Fig. 1: Differentiation of PSCs to DLL4+  AECs and lymphoid haematopoietic cells. | Nature Communications

Fig. 1: Differentiation of PSCs to DLL4+  AECs and lymphoid haematopoietic cells.

From: Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis

Fig. 1

a Sequential stages of PSC differentiation towards DLL4+ AECs, haemogenic endothelium, haematopoietic progenitors, lymphoid commitment, and ILCs at the approximate corresponding days. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licences/by-nc-nd/4.0/deed.en). b Bright-field (BF) image of day 6 monolayer cell culture. Scale bar, 100 μm. c Flow cytometry analysis of day 6 cultures showing co-expression of the AEC markers DLL4, CXCR4, and CDH5 on CD34+ cells. Red dots indicate CD34+ DLL4+ cells. d Bar graph summarizing flow cytometry analysis of CD34 and DLL4 expression in day 6 samples representing independent experiments using nine distinct parental PSC lines (and two subclones of RM3.5 iPSCs and H9 ESCs). Data is shown as the mean ± SD for ≥3 independent experiments, otherwise, only the mean is shown. e Flow cytometry analysis tracing progressive differentiation from CD43+ CD34+ HSPCs (day 12), to CD34+ CD7+ lymphoid progenitors (day 15), to CD34− CD7+ and CD7+ RAG1+ lymphoid cells (day 19). Green dots indicate RAG1:GFP+ cells. f Quantification of RAG1:GFP+ cells showing the frequency of RAG1+ cells increases from day 15 to day 19. Statistics were calculated by a two-sided t-test; data shown as mean ± SEM; n = 5 for RM3.5 day 15, n = 5 for RM3.5 day 19, n = 4 for H9 day 15, n = 6 for H9 day 19. P (H9) = 0.0105, p (RM3.5) = 0.0152. The n values designate independent experiments. g Bright field and fluorescence images showing RAG1:GFP cells at day 15 and day 19. Scale bar, 100 μm. h Flow cytometry of CD45+ cells at differentiation day 23 showing expression of CD7, CD161 and RAG1, and the NK cell marker CD56. i Frequency of the CD7+ CD161+ innate lymphoid cells and CD161+ CD56+ NK-like cells in independent experiments using three distinct PSC lines (mean ± S.E.M). For the values in the left (L) and right (R) panels, these are n = 5 (L) and 6 (R) for H9, n = 7 (L) and 7 (R) for RM3.5, n = 5 (L) and 5 (R) for PB005, (for Fig. 1d, f, and i, n = number of independent experiments). Source data are provided as a Source Data file. Frequencies of each cell fraction is indicated.

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