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.

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.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal