Figure 6.
Cultured Organ-Specific Human Fetal ECs Display Distinct Vascular Functions and Bioenergetics and Support Specific Parenchyma Function
(A) Left panel: Raw data of transendothelial electrical resistance measurement versus time for four types of ECs in monolayer culture. Cell index is calculated as , where fn is the frequency at which the impedance measurement is carried out (fn = 10 kHz), R(fn,t) is the measured impedance at frequency fn at time point t, t0 is the time when the background is measured, and Zn is the corresponding frequency factor of fn (Zn = 15 Ω). Right panel: Electrical impedance measurements (10 kHz frequency) for confluent monolayers of organ-specific ECs (n = 5 cultures).
(B) Spheroid sprouting assay measurements of organ-specific EC angiogenic potential. (i) Representative bright-field images of heart and kidney EC sprouting spheroids, collagen-embedded, upon 24 hr of VEGF stimulation (40 ng/mL) (scale bar: 75 μm) and (ii) quantification of sprouts number at baseline and after VEGF stimulation (40 ng/mL) for organ-specific EC spheroids embedded in collagen matrix (24 hr) (n = 3 donors).
(C) Seahorse measurements of total cellular oxygen consumption rate (OCR) in organ-specific ECs: (i) time course of cellular oxygen consumption upon sequential addition of oligomycin (1 μM), CCCP (1 μM), and rotenone/antimycin A (1 μM each); (ii) quantification of mitochondrial OCR obtained upon electron transport chain inhibition with rotenone and antimycin A; and (iii) quantification of mitochondrial OCR due to ATP synthase activation obtained upon ATP synthase inhibition with oligomycin (n = 3 donors).
(D) Seahorse measurements of total extracellular acidification rate (ECAR) in organ-specific ECs (n = 3 donors): (i) time course of extracellular acidification upon sequential addition of oligomycin (1 μM), CCCP (1 μM), and rotenone/antimycin A (1 μM each) and (ii) ECAR quantification.
(E) ELISA measurement of albumin production from rat hepatocytes when cultured alone and when co-cultured with heart, lung, liver, and kidney ECs after 7 days (n = 4 replicates).
Data information: data are presented as mean ± SEM *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. SEM, standard error of the mean.