Fig 2: pDCs and cDC1s Can Be Efficiently Generated from Human CD34+ Cord Blood Cells(A) CD34+ cord blood (CB) cells were expanded with FLT3L, SCF, TPO, and IL-7 (FST7) for 7 days. Subsequently, they were differentiated on OP9, OP9_DLL1, or OP9+OP9_DLL1 feeder layer cells, with FLT3L, TPO, and IL-7 (FT7), for 14–21 days.(B) On day 18 of differentiation, within the live-cell gate, pDCs were identified as CD206-CD14-CD123+BDCA2+ cells and cDC1s as CD206-CD14-CLEC9A+ cells positive for BDCA3 or CADM1. Plots show one experiment using CB204 donor CD34+ cells for parallel differentiation on the 3 feeder layers. Data are representative of 5 experiments each performed with cells from one or two different donors. Pie charts show mean percentage of pDCs or cDC1s within the live-cell gate from the 5 experiments.(C and D) Frequencies (C) and numbers per well (i.e., per 104 expanded CD34+ cells) (D) of cDC1s (top) and pDCs (bottom) among total live cells on day 18 after differentiation on the 3 different feeder layers. Graphs show individual results for each of the 6 donors.See also Figures S1 and S2 and Table S1.

Fig 3: Flow Cytometric and scRNAseq Analyses Confirm the Identity of the pDCs and cDC1s Generated from Human CD34+ CB Cells and Identify Heterogeneity within cDC1s(A) viSNE plots from live Lin-HLA-DR+ cells harvested from 18 day OP9+OP9_DLL1 FT7 cultures. Color codes indicate relative levels of marker cell-surface expression.(B) Overlay of the viSNE plot shown in (A) with the manually gated populations indicated in the legend. pDCs were gated as BDCA2+CD123+ and cDC1s as BDCA2-CD123neg-to-lowCADM1+CD1c+. In the remaining CADM1- cells, CD1c+ cells were subdivided into BTLA-CX3CR1+ and BTLA+ cells. Plots are representative of 4 donors from 2 experiments.(C) Lineage-HLA-DR+ single cells were index sorted for phenotypes of cDC1s (CD141+CADM1+), pDCs (CADM1-CD123+BDCA2+), putative pre-cDC2s (CADM1-BDCA2-CD1c+BTLA+), or putative cDC2s (CADM1-BDCA2-CD1c+BTLA-), as indicated by the symbols in the graphical legend. scRNAseq was performed using the SmartSeq2 protocol, followed by unsupervised dimensional reduction of the data using t-distributed stochastic neighbor embedding (t-SNE) with graph-based clustering. 7 cell clusters were identified as indicated by the color code in the graphical legend.(D) Violin plots showing mRNA expression profiles of previously known MNP-type-specific genes across all individual cells and in comparison between clusters identified in (A).(E) Enrichment analyses performed on each cell using connectivity map (cMAP), with independently generated and previously published human MNP-type-specific transcriptomic fingerprints. Results are presented as violin plots showing cell densities for the different cMAP scores (y axis) across the cell clusters (x axis) identified in (A). cMAP scores close to 1 versus -1 indicate strong enrichment of the signature of the cell type labeled on top versus bottom of the y axis.(F) Violin plots showing expression profiles of differentially expressed genes between clusters 6 and 7 (cDC1s).Individual dots represent single cells (C, D, and F). Data in (C)–(F) represent one experiment using one donor.(G) Protein expression of CLEC9A and CXCR4 on Lin-HLA-DR+CADM1+BDCA2-XCR1- and XCR1+ cDC1s from FT7 cultures analyzed by flow cytometry. Plots are representative of 3 experiments and 5 donors.(H) Protein expression of CLEC9A and XCR1 on Lin-HLA-DR+CD11c+ CD1c-CADM1+ cDC1s from adult peripheral blood analyzed by flow cytometry. Plots are representative of 3 experiments encompassing 6 different donors.(I) XCR1 protein expression on adult peripheral blood cDC1s sorted as Lin-HLA-DR+CD11c+CD1c-CD141+CADM1+CLEC9A+ and either XCR1+ or XCR1- 14 days after in vitro culture on OP9-DLL1 cells with the FT7+G cytokine cocktail. One experiment representative of three is shown. Dotted line, fluorescent minus one control; plain line, XCR1 staining.(J) Expansion of the sorted XCR1- subset of adult peripheral blood cDC1s after 14 days of in vitro culture on OP9-DLL1 cells with the FT7 cytokine cocktail supplemented with GM-CSF (1 ng/mL). Data shown are from 3 experiments, each with a different donor (symbols), with mean ± Sd shown across the three donors for each experimental condition.See also Figures S3–S6 and Data S1.

Fig 4: GM-CSF Promotes cDC1 Differentiation from CB and Non-mobilized Adult Blood CD34+ Cells(A–C) Expanded CD34+ CB cells were differentiated with FT7 on OP9_DLL1 feeder layer cells in the absence or presence of GM-CSF added at different time points.(A) Top: frequencies of live CD123+ and CD141+ cells in the CD14-CD206- gate. Bottom: frequencies of CD141+CLEC9A- and CD141+CLEC9A+ cells within the CD141+ gate shown on the top. Plots are from one donor (representative of 4). FT7+G, 1 ng/mL GM-CSF added to FT7 on days 0, 7, and 14 of differentiation.(B and C) Frequencies (top) and absolute numbers (bottom) of cDC1s (B) and pDCs (C) generated in the absence or presence of GM-CSF added at the indicated concentrations on days 0, 7, and 14 (w1–3), 7 and 14 (w2–3), or 14 (w3) of differentiation. Absolute numbers of cells obtained were calculated for an initial input of 104 CD34+ CB cells, taking into consideration both the expansion and differentiation phases. Data shown are from 4 experiments, each with a different donor (symbols), with mean ± SD shown across donors for each experimental condition. Statistics were performed comparing each condition of GM-CSF supplementation with the FT7 control culture.(D) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained cells were differentiated with FT7 or FT7+G (0.25 ng/mL) and harvested over time to measure cell expansion. Each symbol represents an individual donor (D1, D2, and D3). The open versus closed formats represent different days of harvest (day [d]6, d12, and d18).(E) Mean CFSE fluorescent intensity at days 6 and 12 on total cells in the same differentiation cultures as shown in (D).(F) Fraction of live (PI-Annexin-V-) versus early apoptotic (PI-Annexin-V+), late apoptotic (PI+Annexin-V+), or necrotic (PI+Annexin-V-/low) cells in cultures over time, for the individual donors (symbols), which mean ±SD shown across donors for each day.(G) Frequency of XCR1- versus XCR1+ cells in the cDC1s generated in vitro from FST7-expanded CB CD34+ cells differentiated for 18 days on OP9_DLL1 feeders with FT7 or FT7+G. Data shown are from 2 experiments, each with 1–3 different donors (symbols), with mean ± SD shown across donors for each experimental condition.(H) Frequency (top) and total numbers (bottom) of CD123+CD45RA+ pDCs and CD141+CLEC9A+ cDC1s generated from 104 non-mobilized FST7-expanded CD34+ cell from adult peripheral blood differentiated on OP9+OP9_DLL1 feeder with FT7 or FT7+G. Data shown are from 3 experiments, each with 2 different donors (6 data points with their mena ± SD shown for each condition).