Fig 1: CD34+ progenitor counting in mobilized peripheral blood involving the use of DNA fluorescent stainsBoolean gating strategy was performed following the ISHAGE guidelines for CD34 and CD45 staining. Region (R) 1 was set in a BSSC vs. DCV-H dot-plot (A) to include nucleated cell events and applied in a DCV-H vs. DCV-A dot-plot (B) for doublet discrimination. R2 including single cells was applied in a FSC-H vs. BSSC-H dot-plot (C) to gate leukocytes (R3). R3 was gated in a BSSC vs. FITC-CD45 dot-plot (D) to select CD45+ events in R4, which was applied to display CD34+ in a PE-CD34-H vs. BSSC-H dot plot (E) and in a PE-CD34-H vs. FITC-CD45-H dot plot (F).
Fig 2: Retinoic Acid Signaling Inhibition Increases Colony-Forming Hematopoietic Progenitor Numbers(A) Micrographs of CFU colony types from iPSC-derived cultures (20,000 cells per sample).(B) Cytospin pictures of various hematopoietic cell types collected from colonies and stained with May Grünwald/Giemsa staining, alternatively benzidine.(C) Fold change in CFUs from cells treated with DEAB (green) normalized to DMSO control (gray) indicated by the dotted line (100%) from seven independent experiments (n = 7), with pie charts showing the distribution of different colony types.(D) Fold change in CFUs after RA treatment, with pie charts showing colony distribution (n = 3).(E) Change in maturation markers CD34 and CD38 of the hematopoietic compartment (CD45/43+) of cells treated with or without RA. Arrow in FACS plot indicates progression of maturing phenotype.Data represent mean ± SEM. Asterisks indicate significant differences (∗∗p < 0.01, ∗∗∗p < 0.001).
Fig 3: Decreased Retinoic Acid Signaling Maintains the Primitiveness of Generated Hematopoietic Progenitors(A) Representative FACS profiles of cells harvested at day 16 and after extended culture period at day 21. Cells were treated with or without DEAB at 10 μM during the whole culture period.(B) Graphs showing fold changes in maturation of the hematopoietic population (CD45/43+) observed between day 16 and day 21 for cells treated with DEAB or DMSO from three independent experiments (n = 3).Data represent mean ± SEM. Asterisks indicate significant differences (∗p < 0.05).
Fig 4: Gating strategy and quality control for droplet scRNA-seq A. Flow sorting of living leukocytes from peripheral blood and decidual tissue using CD45 antibody and 7-AAD. B. Total number of cells that met the requirement of quality control and subjected to droplet scRNA-seq. C. Workflow depicting the dissociation and sorting of CD45+ leukocytes from peripheral blood and decidual to generate scRNA transcriptome profile. D. Quality evaluation on sequencing data including the total number of reads per cell (left panel) and total number of genes per cell (right panel) and in peripheral blood and decidua. 7-AAD, 7-aminoactinomycin D.
Fig 5: CD34+ progenitor counting in mobilized peripheral bloodThreshold was set on FITC-CD45 fluorescence (A) to discriminate erythrocytes, platelets and debris. CD45+ events were subsequently gated on BSSC vs. VSSC (B), FSC vs. SSC (C), FITC-CD45 vs. PE-CD34 (D) and VSSC vs. PE-CD34 (E). Region (R) 2 comprehends CD34+ progenitor cells, which are further gated in VSCC vs. FSC (F) and selected in R3.
Supplier Page from Thermo Fisher Scientific for CD45 Antibody FITC