Putative endothelial progenitor cells were initially discovered in 1997 by Asahara and colleagues. The hypothesis driving their research was that a common precursor may give rise to the hematopoietic cells and angioblasts that condense to form blood islands during the initial phase of vascular development, and that it may be possible to identify an adult progenitor cell using the shared markers between the hematopoietic cells and angioblasts, namely CD34 and Flk-1 (or VEGFR2). CD34+ blood cells were magnetically sorted to a purity of 15.7%, and VEGFR2 cells were sorted to a purity of 20.0%. When CD34+ cells were plated on fibronectin, they adhered and proliferated for 4 weeks, whereas the CD34- cells did not. Plating the positive and negative populations together increased cell proliferation, cluster formation and also formation of tube-like structures. Further, these plated CD34+ cells took up acetylated-LDL and expressed higher levels of endothelial cell markers compared to freshly sorted CD34+ cells. Most importantly, the plated CD34+ cells were found co-localized with CD31+ cells at sites of new blood vessel growth in a rodent model of hindlimb ischemia. Together, these results demonstrated an endothelial phenotype, behavior, and apparent contribution to new blood vessel growth by the isolated CD34+ cells. Further, this was the first evidence of the existence of a circulating endothelial progenitor which could possibly participate in the growth of new vasculature. These findings motivated many researchers to equivalently search for and prove the existence of an endothelial progenitor cell.
In the following years, many papers were published about these putative endothelial progenitor cells, but as findings varied widely, it became clear that different cell types were all being referred to as ‘endothelial progenitors.’ There is still no single, agreed upon definition of an endothelial progenitor by surface markers or gene expression, but the differing characteristics of the various cell types all previously called ‘endothelial progenitors’ have become more clear in recent years. The different cell populations generally fall into three categories: 1) CD34+ cells and subsets, 2) Circulating Angiogenic Cells, and 3) Outgrowth Endothelial Cells. This nomenclature has been adopted here after careful review of the most current literature in the field. The cells in each of these categories exhibit very different roles in terms of how they contribute to the formation of new vasculature, and hence may differ in how they should be used therapeutically.
CD34+ cells and Subsets
Since the initially discovered EPCs were defined based on expression of CD34, many researchers began isolating these cells, and subsets of these cells, to gain further clarity on their role in postnatal vasculogenesis. In early reports, the endothelial phenotype of isolated CD34+ cells was repeatedly demonstrated and compared to Human Umbilical Vein Endothelial Cells (HUVECs), and in vivo CD34+ cells showed the ability to colonize matrigel plugs, to coat vascular grafts, and augment ischemic neovascularization. CD34+ cells were also divided into various subsets, of which CD34+ VEGFR2+ CD133+ cells were heavily investigated. The combination that included CD133, an early hematopoietic marker, was thought to identify a less mature progenitor. These cells had endothelial-like characteristics and lost CD133 expression as they matured. In vivo, CD34+ VEGFR2+ CD133+ cells were found at sites of neovascularization and engrafted on left ventricular assist devices. Alternatively, subjecting these cells to clonal assays revealed that CD34+ VEGFR2+ CD133+ cells also expressed CD45, another hematopoietic marker, and that these cells could form hematopoietic colonies, but not endothelial colonies. Only the CD34+ CD45- population could form endothelial colonies with any proliferative potential, ending the search for an endothelial progenitor within this subpopulation of cells. Other subpopulations of CD34+ cells have also been isolated by flow cytometry, using markers such as CD14 and combinations of cKit, Sca-1, and Lin, to investigate both functionality and origin. While EPCs still cannot be identified by a single surface marker or any combination, it has become clear that CD34+ cells represent a heterogeneous population, mostly of hematopoietic lineage. Even though CD34+ cells may not directly participate in blood vessel regeneration, mounting evidence does suggest they may act as an accessory cell in contributing to neovascularization.
Circulating Angiogenic Cells (CACs)
Rather than using surface molecule expression to isolate endothelial progenitors, the culture protocols employed by Asahara and colleagues have been used to yield the Circulating Angiogenic Cell (CAC) population. CACs are isolated from the mononuclear cell fraction of cord or adult blood by plating on fibronectin coated dishes in endothelial cell growth media for approximately 4 days. The adherent cells are termed CACs, and express endothelial surface markers, such as CD31, VEGFR2 and vWF, and these phenotypic similarities were used as evidence of differentiation into endothelial cells. It was quickly learned that CACs were not actually endothelial cells, but rather monocytes that in some ways mimic endothelial cell expression of surface markers when cultured in these specific conditions. CACs express CD14, a marker for monocytes, maintain monocyte function in culture, and do not incorporate into new blood vessels. But, CACs have been shown to form cord-like structures in a twodimensional assay for angiogenic behavior, and helped promote perfusion recovery after ischemic injury. Interestingly, only CD14+ cells cultured to obtain CACs exhibited this blood vessel growth promoting behavior, whereas freshly isolated monocytes did not. Other researchers have attempted to reduce the monocyte contribution to the CAC population by modifying the culture process. Specifically, the population of cells that initially adheres to fibronectin contains monocytes, which can be eliminated by collecting and re-plating the non-adherent population. This was first done by Ito and colleagues at 24 hours, and later by Hill and colleagues at 48 hours, and resulted in colony formation, with round centrally located cells surrounded by spindle-shaped cells at the periphery. This method has been commercialized into a kit containing specific media and is known as the CFU-Hill assay, in which reduced colony formation is used as an indicator of cardiovascular risk. While quantitatively useful as a diagnostic, too few cells are obtained with this culture method to substantiate their use therapeutically. Furthermore, the colonies do not contain a true endothelial progenitor, but instead still contain monocytes, and also T cells which are necessary for colony formation. Even though the CAC population was mistakenly identified initially as an endothelial progenitor population, these cells have demonstrated their angiogenic promoting ability via paracrine signaling, and are thus still valuable to investigate for therapeutic purposes.
Outgrowth Endothelial Cells (OECs)
The last population of ‘endothelial progenitors’ that has been widely investigated does contain cells that exhibit true progenitor behavior, and are called Outgrowth Endothelial Cells (OECs), or sometimes Endothelial Colony Forming Cells. These cells were named because of their late-outgrowth behavior, with colonies appearing 10-14 days after isolation from cord blood or bone marrow, as opposed to the early outgrowth by CACs. In contrast to the low or absent proliferative potential of the CAC population, OECs exhibited a high-proliferative potential and formed colonies in single cell re-plating assays, as would be expected of a true progenitor. These cells were shown to originate in the bone marrow, but not from a CD133+ or CD45+ population, nor did they express CD14. OECs demonstrated close similarities to endothelial cells in their surface marker expression and genetic profile, and directly participated in blood vessel formation. OECs formed networks of blood vessels ex vivo through the process of vasculogenesis in three-dimensional matrices, and demonstrated superior survival upon implantation in comparison to mature endothelial cells. Ex vivo engineered OEC vascular networks anastomosed with host vessels upon implantation into a mouse, and were robust enough to undergo serial transplantion. OECs have also been directed to migrate out of three-dimensional scaffolds, where they contributed to the formation of new vessels and helped restore perfusion in a mouse model of hindlimb ischemia. Together, these data demonstrate the progenitor cell properties of OECs and their therapeutic potential.