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. 2016 Dec 12;213(13):2861–2870. doi: 10.1084/jem.20161135

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© 2016 Breton et al.

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Figure 2. — Heterogeneous expression of CD172a in human cord blood pre-cDCs identifies populations with CD1c⁺ or CD141⁺ lineage potential. (a) Expression of CD172a receptor on cDCs and monocytes. The flow cytometry histogram shows expression of CD172a on live CD3⁻ CD20⁻ CD335⁻ CD66b⁻–gated monocytes (CD14⁺; orange), pDCs (CD303⁺; green), CD1c⁺ cDCs (blue), and CD141⁺ cDCs (red) from peripheral blood (n = 3). (b) Expression of CD172a receptor on pre-cDCs in human cord blood (CB), bone marrow, and peripheral blood (PB; cord blood, n = 6; bone marrow, n = 2; peripheral blood, n = 3). Flow cytometry plots show that Lin⁻ CD34⁻ CD117⁺ CD123^−/+ CD135⁺ CD115⁻ CD116⁺ pre-cDCs in human cord blood can be divided by CD172a into three populations: CD172a⁻ (red), CD172a^int (green), and CD172a⁺ (blue) pre-cDCs. Gate frequencies from parent population are shown. SSC, side scatter. (c) Heat map showing the top 41 differentially expressed genes selected by unsupervised hierarchical clustering (clustering analysis on all differentially expressed genes with p-value <0.05) between cord blood CD172a⁺ (n = 4), cord blood CD172a⁻ pre-cDCs (n = 4), peripheral blood CD141⁺ cDCs (n = 4), and peripheral blood CD1c⁺ cDCs (n = 4; Table S3). (d) Expression of specific surface markers and lineage-restricted transcription factors involved in DC development in CD1c⁺ cDCs, CD141⁺ cDCs, CD172a⁺ pre-cDCs, and CD172a⁻ pre-cDCs. (e) The graph shows mean output of CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue) from the culture of CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs from five independent experiments. Error bars represent the standard deviation. (f) Gating strategy for culture output after hematopoietic differentiation on MS-5 stromal cells of human cord blood pre-cDCs. Total cord blood pre-cDCs were isolated and then cultured in MS5 + FSG for 7 d. Culture output was assessed by flow cytometry. Cells developing into culture are identified within the live Lin⁻ CD45⁺ cells. Output cells include CD66b⁺ granulocytes (Gran; brown), CD141⁺ cDCs (red), CD1c⁺ cDCs (blue), monocytes (Mono; orange), and CD303⁺ pDCs (green). Gate frequencies from the parent population are shown (n = 5). (g) Differentiation potential of 50–200 purified cells from cord blood CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs in MS5 + FSG cultures for 7 d. Flow cytometry plots show gated Lin⁻ CD45⁺ cells. CD141⁺ cDCs were identified as CD141⁺ CLEC9A⁺ and CD1c⁺ cDCs as CLEC9A⁻ CD14⁻ CD1c⁺ (n = 5). Gate frequencies from the parent population are shown. (h) Single-cell clonal assay of CD172a^int pre-cDCs. Representative flow cytometry of gated Lin⁻ CD45⁺ cells derived from single CD172a^int pre-cDC culture show clonal output of CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue). For this representative experiment, 11 wells gave rise to CD141⁺ cDCs and 33 wells to CD1c⁺ cDCs (120 wells plated; 44 positive wells; n = 3). (i) Proliferative capacity of CD172a⁻, CD172a^int, and CD172a⁺ pre-cDCs. pre-cDC subsets were purified from cord blood, labeled with CFSE, and cultured in MS5 + FSG for 7 d. Proliferation was assessed by flow cytometry. Representative FACS plots show CD141⁺ cDCs (red) and CD1c⁺ cDCs (blue) CFSE dilution (n = 2).