XB-IMG-173439
Xenbase Image ID: 173439
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Figure 3. Id1-induced Kdr+ mesoderm is cardiogenic. (A)
Schematic depicting the prospective differentiation potential
of cryopreserved Id1-induced CMPs to multiple
cardiovascular cell types. (B) mRNA expression profiling
for the spontaneous differentiation potential of mESCs
stably overexpressing Id1 to cardiac (Myh6, Tnnt2, and
Actc1), skeletal muscle (Myod1, Myog, and Actn3), vascular
endothelial (Cdh5 and Pecam1), and fibroblast
(Postn and Vim) markers at days 6 and 15 of differentiation.
(C) mRNA expression profiling for the spontaneous
differentiation potential of h9 hESCs stably overexpressing
Id1 to cardiac (NKX2.5, TNNT2, MYH6, MYL2, and
MYL7), skeletal muscle (MYOD1, MYOG, and ACTN3),
vascular endothelial (CDH5 and PECAM1), smooth muscle
(MYH11 and ACTA2), and fibroblast (POSTN and
VIM) markers at days 5 and 15 of differentiation. (D) Representative
immunofluorescence image of cardiomyocytes
(ACTC1), vascular endothelial cells (CDH5), and
fibroblasts (TAGLN) at day 15 of differentiation in h9
hESCs stably overexpressing Id1. Bar, 50 μm. (E) Diagram
showing quantification of the percentage ofACTC1+ (cardiomyocytes),
TAGLN+ (fibroblasts), and CDH5+ (vascular
endothelial cells) at day 15 of differentiation in h9
hESCs stably overexpressing Id1. (F) Schematic of the
work flowfor the physiological assessment of cardiomyocytes
derived from Id1-overexpressing h9 hESCs using
the calcium-sensitive (Fluo-4) and voltage-sensitive
(VF2.1 Cl) (Miller et al. 2012) dyes. (G) Representative images
illustrating the minimumand maximum changes in
fluorescence of Fluo-4 in cardiomyocytes derived from
Id1-overexpressing h9 hESCs. (H) Representative calcium
transient trace of day 15 cardiomyocytes derived
from Id1-overexpressing h9 hESCs. (I,J) Representative
action potential traces of cardiomyocytes derived from
Id1-overexpressing h9 hESCs in control conditions (I) or in response to isoproterenol (J) measured optically with VF2.1 Cl. (K) Beat rate
quantification of cardiomyocytes derived from Id1-overexpressing h9 hESCs indicating an increase in beating frequency in response to
100 nM isoproterenol treatment as compared with vehicle and measured with VF2.1 Cl. Quantitative data are presented as means ± SD.
All experiments were performed at least in biological quadruplicates. The insets in the top right corners of all immunostaining images
shows corresponding DAPI staining. Image published in: Cunningham TJ et al. (2017) Copyright © 2017. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article. Larger Image Printer Friendly View |