Journal: Frontiers in Immunology

Article Title: The impact of cholesteryl ester transfer protein on the progression of cutaneous leishmaniasis

doi: 10.3389/fimmu.2024.1389551

Figure Lengend Snippet: Histopathological analyses of CD68+ (PANEL 1), CD163+ (PANEL 2), and CD206+ (PANEL 3) cells in footpad lesions of WT and CETP mice at 4 and 12-weeks post-infection with L. (L.) amazonensis . Control, uninfected footpad: WT (A) and CETP (B) . Infected: WT (C, E) and CETP (D, F) in the 4th and 12th week PI analyzed under an optical microscope (100x magnification). Representative images from three independent experiments. Arrows indicate the presence of immunostaining in macrophages. (G) Representative graph of cell density (cells/mm2) with the respective macrophage markers. Results expressed as mean ± SD, n= 4–6 per group, compared by Mann-Whitney test: WT vs. CETP 4th and 12th weeks PI; * p <0.05 and ** p <0.005, *** p <0.001.

Article Snippet: CD206 Mouse Monoclonal , 1/1000 , BioRad/MCA55527.

Techniques: Infection, Control, Microscopy, Immunostaining, MANN-WHITNEY

Journal: STAR Protocols

Article Title: Protocol for differentiation of functional macrophages from human induced pluripotent stem cells

doi: 10.1016/j.xpro.2024.102925

Figure Lengend Snippet: Changes in the morphology and phenotype of HPCs during the hematopoietic commitment phase (A) Representative bright-field images of colonies (upper) and floating HPCs (lower) during the hematopoietic commitment phase of hiPSCs. Scale bars are 200 μm (upper) and 100 μm (lower). (B) Representative FACS dot plots of CD34, CD43, and CD45 expression in generated HPCs. (C) The generated HPCs were analyzed by flow cytometry for the phenotypes of primitive hematopoietic progenitors (CD34 + CD45 + or CD34 + CD43 + ) and mature blood cells (CD34 - CD45 + or CD34 - CD43 + ) during days 15–21. The bars indicate the mean ± SD, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: APC mouse anti-human CD43 (1:500) , BD Biosciences , 560198.

Techniques: Expressing, Generated, Flow Cytometry

Journal: STAR Protocols

Article Title: Protocol for differentiation of functional macrophages from human induced pluripotent stem cells

doi: 10.1016/j.xpro.2024.102925

Figure Lengend Snippet:

Article Snippet: APC mouse anti-human CD43 (1:500) , BD Biosciences , 560198.

Techniques: Recombinant, Knock-Out, Software, Cell Culture, Dissection, Microscopy

Journal: iScience

Article Title: CRISPR-Cas12a-integrated transgenes in genomic safe harbors retain high expression in human hematopoietic iPSC-derived lineages and primary cells

doi: 10.1016/j.isci.2023.108287

Figure Lengend Snippet:

Article Snippet: Mouse anti-human CD43, clone CD43-10G7, APC , STEMCELL Technologies , Cat#60085AZ.

Techniques: Control, Virus, Selection, Recombinant, Knock-Out, Cloning, Plasmid Preparation, Modification, Staining, Reverse Transcription, Generated, Software, Flow Cytometry

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: Hp-spheroid system can induce hematopoiesis from hiPSCs without addition of exogenous factors (A) Schematic of coculturing hiPSCs and hBMSCs as spheroids in 3D conditions. (B) Representative GFP fluorescence microscopy image of Hp-spheroids on day 1. Scale bar: 300 μm. (C) Light microscopy image of Hp-spheroids cultured in 3D culture plates (left) and in bioreactors (right) for 13 days. Scale bar: 600 μm. Red arrows indicate the outline of a single Hp-spheroid. (D) Representative flow cytometry analysis of CD34 + and CD43 + cells in Hp-spheroids on day 13. GFP + cells are gated for analysis. (E) Yield comparison of 3D plate vs. bioreactor. Left: number of GFP + cells in day 13 Hp-spheroids. Graph shows results when 1 × 10 6 hiPSCs were used for differentiation. Right: frequencies of CD34 + CD43 + cells in GFP + cells in day 13 Hp-spheroids. Values represent mean ± SD from 4 independent experiments, ∗∗∗∗p < 0.0001. (F) Number (left) and the percentage (right) of GFP + cells (hiPSC-derived cells) and GFP − cells (hBMSCs) in Hp-spheroids within 13 days of culture. Values represent mean ± SD from 3 independent experiments. Representative flow cytometry analysis is shown in Figure S3 A. (G) Fluorescence microscopy image of Hp-spheroids on day 7. Scale bar: 300 μm. (H) NCRM5-EGFP cells were cocultured with hBMSCs or iSTCs (isolated after 8, 14, or 20 days of differentiation) to form Hp-spheroids. Spheroids were cultured in bioreactors for 13 days, and the percentage of CD34 + CD43 + cells in GFP + cells isolated from each type of Hp-spheroids was analyzed by flow cytometry. Values represent mean ± SD from 3 independent experiments; ns, not significant. (I) Number of GFP + cells (left) and the percentage of APLNR + cells in GFP + cells (right) detected in EBs cultured in conditioned medium or in Hp-spheroids on day 6. The same number of EBs was used for each condition. Horizontal bars represent mean value from 3 independent experiments, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. iSTC, hiPSC-derived stromal cell; CM, conditioned medium.

Article Snippet: APC Mouse anti-Human CD43 (Clone 1G10) , BD Biosciences , Catalog No: 560198, RRID AB_1645460.

Techniques: Fluorescence, Microscopy, Light Microscopy, Cell Culture, Flow Cytometry, Derivative Assay, Isolation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: The Hp-spheroid system induces hiPSCs to self-organize into yolk sac-like organoids (A) NCRM5-EGFP cells and hBMSCs were used for Hp-spheroid formation. The differentiation of GFP + cells in Hp-spheroids was monitored by kinetic flow cytometry analyses. Histological analyses were performed on day 13 Hp-spheroids. (B) Kinetic flow cytometry analysis of APLNR and PDGFRα expression on GFP + cells in Hp-spheroids from days 4 through 7. Values represent mean ± SD from 3 independent experiments. A representative flow cytometry analysis is shown in Figure S4 A. (C) Kinetic flow cytometry analysis of CD144, CD31, and CD73 expression on GFP + cells in Hp-spheroids from days 6 to 10. The percentages of CD144 + CD31 + cells in GFP + cells are shown by blue bars, and the percentages of CD73 + cells in GFP + CD144 + CD31 + are shown by red dots. Values represent mean ± SD from 3 independent experiments. A representative flow cytometry analysis is shown in Figure S4 B. (D) Hematoxylin and eosin staining of day 13 Hp-spheroids. Scale bar: 200 μm. (E) Whole-mount immunostaining analysis of AFP and PDGFRβ in a day 13 Hp-spheroid. Scale bar: 50 μm. (F) Immunostaining of a day 13 Hp-spheroid for CD34 and CD43. Scale bar: 50 μm. (G) Higher magnification of the boxed area in (F). (H) Immunostaining of Hp-spheroids on day 13 for CD31 and CD43. Scale bar: 100 μm. (I) Higher magnification of the boxed area in (H). (J) Schematic outline of bulk RNA sequencing analysis. (K) Gene set enrichment analysis for a human yolk sac gene set (top 196 yolk sac genes from Cindrova-Davies et al. ). NES, normalized enrichment score; FDR, false discovery rate.

Article Snippet: APC Mouse anti-Human CD43 (Clone 1G10) , BD Biosciences , Catalog No: 560198, RRID AB_1645460.

Techniques: Flow Cytometry, Expressing, Staining, Immunostaining, RNA Sequencing Assay

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: HPCs generated in iSTC-Hp-spheroids can undergo maturation to erythroid cells, macrophages, or T lymphocytes using established cell maturation protocols (A) Schematic outline illustrating the use of Mart1-iPSC cells for iSTC-Hp-spheroid formation and subsequent erythroid cell, macrophage, and T lymphocyte differentiation. (B) Frequencies of CD34 + CD43 + and CD34 + CD45 + cells in Mart1-iPSC-derived iSTC-Hp-spheroids on day 13. Values represent mean ± SD from 3 independent experiments. (C–E) Mart1-iPSC-derived HPCs cultured in the erythroid differentiation condition for 15 days were harvested to characterize erythroid cell properties. The color of cell pellets changed to red (C), and typical erythroid lineage markers, such as CD71 and GPA, could be detected by flow cytometry analysis (D). Globin expression patterns were determined by relative RNA expression of α- and ζ-globin (α-globin series) and ε-, γ-, and β-globin (β-globin series) (E). Values represent mean ± SD from 3 independent experiments. (F–I) Macrophage differentiation from Mart1-iPSC-derived CD34 + cells. (F) Macrophage surface markers were analyzed by flow cytometry (gray; isotype control). (G) Giemsa stain of macrophage cytospin. Scale bar: 50 μm. (H) Phagocytosis assay of fluorescein isothiocyanate (FITC)-labeled zymosan A particles (gray; isotype control). (I) DHR assay of reactive oxygen species production by Mart1-iPSC-derived macrophages in response to stimulation with PMA (bottom panel); unstimulated macrophage control is also shown (top panel). (J–L) T lymphocytes generated from Mart1-iPSC-derived HPCs. (J) After 22 days of T cell differentiation, CD4 + CD8 + double-positive T cells could be detected by flow cytometry analysis, most of which were CD3 + Mart1-tetramer + cells. (K) Mart1-iPSC-derived CD4 + CD8 + double-positive T cells could be induced to CD8αβ single-positive T cells by stimulation with Mart1 peptide-primed T2 cells. (L) Mart1-iPSC-derived CD8 + single-positive T cells released interferon γ (INFγ) and tumor necrosis factor α (TNF-α) in response to Mart1 peptide-pulsed T2 cells. T cell responses without T2+Mart1 peptide (no target) or without Mart1 peptide (T2+DMSO) are also shown. Values represent mean ± SD from 3 independent experiments, ∗∗∗∗p < 0.0001.

Article Snippet: APC Mouse anti-Human CD43 (Clone 1G10) , BD Biosciences , Catalog No: 560198, RRID AB_1645460.

Techniques: Generated, Derivative Assay, Cell Culture, Flow Cytometry, Expressing, RNA Expression, Giemsa Stain, Phagocytosis Assay, Labeling, Cell Differentiation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet: The iSTC-Hp-spheroid system can be performed in a xeno-free condition (A) Schematic outline of screening a xeno-free condition for the iSTC-Hp-spheroid system. (B) Representative flow cytometry analysis of CD34, CD43, and CD45 on GFP + cells in iSTC-Hp-spheroids cultured in the xeno-free medium for 13 days. GFP + cells are gated for analysis. (C) Comparison of the frequency of CD34 + CD43 + cells and CD34 + CD45 + cells in iSTC-Hp-spheroids between culturing in FBS-based medium vs. xeno-free medium. GFP + cells are gated for analysis. Values represent mean ± SD from 3 independent experiments; ns, not significant. (D) Representative flow cytometry analysis of T lineage cell markers, including CD5, CD7, CD4, and CD8, on cells differentiated from HPCs generated in xeno-free condition. Cells were harvested on day 22 of T cell differentiation. GFP + cells are gated for analysis. (E) Quantifications of flow cytometry analysis in (D). Values represent mean ± SD from 3 independent experiments. (F) Representative flow cytometry analysis of CD3 + cells in CD4 + CD8 + cells during the T cell differentiation. GFP + cells are gated for analysis. (G) Quantifications of flow cytometry analysis in (F). Values represent mean ± SD from 3 independent experiments. (H) Frequencies of CD3 + cells in CD4 + CD8 + cells on day 32 of T cell differentiation. GFP + cells are gated for analysis. Horizontal bars represent mean value from 3 independent experiments; ns, not significant. (I) TCR sequence analysis showing the diversity of rearranged TCR genes in NCRM5-EGFP-derived CD4 + CD8 + CD3 + T cells isolated from day 32 of the T cell differentiation.

Article Snippet: APC Mouse anti-Human CD43 (Clone 1G10) , BD Biosciences , Catalog No: 560198, RRID AB_1645460.

Techniques: Flow Cytometry, Cell Culture, Generated, Cell Differentiation, Sequencing, Derivative Assay, Isolation

Journal: Cell Reports Methods

Article Title: Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells

doi: 10.1016/j.crmeth.2023.100460

Figure Lengend Snippet:

Article Snippet: APC Mouse anti-Human CD43 (Clone 1G10) , BD Biosciences , Catalog No: 560198, RRID AB_1645460.

Techniques: Recombinant, Giemsa Stain, Modification, Cell Differentiation, Software