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. 2008 Sep 5;112(9):3601–3614. doi: 10.1182/blood-2008-03-144766

Figure 6.

Figure 6

Blast colonies express RAS components that regulate their expansion and differentiation. (A) RNA was harvested from sorted hESC Line H1 day 9 BB9 + hEB (BB9+), day 9 BB9− hEB (BB9−), day 9 blast colonies (Blasts), or control human umbilical vein endothelial cells (HUVECs). Expression was assayed for actin, AGTR1, AGTR2, or angiotensinogen (AGTN) by qRT-PCR, as described in Document S1. Left panel demonstrates agarose gels of samples of PCR products (1/5 sample loaded) at linear amplification ranges for assayed transcripts. In right panel are relative, actin-normalized quantitative comparisons; fold change expression differences indicate levels of transcripts (calculated using the 2−ΔΔT method) of pooled day 9 blast colonies, compared with expression levels from sorted day 9 BB9+ hEB cells. (B) ACE enzymatic activity (units per number cultured cells assayed; mean ± SEM) was determined by colorimetric assay (in triplicate) from supernatants of pooled day 9 hEB blast colonies (BLASTS), or control supernatants from HUVEC (which express high levels of surface BB9/ACE (data not shown). (C) hEB were differentiated for hematopoietic progenitors (Figure S1A) in the presence or absence of specific RAS inhibitors. Captopril (100 μM; ACE inhibitor), 100 μM losartan (AGTR1 inhibitor), or 100 μM PD 123-319 (AGTR2 inhibitor) were included starting at day 4 of hEB culture. Day 14 hEB cell suspensions were assayed in duplicate in CFC assays, as before. (D) BL-CFC assays from days 5 to 9 hEB were conducted in the presence of supplemental angiotensin II peptide (ANG II; 100 μg/mL), captopril (100 μM), losartan (100 μM), or PD 123-319 (100 μM). Shown is the average of 2 independent experiments from day 9 hEB, with a similar pattern of results obtained with inhibitors in days 5 and 7 hEB BL-CFC assays (data not shown). (E,F) Blast colonies generated from day 5 or day 9 hEB cells were expanded in the presence of No inhibitor (control), 100 μM losartan, or 100 μM PD 123-319, and replated for secondary hematoendothelial CFC, as before. Although control blasts generated both hematopoietic and endothelial (HE) secondary progeny, the majority of blast colonies from losartan-treated blasts regenerated (robust) hematopoietic-only (H) progeny, whereas the majority of PD 123-319–treated blasts generated primarily endothelial-only (E) progeny. Shown panel F results are from blasts individually picked and replated blasts (n = 20 per condition), with typical secondary colony morphologies shown in panel E.