Fig. 1: Generation of hiPSC-derived primitive macrophages. | Nature Communications

Fig. 1: Generation of hiPSC-derived primitive macrophages.

From: Medium from human iPSC-derived primitive macrophages promotes adult cardiomyocyte proliferation and cardiac regeneration

Fig. 1

a Schematic diagram of the protocols to generate hiPMs from human iPSCs. Relevant growth factors and duration of differentiation steps are as indicated. Cartoon elements used in the schematic diagram were created using BioRender. b Flow cytometry analysis of cells double positive for KDR and CD235 during hemogenic epithelium (HE) specification at day 5 after the start of hiPM differentiation. c Flow cytometry analysis of cells positive for CD45 during myeloid progenitor generation at day 11 after the start of hiPM differentiation. d Flow cytometry analysis of hiPMs positive for CD11b and CD14 at day 32 after the start of hiPM differentiation. e Immunofluorescence images for CD11b and CD14 expression in hiPMs. iPSCs were used as a negative control. Cells were stained with CD11b (red) and CD14 (green); nuclei were counterstained with Hoechst (blue). Scale bar, 10 µm. The experiment was repeated three times independently with similar results. f Phagocytosis assessment of hiPMs with FITC-conjugated Dextran (green). Human iPSC-derived cardiac fibroblasts (hiCFs) were used as a negative control. Scale bar, 50 μm. g Flow cytometry analysis of CCR2 expression in hiPMs. THP-1Ms were used as a positive control. The experiment was repeated three times independently with similar results. h Principal component analysis of RNA sequencing data revealing in-group clusters with minimal overlap between hiPMs and THP-1Ms. n = 4 biological repeats per group. i Barplot of differentially expressed genes (hiPMs vs THP-1Ms) specific to primitive macrophages. Differentially expressed genes were identified with a cutoff of |Fold change | > 2 and adjusted p-value < 0.05. j Measurement of LYVE1, FOLR2, CD163, and MAF mRNA levels in hiPMs and THP-1Ms via qPCR analysis. β-actin was used as a control. n = 3 biological repeats per group. k Representative immunofluorescence images for LYVE1 expression in hiPMs. THP-1Ms were used as a negative control. Cells were stained with LYVE1 (green); nuclei were counterstained with Hoechst (blue). Scale bar, 20 μm. Quantitative data are presented as the mean ± SEM. Groups were compared using a two-tailed unpaired Student’s t test (j) or ANOVA (i). Source data are provided as a Source Data file.

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