Number of cells of each condition listed above the bar. ( I ) Select feature plots showing expression of markers for epithelial ( CDH1 ), mesenchymal ( EMILIN1 , CSF1 ), endothelial ( CDH5 ), and mono/macro ( PTPRC/CD45 , CSF1R ) cell populations. ( J ) UMAP of reclustered monocyte/macrophage cluster 7 from Day 35 HIO and HIO + Macro datasets. Proportion of cells and relative expression of ( K ) commonly used monocyte and macrophage markers and cytokines and ( L ) macrophage subtype-enriched markers. CSF, colony-stimulating factor; EGF, epidermal growth factor; MHG Agg, mid/hind gut aggregates; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Anti-human CD45 FITC , Tonbo Biosciences (35-9459-T025).

Techniques: Derivative Assay, Immunofluorescence, Staining, Expressing, Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Deriving Human Intestinal Organoids with Functional Tissue-Resident Macrophages All From Pluripotent Stem Cells

doi: 10.1016/j.jcmgh.2024.101444

Figure Lengend Snippet: Antibodies for Immunofluorescence and Flow Cytometry

Article Snippet: Anti-human CD45 FITC , Tonbo Biosciences (35-9459-T025).

Techniques: Immunofluorescence