Technical notes on endothelial progenitor cells: Ways to escape from the knowledge plateau

Abstract

In the last 10 years an increasing interest has been devoted to the study of endothelial progenitor cells (EPCs), a subtype of immature cells involved in endothelial repair and neoangiogenesis. EPCs have been discovered as a novel integrated part of the cardiovascular system, which plays a comprehensive role in tissue homeostasis. Consistently, alterations and/or reduction of the circulating EPC pool have been associated with different manifestations of cardiovascular disorders and atherosclerosis. This is why, the extent of the EPC pool is now considered a mirror of vascular health, while EPC reduction has become a surrogate biomarker of cardiovascular risk and of the ongoing vascular damage. Unfortunately, the methods used to study EPCs still lack standardization, and this is significantly decelerating progress in the field. In this review, we focus on some aspects related to the two methods used to assess circulating EPCs: flow cytometry and cell culture. We uncover the many traps hidden in the choice of the right protocol, and suggest the best solutions on the basis of evidence and background theories.

Introduction

Since 1997 [1], a huge amount of literature has described the involvement of endothelial progenitor cells (EPCs) in cardiovascular disease. EPCs were originally thought to derive exclusively from the bone marrow, but recent studies have indicated alternative sources of EPCs, including parenchymatous organs and blood vessels [2], [3]. We have learned from animal studies that EPCs participate in endothelial homeostasis and stimulate the formation of new blood vessels [4], [5]. Clinically, EPCs have been found to be reduced in peripheral blood of subjects with cardiovascular risk factors and/or established atherosclerosis [6], [7], [8], [9], [10], [11], [12]: this is why depletion of circulating EPCs is considered a marker of the ongoing vascular damage [13]. Additionally, EPC reduction is an independent predictor of future cardiovascular events [14], [15], a notion that strengthens the concepts that EPCs are at the same time markers and actors in the entire atherogenetic process. Most importantly, cell therapies that restore the EPC pool have proven beneficial in patients with coronary and peripheral artery disease [16], [17]. Really, this has been a revolution, as EPCs are now considered an integrated part of the cardiovascular system.

Unfortunately, expanding interest and lack of a consensus have multiplied the number of different methodologies to study EPCs. A myriad of small studies are now available, each using slightly or profoundly different methods. Likely, this will ingenerate a sort of “knowledge plateau”: because of mutual inconsistencies, new studies often raise more questions than they answer. Herein, we would like to focus attention on a few simple technical aspects that all researches should consider when approaching the study of EPCs.

To start, let us define what an EPC is: a cell that derives from the bone marrow and probably from other sources [18], circulates in the bloodstream, proliferates, and can differentiate into a mature endothelial cell [19]. Basically, there are two techniques to study EPCs: (i) flow cytometry of fresh samples and (ii) cell culture.