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. 2021 Dec 20;219(3):e20211924. doi: 10.1084/jem.20211924

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© 2021 Atkins et al.

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Figure 4. — The day 6 CD34⁺CD43⁻ population give rise a unique T lymphoid lineage. (A) Representative flow cytometric analysis of CD45, CD56, CD5, CD7, CD4, and CD8 expression on cells derived from the day 6 CD43⁺ or CD34⁺CD43⁻ populations following 30 d of culture with OP9-DL4 cells. FSC-A, forward scatter area; SSC-A, side scatter area. (B) Gating strategy used for the FACS-based isolation of day 4 KDR⁺CD235a/b⁺ cells, which give rise to CD43⁺ and CD34⁺CD43⁻ populations when cultured as aggregates for 3 d. Transfer of these cultures to OP9-DL4 cells generated T lymphoid progenitors marked by the presence of CD45⁺CD56⁻CD4⁺CD8⁺ cells after 30 d. (C) Colony-forming progenitor number after 5 d of culture of 62,500 day 4 KDR⁺CD235a/b⁺ cells (n = 3). Colonies: E, erythroid (red); M, myeloid (blue); E/M, mixed erythro-myeloid (gray). (D) Quantification of the frequency of γδ (gray) and αβ (green) CD3⁺ T cells between days 25 and 42 of T lymphoid differentiation in cultures derived from day 6 CD34⁺CD43⁻ or cord blood CD34⁺ cells (n = 3). ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 versus the stage and population-matched sample or versus day 25 of T lymphoid differentiation (arrow), as indicated. #, P < 0.05; ##, P < 0.01; and ####, P < 0.0001 between stage-matched γδ and αβ T cells for the indicated sample. (E) Quantification of the frequency of Vδ1⁺ (blue) and Vδ2⁺ (purple) CD3⁺ T cells between days 25 and 42 of T lymphoid differentiation in cultures derived from day 6 CD34⁺CD43⁻ or cord blood CD34⁺ cells (n = 3). ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 versus the stage- and population-matched sample or versus day 25 of T lymphoid differentiation (arrow), as indicated. #, P < 0.05; ##, P < 0.01; and ####, P < 0.0001 between stage-matched Vδ1⁺ and Vδ2⁺ T cells for the indicated sample. (F) Representative flow cytometric analysis of TCRγδ⁺, TCRαβ, Vδ1, Vδ2, and Vγ9 expression on day 35 of T lymphoid differentiation in cultures derived from day 6 CD34⁺CD43⁻ or cord blood CD34⁺ cells. (G) Representative flow cytometric analysis of CD34, CD7, and CD5 expression on cells derived from day 6 CD34⁺CD43⁻ or cord blood CD34⁺ cells following 12 d of culture with OP9-DL4 cells.