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. 2020 May 12;31(6):107627. doi: 10.1016/j.celrep.2020.107627

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© 2020 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

All Functional AGM HSCs Are within the HC1 CD27^med Fraction

(A) Violin plots of Tnfrsf7 (CD27) expression in the 5 major transcriptomic clusters. EC, endothelial-like cluster; HC1, hematopoietic cluster 1; HC2, hematopoietic cluster 2; UC1, unknown cluster 1; UC2, unknown cluster 2.

(B) Enrichment sorting parameters for CD27⁺ HC1 cell isolation. Side scatter (SSC-A), CD31, cKit, Gata2 (G2), and CD27 sorting gates and percentage (±SEM) of cells. CD27⁺ cells (%) are indicated within a wide (+W) and a narrow (+N) mean fluorescent intensity (MFI) gate.

(C) Analysis of HSC function within CD27 fractions (CD27^+W, CD27^+N, and CD27^−/lo) of HC1 cells by in vivo transplantation. Open squares denote CD27^+W HC1 cell recipients (n = 2) of 442 cells (2.5ee) and 479 cells (3.3 ee) from 15 and 9 (42–50 somite pairs, sp) embryos, respectively. Closed squares (n = 4; 42–49 sp; embryos/experiment: 25, 11, 22, 28) denote recipients injected with CD27^+N HC1 cells (238–957; 3.7–9.8 ee), EC cells (899–2,333; 3.9–8.6 ee), CD27^-/lo HC1 cells (1,042–4,325; 4.1-9.8 ee), and UC1+2+HC2 cells (1,361–3,741; 4.2–9.9 ee). Percentage of donor cell chimerism in peripheral blood of recipients at 4 months post-injection. Black line indicates mean.

(D) Single-cell RNA-seq of 119 CD31^hiSSC^locKit^hiGata2^medCD27^+N sorted cells (all passed quality control) from 6 embryos (41–47 sp). These data combined with the previous dataset (gray) resulted in an extended dataset SPRING plot of 1,087 cells in total. Overlay of CD27^+N cells (red) showing localization to one HC1 subcluster (circled).

(E) Single CD27^+N HC1 cells from E11 G2V IAHCs (50 embryos; 42–49 sp) deposited into methylcellulose and colony-forming unit-culture (CFU-C) counts at 8–10 days (138 total CFU-Cs). Colony type (individual dot) plotted against log₁₀-normalized mean fluorescence intensities (MFIs) for (Ei) CD27, (Eii) G2, (Eiii) cKit, and (Eiv) CD31 expression. GEMM, granulocyte-erythroid-macrophage-megakaryocyte; GM, granulocyte-macrophage. Red lines indicate mean ± SEM.

(F) Representative FACS plot showing newly defined CD27^med MFI range (red dotted line) within the CD27^+N fraction. Percentage ±SEM.

(G) Summary of enrichment strategy. The HC1 fraction containing all HSCs is 10.5-fold enriched compared to previous sorting strategies (Eich et al., 2018). Further enrichments are based on gating of specific CD27^+N and CD27^med fractions to give an additional 6.5-fold enrichment. After iterative analyses and refinement of sorting gates, a total combined HSC enrichment of 68.3-fold is achieved.