Fig 2: 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 3: Retinoic Acid Inhibition Increases the Differentiation Capacity of Hematopoietic Progenitors toward Myeloid and Lymphoid Lineage(A) CFU potential of 500 sorted progenitors (CD45/CD43+CD34+) from DEAB-treated cultures (green) and DMSO control (gray) from four independent experiments (n = 4). CFU potential of progenitors similarly sorted from cord blood (CD45/CD43+CD34+) is shown in red.(B) Lymphoid/myeloid differentiation assay performed on iPSC-derived hematopoietic cells. FACS plots and bar graphs showing non-myeloid (CD33/CD14-), CD19-, CD56+, and CD56+CD16+ NK fraction from wells seeded with 50 HSC-like cells (CD45/43+CD34hiCD38loCD90+CD45RA-) (17 individual wells) cultured on OP9 stroma for 4 weeks. Left bar graph shows the cluster size of the NK fraction. Right bar graph shows the frequency of this population in the viable non-stroma fraction. See also Figure S2.(C) FACS plots showing non-myeloid, CD7+, CD45RA+ multipotent early lymphoid progenitors from 3,500 sorted CD34+ cells cultured on OP9DL1 stroma for 3 weeks from one out of two independent experiments (n = 2). FACS plots are gated on the viable non-stroma fraction. Bar graph show the total output of differentiated cells from progenitors generated with DMSO, DEAB, or RA. FACS gates are based on FMO controls.Data represent mean ± SEM. Asterisks indicate significant differences (*p < 0.05).

Fig 4: Retinoic Acid Signaling Inhibition Increases the Yield of Hematopoietic Progenitors with an HSC-like Phenotype from Human Pluripotent Stem Cells(A) Schematic of pluripotent stem cell differentiation system used to model human hematopoietic development through mesoderm specification and blood commitment. RA inhibitors or RA was present continuously during the 16-day differentiation, except where otherwise stated.(B) Representative FACS plots showing the hematopoietic population derived from pluripotent stem cells at day 16 of differentiation. FACS gates show blood (CD45/43+), hematopoietic progenitors (CD45/43+CD34+), and HSC-like immature progenitors (CD45/43+CD34hiCD38loCD90+CD45RA-). Gates are based on FMO controls with more stringent CD34hi and CD38lo gating based on cord blood hematopoietic stem and progenitor cell standards. Doublet exclusion and dead cell exclusion were done before applying the gates.(C) Frequency (%) of blood subfractions derived from cells treated with DEAB (green) at 10 µM from seven independent experiments (n = 7) (see also Figures S1A and S1B) or RA (orange) (0.01, 0.1, 1.0 µM; n = 3). All values are normalized to the DMSO control (gray) as indicated by the dotted line (100%). X = no events. Bar graphs show change in cell viability as measured by 7AAD for tested conditions.(D) mRNA expression level of RA target gene RARß at day 8 of differentiation (n = 3).(E) Frequency of the HSC-like fraction in cells treated with DEAB (n = 7) or an alternative RA signaling inhibitor, LG1506 (1 µM) (blue, n = 3).Data represent mean ± SEM. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 5: Mass Cytometry Reveals Immune Cell Heterogeneity between Fetal and Adult SkinTwenty-three weeks gestational age (g.a.) fetal torso skin along with healthy adult skin torso samples were analyzed in parallel for 22 markers using mass cytometry.(A) Uniform manifold approximation and projection (UMAP) plots of viable CD45+ cells in fetal versus adult skin, cells colored by cluster identity, and plots annotated with cluster numbers and assigned identities.(B) Heatmap demonstrating relative expression by cluster of 22 markers in panel, inclusive of both fetal and adult cells.