Endothelial Progenitor Cells—Potential New Avenues to Improve Neoangiogenesis and Reendothelialization - International Review of Cell and Molecular Biology - page 48

The harvested MNCs are further processed by CD34 magnetic beads sep-
aration. CD34 þ cells are plated on fibronectin-coated culture plates in an
endothelial culture medium. Further identification is achieved by (endothe-
lial) cell markers such as flk-1, uptake of acetylated low-density lipoprotein
(acLDL)-DiI or CD31. Cells isolated according to this protocol are generally
termed as EPC. Based on the fact that this procedure does not bring a high
specificity and different cell populations can be found in culture, the use of
the marker AC133 has been advocated for the magnetic bead isolation
instead of the use of CD34 ( Kawamoto et al., 2002; Yu et al., 2007 ). Since
this early hematopoietic marker is lost during transition from BM to PB and
therefore is found scarcely in peripheral venous blood, other isolation pro-
cedures have become more popular.
The original method as described by Asahara also leads to a large amount
of CD45 þ , CD16 þ , or CD68 þ cells, which are typical markers for macro-
phage cells. Therefore, a preculture step has been introduced to reduce
the amount of possible macrophages. Through this method, two distinct
types of EPC were defined:

the early-outgrowth EPC and the late-

outgrowth EPC.

2.1.2 Early EPC/circulating angiogenic cells
Early-outgrowth EPCs, which can be found after 3-5 days of culture and are
also termed as circulating angiogenic cells (CACs), have a low proliferation
potential and are unable to form tubular networks ( Hur et al., 2004 ). In
culture, they have an elongated, spindle-like appearance. Furthermore, it
is known that they produce large amounts of cytokines and angiogenic fac-
tors and therefore promote angiogenesis in a paracrine fashion ( Sieveking
et al., 2008 ).

2.1.3 Late EPC/ECFC
Late-outgrowth EPCs were first described by Lin et al. (2000) ,who attrib-
uted high proliferative potential to these cells. These cells arise after long-
time culture (about 2 weeks) in vitro and form colonies with a cobblestone
appearance. Therefore, the term “endothelial colony-forming cells”
(ECFCs) is also used. They have a high proliferative potential with up to
over 30 population doublings ( Lin et al., 2000 ).

2.1.4 Colony-forming units according to Hill et al.
The colony-forming units (CFUs) should not be mixed up with ECFCs as
mentioned earlier. Hill et al. described a method that uses PBMC cultured in