Abstract
Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.
Highlights
Angiogenic assays are commonly used to determine the ability of specific compounds to promote or inhibit angiogenesis
It is well-documented that the decline of endothelial progenitor cells (EPCs) in the circulation is linked to poor outcomes for patients with chronic kidney disease (Povsic and Goldschmidt-Clermont, 2008; Goligorsky et al, 2010)
For expansion of Endothelial colony forming cells (ECFCs) into appropriate numbers for investigation of angiogenic potential it was important to first determine if the number of cells in the colony were appropriate for harvesting
Summary
Angiogenic assays are commonly used to determine the ability of specific compounds to promote or inhibit angiogenesis. Whilst angiogenesis is stimulated in the early stages of kidney disease, there is a rapid switch to a hypoxic state which denudes the angiogenic processes (Long et al, 2012) It is well-documented that the decline of endothelial progenitor cells (EPCs) in the circulation is linked to poor outcomes for patients with chronic kidney disease (Povsic and Goldschmidt-Clermont, 2008; Goligorsky et al, 2010). Since their discovery in 1997 (Asahara et al, 1997), EPCs have been suggested as a potential therapeutic target for a range of vascular disorders, yet translation to clinical therapies has shown limited success due to difficulties in the expansion of EPCs into appropriate numbers due to senescence of the isolated cells (Chong et al, 2016). In going forward it is important to have consensus on the methodology used to isolate and culture cells to be used as therapies in patients
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call