Introduction:
Features of an ideal cell type, that would be conducive to vascular regeneration include (1) expression of pro-angiogenic genes (2) secretion of factors that promote developing or regenerating vasculature, and (3) maintenance of pro-angiogenic properties in the microvascular niche.
Hypothesis:
FTM HUCPVCs, a young rich source of mesenchymal stromal-like cells (MSCs) are ideal candidates for vascular regeneration due to their high expression of pro-angiogenic factors.
Methods:
The paracrine angiogenic potential of 3 types of MSCs was evaluated and compared using
ex vivo
tissue culture of rat aortic rings. Aorta sections were embedded into Matrigel™, cells were added to transwell membranes (pore=0.1μm, EBM 2% FBS) and combined with aortic rings (Day 0). Radial network growth and total loop formation were monitored by microscopy. Endothelial networks were quantified by ImageJ
TM
software. p values were calculated using ANOVA. (N=3 experiments, n=3 replicates). At day 7, the MSCs were isolated from transwells, human cytokine gene expression levels were measured using human growth factor profiler qPCR array (Qiagen, normalizers: GAPDH, βACT). Ct>35 considered negligible.
Results:
In the transwell aortic ring assay, FTM HUCPVCs induced significantly greater network growths when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated rings (p≤0.05). Quantification of network loop formation showed that FTM HUCPVCs induced greater numbers of closed loops when compared to term HUCPVCs (p≤0.0001), BMSCs (p≤0.0001) and untreated networks (p≤0.0001). Human growth factor qPCR array showed a high expression of angiogenic factors (Ct<25 cycle) both at day0 and day7 of co-culture, including BMP1, GDNF, MDK, NRG1, PDGFc, VEGFa, and VEGFc. Most cytokine expression levels were maintained up to 7 days in co-culture with 1 gene upregulated (BMP6 ΔCt>3), and 3 downregulated genes (FGF19, FGF9, NRTN ΔCt>3) at day 7 when compared to day 1.
Conclusion:
Compared to older sources of MSCs, FTM HUCPVCs promote developing endothelial networks
in vitro
via paracrine mechanisms and maintain the high expression of pro-angiogenic factors when cultures with endothelial cells.
Mouse aortic ring assay: A new approach of the molecular genetics of angiogenesis