Figure 2.

Generation of in vitro differentiated CB-pDCs from CD34⁺ HSCs. (A) Schematic overview depicting the generation of CB-pDCs from CB-derived CD34⁺ HSCs. 5,000 HSCs were expanded for 7 days in expansion media and cryopreserved. Expanded, thawed HSCs were differentiated for 12 days on a monolayer of Mitomycin C-treated MS-5 stromal cells before being harvested on day 19 or used for subsequent experiments. (B) Graph displaying the frequencies of CB-pDCs as percentage of living cells. (C) Graph displaying the total cell proliferation in the mixed culture during differentiation by fold increase and (D) absolute CB-pDC numbers corrected on the frequency of differentiated CB-pDCs on day 19 comparing supplementation of SR-1 and GM-CSF separately or in combination. (E, F) Quantification of IFN-ɑ release from (E) TLR9-activated and (F) TLR7-activated cells in the mixed culture comparing unprimed and primed cells of the different conditions with LLOD at 1.95 pg/mL. (G) Pie chart showing the mean frequencies of CB-cDC1s, CB-cDC2s, CB-pDCs, and other not further characterized cells from 6 independent CB donors as defined by flow cytometry phenotyping. (H) Quantification of CB-pDCs and CB-pre/AS-DC frequencies (from CD45⁺Lin^-CD123⁺CD45RA⁺) from 5 independent CB donors and dot plot displaying CB-pDC and CB-pre/AS-DC gates from one representative donor. (I) Overlaid histograms comparing expression of CD4, CD45RA, ILT-7, and CD304 of CB-pDCs and primary pDCs of one representative donor. Graph depicts GMFI of CD4, CD45RA, ILT-7, and CD304 as determined by flow cytometry shown as mean ± SEM of 2 independent experiments, 8 independent CB donors, and 3 independent pDC donors. (B–F) Results are shown as mean ± SEM of 4 independent experiments and 12 independent CB donors. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant (B–D) one-way ANOVA with Tukey’s post-hoc test or (E, F) Wilcoxon test. See also Supplementary Figure S1 .