Fig. 1. The presence of DLL4 and VCAM1 during the endothelial to hematopoietic transition supports development of HSPC with robust T cell potential.

(A) Schematic overview of chemically defined platform for producing multipotent hematopoietic progenitors and T cell progenitors from pluripotent stem cells. (B) Flow cytometry analysis of progenitor T cell output after transitioning cells from 7 days in each EHT coating condition into a common defined thymic niche for an additional 7 days. (C) Quantification of the frequency and yield of CD7⁺ lymphoid progenitors and CD7⁺, CD5⁺ T cell progenitors after 7 days in the thymic niche (mean ± SD, n = 6). Indicated P values reflect result of Mann-Whitney test. (D) Experimental design to assess the effect of adding or omitting the EHT culture phase before transferring cells into the thymic niche. (E) Immunophenotype of cells generated with or without the EHT culture stage. Numerals in (E) correspond to the schematic in (D). (F) Results of limiting dilution analysis to assess the frequency of cells with CD7⁺ NK/T lymphoid potential within the CD34⁺ population with or without an EHT step. Indicated cell numbers were seeded in 96-well plates and cultured for a total of 14 days (either 7 days EHT + 7 days pro-T differentiation medium or 14 days pro-T differentiation medium). Wells were scored as positive if they contained >25 viable CD7⁺ cells. Frequencies were modeled as the number of cells required to achieve a failure rate of 0.37. (G) Quantification of the yield and frequency of T cell progenitors generated with or without an EHT culture phase. Indicated P value is for the effect of EHT on CD5⁺, CD7⁺ yield, two-way ANOVA.