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. 2018 Feb 15;10(3):875–889. doi: 10.1016/j.stemcr.2018.01.009

Figure 2.

Figure 2

Formation of HE Colonies Is Promoted by HOXB4

(A) During co-culture on OP9 cells, circular sheet colonies were formed by the dissociated CCE-ESC-derived EBs (eGFP-HOXB4 transduced), which were commonly associated with hematopoietic suspension cell clusters. Left panel: phase contrast; right panel: eGFP-fluorescence. Scale bars, 100 μm.

(B) The observed endothelial colonies expressed VE-cadherin, CD31, and were capable of acetylated low-density lipoprotein (LDL) (DilAcLDL) uptake. Scale bars, 100 μm.

(C) The number of endothelial CD31+ and DilAcLDL+ colonies strongly increased when HOXB4 was ectopically expressed. Average colony numbers per 105 seeded cells are represented as columns, error bars represent SD of n = 3 independent experiments.

(D) iRunx-ESCs with and without a 4-hydroxytamoxifen (Tam) inducible form of HOXB4 (vector FMEV-tdTomato-2A-HOXB4ERT) were differentiated as EBs for 6 days, dissociated, and co-cultured on OP9 stroma cells for further 4 days without Runx1 induction (no addition of doxycycline); n = 9 and 4 independent experiments for controls, n = 7 for HOXB4. Without HOXB4 induction, the number of HE colonies per 105 seeded EB cells was comparable with unmanipulated controls. When HOXB4 was induced throughout differentiation, the number of HE colonies increased approximately 30-fold (p < 10−4). The p values were calculated using the two-sided, unpaired Student's t test with a significance level defined as 0.05.

(E) Flow cytometric analysis showing the proportion of CD41+ and CD45+ cells in OP9 co-cultures after 5 and 12 days. Dissociated iRunx EBd6 were co-cultured on OP9 cells with or without addition of doxycycline (0.1 μg/mL) to induce Runx1 expression and with or without addition of 100 nM Tam for induction of HOXB4ERT (FMEV-tdTomato-2A-HOXB4ERT). Induction of Runx1 and HOXB4ERT started from day 3 of EB development on until day 5 of OP9 co-culture. Cells were harvested after 5 or 12 days of OP9 co-culture, and the proportion of CD41- and CD45-expressing cells determined by flow cytometry. OP9 cells were removed with an anti-CD140b antibody.