Fig. 3: MS5_FS12 stromal cells support pDC and pre/AS-DC development in vitro.

a Representative FACS plots and absolute number of CD123⁺CD303/4⁺ cells generated in vitro from CD34⁺ HSPCs co-cultured with MS5 expressing human FLT3L (MS5_F) in combination with human SCF (S), TPO (T), and CXCL12 (12). Day 15 flow cytometry analysis of n = 3 cord blood donors in three independent experiments. *p < 0.05, one-way ANOVA test with Tukey’s multiple comparisons. b Gating strategy used to identify AXL⁻CD327^lo/− pDC and AXL⁺CD327⁺ pre^/AS-DC within CD123⁺CD45RA⁺ cells generated in vitro using MS5_FS12. Graph illustrates the frequency of each subset in CD45⁺ cells (n = 4 cord blood donors). c GSEA comparing in vitro-differentiated pDC and pre/AS-DC using published human pDC and AS-DC gene signatures²⁰ (FDR false detection rate, NES normalized enrichment score). Statistical significance is defined by the FDR q-value calculated by the GSEA software (www.broad.mit.edu/gsea) using default parameters. d Intracellular flow cytometry analysis of IFNα production in pDC and pre/AS-DC in response to 16 h of TLR stimulation (lipopolysaccharide (LPS) 10 ng/ml, R848 1 μg/ml, Poly(I:C) 25 μg/ml). Bar graph shows the frequency of IFNα⁺ cells in each subset with (+TLR) or without (NT) stimulation (n = 4 cord blood donors). *p < 0.05, one-way ANOVA test with Holm–Sidak’s multiple comparisons. Data are presented as floating bars ranging from min to max and line represents median (a, b, d).