Biology and Flow Cytometry of Proangiogenic Hematopoietic Progenitors Cells

Jonathan A Rose; Serpil Erzurum; Kewal Asosingh

doi:10.1002/cyto.a.22596

. Author manuscript; available in PMC: 2016 Jan 1.

Published in final edited form as: Cytometry A. 2014 Nov 21;87(1):5–19. doi: 10.1002/cyto.a.22596

Biology and Flow Cytometry of Proangiogenic Hematopoietic Progenitors Cells

Jonathan A Rose ^1,², Serpil Erzurum ^1,³, Kewal Asosingh ^1,^*

PMCID: PMC4356492 NIHMSID: NIHMS667752 PMID: 25418030

Abstract

During development hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative “endothelial progenitor cells” that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multi-disciplinary expertise in flow cytometry, hematology and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and bone marrow.

Key terms: progenitors, proangiogenic cells, hematopoiesis, angiogenesis

Introduction

Angiogenesis and vasculogenesis are two distinct processes by which new blood vessels are formed in order to provide distant tissues with access to oxygen, nutrients, and metabolic waste removal. Physiologically, blood vessel growth is necessary for both organ development during embryogenesis as well as the repair of wounded tissue in adults. A switch in angiogenic balance, however, is involved in the pathophysiology of many human diseases including a relative deficit in ischemic disorders and unregulated excess in tumor formation (1,2).

Until the late 1990s, postnatal blood vessel formation was generally believed to result exclusively from angiogenesis, a process of vessel sprouting due to the proliferation of fully-differentiated endothelial cells present in pre-existing blood vessels. Asahara’s seminal paper demonstrating the presence of circulating ‘endothelial progenitor cells’ (EPC) introduced the concept of adult vasculogenesis, a process of de novo blood vessel formation from recruited progenitor cells, and spawned a new era in vascular biology (3). Since this time, there has been considerable debate over what defines an EPC, driven by a diversity of methods used to characterize and isolate putative EPCs. Although the term EPC has been used to describe many cell types, Asahara’s original work has later been found to involve a heterogeneous group of cells including a subset of EPC termed proangiogenic hematopoietic progenitor cells (4), which have been shown to be an integral part of vascular repair and regeneration (4). In line with subsequent literature reporting on these cells, we abbreviated proangiogenic hematopoietic progenitor cells in this review as PAC.

There is emerging evidence that PAC result from the differentiation of hematopoietic stem/progenitor cells (5,6) that are mobilized from the bone marrow by angiogenic factors (7–10) and home to sites of neovascularization (10–13). Here they contribute to angiogensis by temporarily incorporating in the vessel wall (14–16) and elaborating potent paracrine factors (5,17–19). Their contribution to vascular health has also been highlighted by their strong association with many pathologic processes (20–23). This review will focus on the literature elucidating the identification and biology of PAC with special emphasis on flow cytometric monitoring of these cells.

Defining Proangiogenic Hematopoietic Progenitor Cells (PAC)

The term EPC has been used to describe a vast array of cell types participating in angiogenesis (24,25). This confusion has been driven by a diversity of methods used to characterize and isolate putative EPCs, which has made it difficult to directly compare studies, and as a result, there is no consensus on how to exactly define EPC or individual subsets (4,24). Although the existence of true endothelial progenitor cells during post natal life continues to remain a subject of debate, the term EPC is now commonly used to describe two functionally distinct cell types: those cells of hematopoietic lineage that promote angiogenesis via paracrine effects called proangiogenic hematopoietic cells (PHC) and the cells that proliferate to form new endothelium called endothelial colony forming cells (Table 1) (26).

Table 1.

Defining subsets of endothelial progenitor cells (EPCs)

Type of EPC	Surface Markers	Function	Source	References
I. Proangiogenic hematopoietic cells (PHC)	CD45, CD31, VE-cadherin, vWF, E-selectin, UEA-1, acLDL	Paracrine angiogenic effects		(125)
A. Mature PHC (e.g. Tie2-expressing monocytes)	CD14, CD11c, Tie2		Circulation	(126)
B. Proangiogenic hematopoietic progenitor cell (PAC) or CFU-Hill	CD34, (Sca-1 in murine), CD133, VEGFR2 (KDR in humans, Flk-1 in murine), C-kit, CXCR4	In addition to paracrine effect, temporarily engraft into injured endothelium	Bone marrow, circulation, blood vessel wall	(5,62), see Table 2
II. Endothelial colony forming cell (ECFC) or late outgrowth cell or blood outgrowth cell	CXCR4, VEGFR1, endothelial markers, not CD133 or CD45	Reconstitute endothelium	Circulation and endothelium	(6,127,128)

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In most early studies, proangiogenic hematopoietic cells (PHC) were defined by culture technique, which has been shown to isolate a diverse mixture of cell types including both mature hematopoietic cells such as monocytes and macrophages and hematopoietic stem/progenitor cells, each with the ability to secrete proangiogenic factors (26). Although much of the literature has treated all proangiogenic hematopoietic cells (PHC) the same, there are key differences (27). PACs express traditional stem cell markers such as CD34, CD133, and c-kit, and more importantly, retain the ability to form colonies in vitro (20,26). Therefore, PAC are of great interest for their potential to respond to the local milieu and proliferate as part of the angiogenic process. For the purposes of this review, we will include studies involving hematopoietic cells containing stem cell markers that demonstrate proangiogenic functionality as assessed by in vitro or in vivo assays such as the Matrigel plug assay or the hindlimb ischemia murine model. We will also include those studies using colony forming unit-endothelial cells (CFU-EC) or colony forming unit-Hill (CFU-Hill) due to the presence of PAC in these mixtures (6).

PAC include a phenotypically heterogenous group of cells comprising hematopoietic stem cells (6) and lineage progenitors (5) with the capacity to promote neovascularization (27). Table 2 provides an overview of the various phenotypes and nomenclature used to describe these cells. PACs can be further sub-classified based on their proangiogenic potential (5). The vast majority of the literature, however, has defined PAC using different combinations of CD34, CD133, and VEGFR2 (KDR in humans, Flk-1 in mice) (26). These cells also express endothelial markers such as CD31 (3,28), Tie2 (9,29), vWF (30,31), and E-selectin (31). Functionally, PAC are identified by their ability to possess endothelial properties such as adherence to fibronectin plates, uptake of acetylated low density lipoprotein (AcLDL), and binding of ulex-lectin. They do not, however, contain more robust endothelial properties such as the ability to form capillary-like structures in vivo. PAC are also unique in their ability to form “early outgrowth” colonies when cultured on fibronectin (20).

Table 2.

Various Phenotypes of Proangiogenic Hematopoietic Progenitor Cells

Phenotype	Terminology	Source	Angiogenic Activity	Reference
CD34⁺ VEGFR2⁺ CD45⁺ CD31⁺ Tie-2⁺ E-selectin⁻ DiI-acLDL⁺ UEA-1⁺	Endothelial progenitor cells	Human, murine, rabbit peripheral blood	Incorporation into neovasculature in hindlimb ischemia model	Asahara, 1997 (3)
BM-derived VEGFR2⁺ Tie2⁺	Endothelial progenitor cells	Murine tissue	Incorporation into neovasculature in hindlimb ischemia model	Asahara, 1999 (29)
Sca-1⁺ CD31⁺ DiI-acLDL⁺	Endothelial progenitor cells	Murine peripheral blood	Incorporation into neovasculature in hindlimb ischemia model	Takahashi, 1999 (59)
CD34⁺ AC133⁺ VEGFR2⁺	Circulating endothelial progenitors	Human peripheral blood	Neo-intima formation	Peichev, 2000 (87)
AC133⁺ VEGFR2⁺ VE- cadherin⁺	Circulating endothelial progenitors	Human peripheral blood	Not tested	Gill, 2001 (8)
VEGFR1⁺ CD11b⁺	Hematopoietic precursor cells	Murine BM	Increased vascularity in mouse tumor model	Lyden, 2001 (39)
Side population⁺ CD34^−/low c-Kit⁺ Sca-1⁺	Hematopoietic stem cells	Murine BM and cardiac tissue	Incorporation into coronary vessels in acute myocardial infarction model	Jackson, 2001 (121)
CFU-EC, VEGFR2⁺ DiI-acLDL⁺	Circulating endothelial progenitors	Murine peripheral blood	Not tested	Heissig, 2002 (10)
Lin⁻ Sca-1⁺ c-Kit⁺	Hematopoietic stem cells	Murine BM	Increased vascularity in retinal ischemia model	Grant, 2002 (34)
Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁺ Flk-1⁺	Hematopoietic stem and progenitor cells	Murine BM and peripheral blood	Increased vascularity in hindlimb ischemia model	Aicher, 2003 (41)
CD34⁺ CD133⁺ VEGFR2⁺ CD45⁺ vWF⁺	Endothelial progenitor cells	Human umbilical cord blood	Not tested	Eggermann, 2003 (30)
CD34⁺ CXCR4⁺	Endothelial progenitor cells	Human peripheral blood	Incorporation into neovasculature in hindlimb ischemia model	Yamaguchi, 2003 (12)
CXCR4⁺ CD31⁺ UEA-1⁺ DiI-acLDL⁺	Endothelial progenitor cells	Human peripheral blood	Incorporation into neovasculature in hindlimb ischemia model	Ceradini, 2004 (13)
VEGFR1⁺ CD45⁺ CD31⁺ eNOS⁺ vWF⁺ VEGFR2⁺ VE-cadherin⁺	Early endothelial progenitor cells	Human peripheral blood	Increased vessel formation in tube formation assay and hindlimb ischemia model	Hur, 2004 (125)
Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁺ CXCR4⁺	Bone marrow progenitor cells	Murine peripheral blood	Increased vascularity in Matrigel plug assay and hindlimb ischemia model	De Falco, 2004 (44)
CFU-Hill	Endothelial progenitor cells	Human peripheral blood	Not tested	Ghani, 2005 (60)
CD34⁺ CD133⁺ VEGFR2⁺	Immature endothelial progenitor cells	Human peripheral blood	Not tested	Werner, 2005 (61)
CD34⁺ CD133⁺ cKit⁺ VEGFR1⁺	Hematopoietic progenitor cells	Murine BM	Increased tumor metastasis in lung carcinoma xenograft model	Kaplan, 2005 (11)
CFU-Hill	Early endothelial progenitor cells	Human peripheral blood	Improved perfusion in hindlimb ischemia model	Yoon, 2005 (17)
Lin⁻ Sca-1⁺ c-Kit⁺	Hematopoietic stem/progenitor cells	Murine tumor tissue	Localized to neovasculature in prostate tumor xenograft model	Okamoto, 2005 (15)
CD34⁺ VEGFR2⁺	Endothelial progenitor cells	Human peripheral blood	Not tested	Fadini, 2005 (55)
CXCR4⁺ VEGFR1⁺ Sca-1⁺	Hemangiocytes	Murine peripheral blood	Incorporation into neovasculature in hindlimb ischemia model	Jin, 2006 (9)
CD34⁺ CD133⁺	Endothelial progenitor cells	Human choroidal membranes	Incorporation into vasculature of choroid membrane	Sheridan, 2006 (63)
CD34⁺ CD133⁺	Endothelial progenitor cells	Human peripheral blood	Not tested	Palange, 2006 (64)
CD34⁺ AC133⁺ VEGFR2⁺ CD45⁺	Primitive hematopoietic progenitor cells	Human peripheral and cord blood	Not tested	Case, 2007 (89)
CD34⁺ Sca-1⁺ VEGFR1⁺ VEGFR2⁺ E-selectin⁺ CD31⁻	Endothelial progenitor cells	Murine BM	Improved perfusion in hindlimb ischemia model	Oh, 2007 (31)
CFU-Hill (CD45⁺ CD14⁺ CD31⁺ CD105⁺ CD144⁺ CD146⁺ vWF⁺ UEA⁺)	Proangiogenic hematopoietic progenitor cells	Human peripheral blood	Incorporation into neovasculature in gel plug model	Yoder, 2007 (45)
CFU-Hill	Endothelial progenitor cells	Human peripheral blood	Not tested	Sobrino, 2007 (23)
CD34⁺ CD133⁺ CD45⁺	Hematopoietic stem/progenitor cells	Human BM	Not tested	Kissel, 2007 (58)
CFU-Hill, CD34⁺ CD133⁺	Bone marrow-derived proangiogenic precursors	Human peripheral blood	Cell clusters in Matrigel plug assay	Asosingh, 2008 (21)
CFU-Hill, CD34⁺ CD133⁺; SCA-1⁺ c-Kit⁺ VEFGR2⁺	Circulating endothelial progenitor cells	Human and murine peripheral blood	Tubulogenesis in tube formation assay; Increased vascularity in asthma mouse model	Asosingh, 2007 (22)
c-kit⁺ VEGFR2⁺ CD11b⁻	Endothelial progenitor cells	Murine peripheral blood and lung tissue	Increased vascularity in mouse model of pulmonary metastasis	Gao, 2008 (16)
CFU-Hill, CD34⁺ AC133⁺ VEGFR2⁺	Endothelial progenitor cells	Human peripheral blood	Increased tubulogenesis in tube formation assay	Diller, 2008 (71)
CFU-Hill, CD45⁺ CD31⁺ VEGFR2⁺	Early endothelial progenitor cells	Human peripheral blood	Increased tubulogenesis in in vitro co-culture assays	Sieveking, 2008 (51)
CD34⁺ CD133⁺ VEGFR2⁺	Circulating angiogenic progenitors	Human peripheral blood and lung tissue	Not tested	Toshner 2009 (72)
ALDH^hi	BM-derived stem and progenitor cells	Human BM	Increased vascularity in hindlimb ischemia model	Capoccia, 2009 (110)
CD34⁺ VEGFR2	Endothelial progenitor cells	Human peripheral blood	Not tested	Sala, 2010 (66)
ALDH^hi Lin⁻	Stem and progenitor cells	Human cord blood	Increased vascularity in acute myocardial infarction mouse model	Sondergaard, 2010 (111)
CD34^bright CD45^dim AC133⁺ CD31⁺ CD14⁻	Circulating hematopoietic stem/progenitor cells	Human peripheral blood	Increased vascularity in melanoma xenograft model	Estes, 2010 (28)
CD34^bright CD45^dim AC133⁺ CD31⁺ CD14⁻	Circulating progenitor cells	Human peripheral blood	Not tested	Pradhan, 2011 (62)
Sca-1⁺ c-Kit⁺ VEGFR2⁺	Vascular endothelial progenitor cells	Murine BM and lung tissue	Increased vascularisation of airway tissue	Doyle, 2011 (70)
CD34⁺ CD133⁺	Proangiogenic progenitor cells	Human peripheral blood and BM	Not tested	Farha, 2011 (35)
Lin⁻ Sca-1⁻ c-Kit⁺ CD34⁺ CD133⁺ CD45⁺ VEGFR2⁺ CD31⁺ Tie2⁺ vWF⁺	Proangiogenic cells	Murine BM	Increased vascularity in hindlimb ischemia model	Wara, 2011 (5)
KLF10⁺ CD34⁺ VEGFR2⁺	Proangiogenic cells	Murine and human peripheral blood	Improved perfusion in mouse hindlimb ischemia model	Wara, 2011 (18)
KLF10⁺ Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁺ VEGFR2⁺	Proangiogenic cells	Murine BM and peripheral blood	Improved reendothelialization in arterial injury model	Wara, 2013 (19)

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Origin of Proangiogenic Hematopoietic Progenitor Cells

Many have argued for the existence of a postnatal hemangioblast, a common progenitor for hematopoietic and endothelial lineages (32,33). This is an attractive theory given the synergistic function of PAC and endothelial colony forming cells in vessel growth and overlap in surface phenotype (17), but there has been a relative paucity of conclusive evidence. Regardless of more remote ancestry, the direct origin of PAC is likely hematopoietic stem/progenitor cells (5,6), which differentiate to acquire proangiogenic capabilities. In support of this, several reports documented that labeled hematopoietic cells localized to sites of vascular injury after bone marrow transplant (29,34). Several other studies have demonstrated that PAC are enriched in the myeloid lineage (5,21,35).

Three pools of PACs that are important in the context of vascular biology—a large reserve located in the bone marrow, the site where active hematopoiesis goes on during postnatal life (11,36,37), a small group circulating throughout the periphery in blood (38,39), and a subset residing in blood vessel walls in close proximity of the endothelium (11,35,39). It has been shown that this is the same population of progenitors, in which some cells migrate from the BM to tissues through the circulation and can later return to the BM (38). PACs may be pre-committed as shown by the presence of VEGF receptor type 2 (VEGFR2) (8), however, they likely lack proangiogenic ability until recruited to sites of neovascularization and stimulated by an angiogenic milieu (5). Hematopoietic stem/progenitor cells differentiate into PAC through upregulation of endothelial cell antigens and functional angiogenic properties (40).

Mobilization from the Bone Marrow

Under conditions of ischemia and other angiogenic states, PACs are mobilized from the BM and enter the circulation (7). Considerable work has been done in elucidating the mechanisms that lead to mobilization of PACs from the bone marrow under these conditions, and several factors that have been implicated in this process include matrix metalloprotease 9 (MMP-9), soluble kit-ligand (sKitL), vascular endothelial growth factor (VEGF), and stromal-derived factor 1 (SDF-1). The protease MMP-9 has been shown to be key to the mobilization of progenitors, most likely through the breakdown of the extracellular matrix anchoring these cells to the BM (9)(10). MMP-9 knockout mice (Mmp9^−/−) showed decreased numbers of circulating CXCR4⁺VEGFR1⁺ Sca-1⁺ progenitor cells, resulting in impaired revascularization in a hindlimb ischemia mouse model (9). MMP-9 may also act via the generation of sKitL (10), a stem cell active cytokine that is cleaved from a membrane bound version (mKitL) and binds to c-Kit (CD117) receptor on hematopoietic stem/progenitor cells. Mmp9^−/− mice displayed low levels of sKitL, and adenoviral delivery of sKitL increased Sca-1⁺ c-Kit⁺ progenitor cell mobilization and reversed the revascularization defect.

VEGF-A contributes to the mobilization of progenitor cells (8–10), and has proven to be one of the most potent mediators of the angiogenic switch in tumorigenesis (1). VEGF-A works through two receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR, Flk-1 in mice), which are both expressed on PACs (3,8,9,11). Under conditions of vascular injury, plasma VEGF-A levels are elevated and this was found to promote the rapid mobilization of PAC from the bone marrow to peripheral circulation (8). Another study showed that circulating VEGF induced MMP-9 expression in the BM (10), suggesting that VEGF may have promoted egress of PAC from the BM through upregulation of MMP-9. Another angiogenic factor in the VEGF family, placental growth factor (PlGF) signals through VEGFR1 and was shown to upregulate MMP-9 within the BM and result in subsequent release of sKitL (9). Factors like VEGF and PlGF may be released by hypoxic tissues and act on the BM to result in release of PAC. Expression of eNOS in the BM may also mediate the effect of these angiogenic factors as mice deficient in eNOS (Nos3^−/−) had significantly decreased VEGF-induced mobilization of CD34⁺Flk-1⁺ PAC, which corresponded with a decrease in BM expression of MMP-9 (41). These mice also had severely impaired neovascularization in a mouse hind-limb ischemia model. Interestingly, intravenous infusion of wild-type CD34⁺Flk-1⁺ PAC but not BM transplantation, rescued the defective neovascularization of Nos3^−/− mice in a hind-limb ischemia model, indicating that VEGF-induced mobilization acted through upregulation of eNOS in BM stromal cells.

One of the more recently studied, but perhaps most important, mechanisms involved in PAC mobilization is the SDF-1 (CXCL12)-CXCR4 axis (42). SDF-1/CXCL12 is a CXC-type chemokine that signals through binding to the seven-transmembrane G protein-coupled receptor CXCR4. The role of SDF-1-CXCR4 in mobilization was originally proposed based on work that showed treatment by G-CSF induces increased circulating PACs by down-regulation of bone marrow SDF-1 and up-regulation of CXCR4 on hematopoietic stem cells (43). A later study demonstrated an inverted SDF-1 gradient with elevated plasma concentrations and simultaneous decrease in bone marrow levels (44). In parallel, circulating c-Kit+ PACs were increased. This strongly implicated SDF-1 as a chemoattractant for PAC that both tethers these cells to the BM in physiological conditions and mobilizes them to the periphery in conditions of ischemia.

Recruitment to Sites of Neovascularization

Once present in the peripheral circulation, PAC migrate to tissue sites of neovascularization, and many of the same factors that contribute to mobilization from the bone marrow also play a role in local recruitment (10,12,13). For instance, MMP-9 was shown to participate in homing of PAC, possibly through the breakdown of the extracellular matrix allowing invasion into the surrounding tissue (10). One study showed that neutralizing antibodies against c-Kit, the receptor for sKitL, blocked migration of PAC in a prostate tumor xenograft model (15). In another report, the increased release of pro-MMP-9 by VEGF stimulation caused in vitro migration of human CD34⁺ PACs which was blocked by addition of a synthetic metalloproteinase inhibitor (10).

VEGF also contributes to the migration of PACs to areas of neovascularization, possibly through direct chemoattraction of PAC (10,11). Mice inoculated with Lewis lung carcinoma cells and treated with antibodies inhibiting VEGFR1 resulted in reduced PAC proliferation and tumor metastasis in the lung (11). Interestingly, antibodies against VEGFR2 did not prevent proliferation of PAC but did limit their recruitment to sites of tumor metastasis. This suggests that VEGF-A may have dual action on PAC by regulating cell proliferation via VEGFR2 and recruitment via VEGFR1. Other studies showed that VEGF induced migration of PAC via indirect ways such as upregulation of MMP-9 and sKitL (10). Taken together these results underscore the importance of VEGF in PAC recruitment via a multitude of mechanisms of action.

The role of SDF-1-CXCR4 in PAC homing has been shown by SDF-1 induced migration of human CD34⁺ PAC in an in vitro transwell migration assay (10). This was confirmed in vivo by a later study showing that injection of SDF-1 to the ischemic muscle of mice resulted in increased accumulation of CD34⁺ PAC as well as increased perfusion and capillary density compared to control mice (12). SDF-1 levels in ischemic tissues are upregulated after femoral ligation in a hindlimb ischemia mouse model (44) and in vitro studies demonstrated increased SDF-1 in hypoxic conditions (13) showing that SDF-1 is a factor secreted by tissues in angiogenic demand. In another study, ischemia-induced homing and engraftment of CXCR4⁺ PAC was decreased by administration of either CXCR4 or SDF-1 neutralizing antibodies in a mouse hindlimb ischemia model, resulting in decreased tissue reperfusion (13). This work suggests that homing of PAC and subsequent neovascularization is dependent upon SDF-1 secreted by hypoxic tissues binding to CXCR on PAC. Another study, however, suggested that SDF-1 may act indirectly as well by mediating the release of angiogenic factors through upregulation of MMP-9 (10).

The mechanism of ischemia-induced upregulation of SDF-1 likely involves HIF-1α activation in hypoxic tissues based upon in vitro work in which human umbilical vein endothelial cells (HUVECs) preconditioned in hypoxic conditions were found to adhere a greater number of CXCR4⁺ PAC than those cultivated in normoxia (13). This effect was blocked by either SDF-1 or CXCR4 neutralizing antibodies and also by silencing of HIF-1α expression in HUVECs with siRNA (13). Thus, HIF-1α upregulation in ischemic conditions may lead to elevated levels of SDF-1 that signals through CXCR4 on PAC to induce migration to sites of neovascularization.

Ex vivo studies have shown that Kruppel-like factor 10 (KLF10), which is a subclass of zinc-finger transcription factors, in PAC may mediate the role of SDF-1α-induced migration (18). PAC from KLF10 knockout (KLF10^−/−) mice exhibited reduced adherence to fibronectin-coated plates and reduced transwell migration compared with cells from wild type mice (18)(19). Interestingly, KLF10^−/− PAC had reduced cell surface expression of CXCR4 compared to wild type, suggesting that KLF10 is important for CXCR4 upregulation. In vivo studies showed that carotid arteries from KLF10^−/− mice had reduced numbers of PAC after carotid artery endothelial injury compared to wild type, a result that was reversed by BM transplant of PAC from wild type into KLF10^−/− mice (19). These studies showed the importance of KLF10 in contributing to SDF-1-induced migration through binding to CXCR4 on PAC.

As with mobilization, recruitment of PAC to neovascularizing tissues most likely involves a multitude of mechanisms that are still being investigated. The vast majority of studies thus far have been done in mice and it remains to be seen how much of these pathways contribute to human disease. Likewise, the question of whether these pathways other than VEGF can be targeted pharmacologically in conditions of excess angiogenesis remains to addressed in humans.

Differentiation

There is increasing evidence that hematopoietic progenitors in a proangiogenic microenvironment differentiate into PAC by upregulation of endothelial markers and adoption of proangiogenic functionality (5,18). Early work showed that colony forming unit-Hill, cultured from human peripheral blood, exhibited myeloid progenitor colony-forming activity when plated in colony forming unit-granulocyte monocyte methylcellulose assays (45), suggesting that PAC likely arise from hematopoietic progenitor cell differentiation along myeloid lineage. This was further supported by the presence of myeloid cell surface markers on some populations of PACs (21,46)(21)(21)(21). In addition, Wara et al. showed that murine bone marrow-derived common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) upregulated VEGFR2 and preferentially differentiated into PAC when cultured in an angiogenic medium, endothelial growth media-2 (EGM-2), in contrast to hematopoietic medium, IMDM (5). VEGFR2 has been previously shown to be essential for vasculogenesis and hematopoiesis during development in mice (47,48) and has been used as a key marker for defining PAC (26). The upregulation of VEGFR2 on myeloid progenitor-derived PAC translated to increased functional angiogenic properties as cells cultured in EGM-2 had significantly increased angiogenesis in a Matrigel plug assay and hindlimb ischemia model compared to those grown in hematopoietic IMDM (5). This suggests that once present in ischemic tissues, surrounded by a proangiogenic milieu, PAC fully differentiate by upregulation of endothelial markers and adoption of proangiogenic functions.

Additional work by Wara et al. to elaborate mechanisms of differentiation showed that TGF-β1 mediated the conversion of CMP and GMP into PAC (18), via increased expression of VEGFR2 protein and elevated VEGFR2 mRNA (18). In searching for possible downstream effectors, exogenous TGF-β1 was found to drastically increase KLF10 expression in GMP-derived PACs. Culture of purified CMP or GMP in angiogenic EGM-2 medium resulted in markedly higher levels of KLF10 compared with cells grown in hematopoietic IMDM medium. Furthermore, KLF10 over-expression in progenitor cells increased VEGFR2 cell surface expression suggesting that TGF-β-induced upregulation of VEGFR2 is mediated by KLF10. In a hindlimb ischemia model, KLF10^−/− mice developed significantly more autoamputation or severely impaired blood flow recovery compared to wild type mice, and injection of WT PAC rescued blood flow recovery in KLF10^−/− mice. While much of the impaired angiogenesis in KLF10^−/− mice is likely due to defective KLF10-induced differentiation of progenitor cells into PAC, these cells also displayed impaired mobilization and recruitment as described above, revealing a significant overlap in the mechanisms regulating recruitment and differentiation of PACs.

Although ex vivo studies demonstrated that myeloid progenitors stimulated by VEGF obtain a proangiogenic phenotype, some work suggests that the process of differentiation starts during the process of mobilization from the bone marrow. More immature cells were found in the bone marrow and expressed CD133⁺CD34⁺KDR⁺CD31⁻VE-cadherin⁻vWF⁻ (49), whereas upon entering circulation, CD133⁺CD34⁺KDR⁺ cells started to express more endothelial cell markers such as CD31, VE-cadherin, and vWF (3,50). Although to the extent of the authors’ knowledge, no study exists to directly compare equivalent populations in the BM, circulation, and peripheral tissue.

Mechanisms of Proangiogenic Activity

Angiogenesis involves a sequence of steps that results in the sprouting of pre-existing blood vessels. Initially, upon stimulation with angiogenic factors, an endothelial cell becomes the tip cell, which invades the basement membrane and leads the newly formed sprout. The tip cell migrates along the increasing gradient of angiogenic signals. Other endothelial cells called stalk cells fall in behind the tip cell and proliferate and elongate to form the new vessel (2). PACs assist in this process through various mechanisms (Figure 1).

Bone marrow-derived hematopoietic stem cells (HSC), common myeloid progenitors (CMP) and granulocyte monocyte progenitors (GMP) commit to proangiogenic lineage via transcription factor KLF10-induced upregulation of VEGFR2 on their cell surface. Common lymphoid progenitors (CLP) and megakaryocyte/erythroid progenitors (MEP) exhibit little, if any, angiogenic activity. The proangiogenic hematopoietic progenitors proliferate and fully differentiate into potent angiogenic cells in peripheral hypoxic microenvironment. The release of angiogenic factors and matrix metalloproteinases (MMPs) promote onset of vascularization by inducing local basement membrane degradation, endothelial cell proliferation and migration. Some proangiogenic cells may temporarily incorporate into the newly formed blood vessels. Reprinted with permission from Cleveland Clinic Center for Medical Art and Photography. ©2014. All rights reserved.

PAC facilitated angiogenesis through the breakdown of the basement membrane allowing invasion by the tip cell and release of extracullar matrix bound proangiogenic factors. Several studies have demonstrated the importance of MMP-9 in neovascularization(9,11). Certain subsets of PAC may also contribute to neovascularization by temporarily engrafting into the endothelium through a process known as vascular mimicry (14). Early studies showed that ex vivo expanded CFU-Hill formed capillary-like structures in vitro (17), while later studies showed bone marrow-derived PAC incorporate into newly forming blood vessels in a mouse model of pulmonary metastasis (16). Another study showed that in nude mice inoculated with PC3 human prostate cancer cells, small capillaries begin to form near c-Kit⁺ PACs (15). As the tumor grew these angiogenic cells accumulated around the newly formed blood vessel and the caliber of the capillaries increased. This led the authors to conclude that PACs stabilize and support the newly forming vessels. It may be that these cells form a temporary structure until a bone fide vessel can be formed or they may stabilize developing structures by exerting a strong paracrine effect through secretion of proangiogenic factors.

Many soluble factors have been shown to be secreted by PAC in mediating their proangiogenic effect. One of the first works to suggest PAC’s role as paracrine showed that early outgrowth colonies had higher levels of angiogenic cytokines VEGF and IL-8 in supernatant than late outgrowth colonies generated by endothelial colony forming cells (17). Others demonstrated direct in vitro paracrine effect of PACs (51), and a more recent study corroborated this by showing that myeloid-derived PACs produced more VEGF than other PACs, and when injected intramuscularly into an ischemic limb, these cells increased blood flow recovery (5). Conditioned media of myeloid-derived PACs from KLF10^−/− mice had impaired wound healing in an in vitro scratch wound assay of HUVECs as compared to media from wild type PAC (18), attributable to reduced PDGF-AA and PDGF-AB, which have been shown to play a role in stabilizing newly formed blood vessels (52–54). Media from cultured KLF10^−/− BM progenitors was markedly inefficient in promoting in vitro endothelial cell growth and migration (19). This suggested that KLF10 may be important in PAC elaboration of angiogenic factors for vascular repair. In vivo analysis further demonstrated that KLF10^−/− mice had significantly reduced reendotheliazation in a wire-induced carotid endothelial injury model, which was reversed with BM transplant from wild type mice (19).

Although significant research has been done to elucidate the mechanisms underlying the proangiogenic effect of PACs additional questions remain. For instance what happens to these cells once the stimulus for angiogenesis abates? This is one area for further investigation as there could be significant long-term effects on the tissue surrounding new vessels. For instance PAC could undergo further differentiation to become fibroblasts and lead to scar formation. Other possibilities include becoming senescent vascular pericytes, undergoing apoptosis, or possibly de-differentiating and returning to the BM to await further stimulus.

Monitoring PAC in Human Disease

The levels and function of PACs are associated with many pathologic processes (20–23,55–57). Reduced numbers and function of PACs have been shown in several ischemia disorders including cardiovascular disease (20,58), cerebrovascular disease (23,59,60), and peripheral artery disease (28,55). In many instances level and function have been shown to inversely correlate with disease severity and prognosis, suggesting a protective role in vascular disorders (23,28,61). Most diseases that are characterized by pathologic vascularization such as neoplasia (56,62) and macular degeneration (57,63) have shown increased numbers or function of PAC indicating a contributory role in the pathophysiology. There have been many correlations found between pulmonary diseases characterized by endothelial injury and degeneration, but it has been controversial whether these cells are contributing to the disease or protective under these circumstances. For instance in COPD, several reports have shown decreased levels of circulating PACs (64,65), while others have shown increased numbers (66). Patients with acute lung injury have increased number of PACs (67–69), and increased levels correlate with improved survival, indicating a repairing effect under conditions of endothelial injury (69). In pulmonary diseases of vascular remodeling such as PAH and asthma, reports indicated that PACs play a contributory role in the pathological angiogenesis (21,22,35,70–72). Whether contributory or protective, it has been clearly shown that PACs are important in many human diseases, and therefore monitoring these cells has many broad clinical implications. It allows for an easily accessible biomarker in peripheral blood of patients to enable diagnosis, assess prognosis, guide management, and follow response to treatment. Detecting these cells is also vital to continuing to study and understand the underlying mechanisms of these many human diseases.

Flow Cytometric Methods to Detect PACs in Peripheral Blood and Bone Marrow

From the time of the initial discovery by Asahara et al., flow cytometry has been central to defining PACs and understanding their function (3). Part of this stems from the ease of identifying cell populations based on surface markers directly from peripheral blood, which can provide a wealth of information about the health status of the organism. Over the last decade and a half, many advances have taken place in the area of flow cytometry, leading to the refinement in our understanding of the nature of these cells and the basis for their biologic function. However, despite these strides, there is still no single method for their detection that has proven to be superior above all others. This section will review these techniques, how they have contributed to the understanding of PACs, and will end with a practical illustration of the technicalities for one method of detecting PACs by flow cytometry.

Cell Surface Markers

The most common method of identifying PACs has been to utilize the presence of cell surface markers. In the vast majority of reports, these have included hematopoietic stem/progenitor cell antigens in combination with one or more proangiogenic markers (see Table 2). Once cells have been isolated based on cell surface antigens, however, functional assays must be conducted in order to prove their proangiogenic nature. These assays include the in vitro tube formation assay (73), in vivo Matrigel plug assay (74), hindlimb ischemia mouse model (17,75), and tumor xenograft mouse model (15,76). Hematopoietic markers that have commonly been used include CD34 and CD133 in humans and stem cell antigen-1 (Sca-1) and c-kit in mice (26). CD34 is a transmembrane sialomucin expressed on the surface of proliferating hematopoietic stem/progenitor cells and is likely important for proliferation and migration (77). However this has been made more complicated by the fact that endothelial cells as well as others have significant CD34 expression (78). CD133 is also a transmembrane glycoprotein that was initially described as an hematopoietic stem/progenitor cells marker (79), but while specific epitope AC133 may be more specific in hematopoietic stem/progenitor cell populations, CD133 has been shown to be present on many other cell types including somatic cancer stem cells (80) and epithelial cells (81). Sca-1 is required for self-renewal in stem cells and development of committed progenitor cells (82). C-kit, also known as CD117, is a tyrosine kinase receptor for ligand stem cell factor (SCF) and is involved in stem cell proliferation, survival, and differentiation (83). Proangiogenic markers for PACs include those typically associated with endothelial cells including CD31 (PECAM-1), CD105 (Endoglin), VEGFR-2 (KDR in humans and Flk-1 in mice), CD144 (VE-cadherin), CD202b (Tie-2), UEA-1, and vWF, as well as others. Most commonly FACS is used for characterization of these cells based on surface markers (84). Recently laser scanning cytometry or imaging cytometry have also proven to be useful tools in the phenotypical analysis of PACs (85,86). A major advantage of the imaging technology is that it provides morphological information together with cytometric data at the single cell level (85,86).

Early on in the discovery of circulating cells that contribute to neoangiogensis, Peichev et al argued that the fraction of CD34⁺ cells that were also VEGFR2⁺ and AC133⁺ represented a proangiogenic fraction based on the observation that they formed neointima on the surface of left ventricular assist devices (LVADs) in humans (87). This protocol used dual-color flow cytometry and estimated the percentage of VEGFR2⁺ CD34⁺ and VEGFR2⁺AC133⁺ cells from mononuclear cells (MNCs) recovered from explanted LVAD specimens. Nonviable cells detected by propidium iodide staining and monocytes identified by CD15 and CD14 expression were excluded as part of the analysis. After this work, expression of CD34, CD133 and VEGFR2 quickly became the model for identifying PACs by flow cytometry (84). Many studies have shown that CD34⁺CD133⁺ cells are enriched in colony-forming activity and engraft into immunodeficient mice (28,88,89). The importance of CD133 for identifying the proangiogenic fraction of CD34⁺ cells has been further supported by later studies (28). In one study by Estes et al, human CD14⁻CD235a⁻CD31⁺CD45⁺CD34⁺CD133⁺ cells were shown to display significantly greater proangiogenic activity than those cells without CD133 expression using a NOD/SCID mice model bearing human melanoma xenografts (28).

As the technology and methods for detecting rare events by flow cytometry improved (90–94), protocols for identifying PACs began to incorporate these advancements. With the expansion of multi-color flow cytometry, methods began to incorporate “dump” channels to exclude analysis of cells with known phenotypes such as lymphocytes identified by CD3 and CD19 and myeloid cells with CD33 expression (84). This practice, however, may no longer be recommended as recent work by Wara has shown that myeloid progenitors possess some of the most proangiogenic phenotypes (5) and lymphocyte antigens may be expressed at dim levels on proangiogenic hematopoietic stem cells. After it became appreciated that there are likely two different types of “endothelial progenitor cells” in circulating blood contributing to neoangiogensis, CD45 became essential for isolating the hematopoietic cells and not circulating endothelial cells that are also CD34⁺VEGFR2⁺ (89). Procedures began to reflect this change, and one important one advocated for identifying a CD34⁺CD133⁺CD45^dim population using four-color flow cytometry to also quantify CD31 expression (95). Protocols since this time have reinforced the need to identify CD34⁺CD133⁺CD45^dim and have also used endothelial markers such as VEGF2 and CD31 to detect proangiogenic differentiation (96,97). The protocol by Estes et al. used an eight-color panel with several dump channels including CD235a, CD41a, CD14, and LIVE/DEAD Violet to identify RBCs, platelets, monocytes, and non-viable events, respectively.

When analyzing bone marrow or the peripheral circulation, selecting for endothelial markers may be less important and actually limit the number of PACs quantified or collected. Wara’s group has shown the ability of hematopoietic stem/progenitor cells to respond to a proangiogenic milleu and differentiate into PACs, with myeloid progenitors giving rise to the most proangiogenic cells (5). The majority of circulating hematopoietic stem/progenitors in both health and disease are of myeloid lineage (98) and probably have the capacity to become PAC under the right conditions. This is supported by the notion that there is a functional exhaustion of progenitor cells in the BM of patients with ischemic cardiovascular disease (58). Therefore selecting for hematopoietic stem/progenitor markers such as CD34 and CD133 should suffice for identifying cells with proangiogenic potential in the peripheral circulation.

Although expression of cell surface markers has provided the ability to detect PACs, there are several drawbacks to this method. As PACs are not a homogenous population of cells, cell surface markers vary depending on the stage of differentiation and cell source, inherent to the fact that PACs originate from a heterogeneous population of hematopoietic stem/progenitors. Cell surface markers also vary among species. Lastly, surface phenotype may remain stable even as functionality changes. There may be a relative stability in the expression of cell surface molecules even as PACs gain or lose proangiogenic function, making it difficult to predict angiogenic potential based on surface phenotype alone.

Functional Probes

Aldehyde Dehydrogenase Activity

With the difficulty in identifying PACs by cell surface antigens, other methods have been employed that allow detection of PACs based on functional activity. This has also allowed for the replacement of multiple cell surface markers with a single molecular probe. One of the most successful methods in this pursuit has used expression of high levels of aldehyde dehydrogenase (ALDH) activity. ALDH is a family of intracellular enzymes that catalyze the oxidation of aldehydes into carboxylic acids and are central to the metabolism of alcohol, catecholamines, and conversion of Vitamin A to retinoic acids (99,100). ALDH is expressed in high levels within human and mouse hematopoietic stem/progenitor cells, and a fluorescent substrate for ALDH was first used to purify these cells by flow cytometry in 1995 (101). This method was later improved by Storm et al. who developed a new fluorescent aldehyde substrate capable of binding ALDH that consists of an amino acetaldehyde conjugated to a BODIPY (4,4-difluroro-5,7-dimethyl-4-bora-3a,4a-diaza-5-proprionic acid) fluorochrome. The substrate is metabolized to an amino acetate anion by ALDH, which is negatively charged at physiologic PH and thus retained within the cell. Therefore, the amount of fluorescent product that accumulates in each cell correlates with ALDH activity. This substrate was used in combination with low side scatter (SSC^loALDH^hi) to efficiently isolate hematopoietic stem/progenitor cells from human umbilical cord blood (UCB) (102), and this method was later commercialized and referred to as Aldefluor^™. Since that time, ALDH activity has been used to enrich populations of hematopoietic stem/progenitor cells from a variety of human and mouse tissues, including BM and peripheral blood, to select cells with proangiogenic potential (103–106).

Many works have shown that populations of human cells from BM, UCB, and peripheral blood with high ALDH activity (ALDH^hi) are highly enriched in early hematopoietic stem/progenitor cell surface markers such as CD133 and CD34 (103,104,107–109). These cells have been shown to possess more long-term proliferative and reconstituting ability both in vitro and in vivo (102,104–106). Other work has shown that the ALDH^hiCD34⁺ cell fraction enriched short-term myeloid progenitors detected in vitro, which in other studies have been shown to produce highly proangiogenic cells (5). Cell populations with high ALDH activity have also been shown to possess proangiogenic activity directly. Transplantation of human BM-derived ALDH^hi cells into a hindlimb ischemia mouse model showed significantly improved perfusion and capillary density compared to ALDH^lo and BM cells (110). ALDH^hi cells were also shown to be transiently recruited to ischemic regions without fully integrating into the tissue, much in the same way as CD34⁺ PACs (12). Another study showed that transplantation of UCB-derived ALDH^hiLin⁻ cells into NOD/SCID mice after experimentally-induced acute myocardial infarction resulted in higher frequency of localization to site of injury as well as long-term engraftment compared to ALDH^loLin⁻ cells (111). There was also a significant increase in vascular density in the central infarct zone of ALDH^hiLin⁻ cell-treated mice. Together these works show the angiogenic potential of cells expressing high ALDH activity. Other studies have validated that these cells act through paracrine mechanisms. Smith et al. reported that human BM-derived ALDH^hi cells expressed angiogenic cytokines that protect endothelial cells from ischemic damage (112), and another study demonstrated that coculture of ALDH^hi cells promoted the survival of HUVECs under growth serum-free conditions as opposed to coculture with ALDH^lo cells, which resulted in death of HUVECs. In a hindlimb ischemia mouse model, transplantation of ALDH^hi cells enhanced recovery of perfusion and capillary density. These results demonstrate that isolating cells based on ALDH activity selects for a proangiogenic cell compartment. Though never done directly, it would be interesting to compare the proangiogenic capacity of ALDH^hi cells with those CD34⁺CD133⁺ PACs isolated in other studies (21,87).

Hoechst Uptake

Exclusion of Hoechst 33342 dye uptake is a characteristic common to stem cells, as well as chemotherapy-resistant cancer cells (113,114). Expression of the ATP binding cassette transporter ABCG2 (or breast cancer resistance protein 1, BRCP1), is a cell surface channel that allows for efflux of the dye, and these dye-excluding cells can then be sorted with a UV laser equipped flow cytometer (115). This method has been used to isolate hematopoietic stem cells, and cells with the highest dye exclusion have been shown to contain the highest long-term reconstituting ability (116,117). These cells are also remarkably homogeneous for expression of hematopoietic stem cell surface markers, including Sca-1 and c-Kit (118–120). One study showed that ABCG2 is expressed at high levels in primitive hematopoietic stem cells (CD34⁺CD38⁻ or CD34⁺KDR⁺) and drops sharply in committed progenitors (CD34⁺CD38⁺, CD34⁺CD33⁺, or CD34⁺CD10⁺) (118). These cells have also shown proangiogenic potential in one study in which Hoechst 33342-excluding cells transplanted into mice that later underwent coronary artery occlusion showed engraftment into the coronary vasculature as compared with controls who underwent sham surgery (121). To these authors’ knowledge, however, this has been the only study demonstrating a proangiogenic function of Hoechst-excluding cells. This method, however, may not be the most efficient way to isolate PACs as it excludes multipotent progenitor populations (118), which have been shown to be highly proangiogenic (5).

Intracellular Detection of Transcription Factor KLF10

The importance of the transcription factor KLF10 in regulating proangiogenic progenitor cell differentiation from hematopoetic stem/progenitor cells has been reported recently (18,19). Using quantitative PCR KLF10 expression was shown to be increased in bone marrow and circulating progenitor cells after endothelial injury (19). This same group has recently developed a method for quantifying intracellular KLF10 expression using flow cytometry that employs permeabilization and fixation techniques of CD34⁺VEGFR2⁺ cells and intracellular staining using a KLF10 polyclonal antibody (18). Detection by KLF10 expression, therefore, allows precise enumeration of this proangiogenic subset in ischemic disease and can serve as a single marker rather than multiple cell surface antigens. In addition to this procedure using intracellular antibodies against the KLF10 protein, new technology could be utilized that takes advantage of quantitative RNA detection. For instance, a novel in situ hybridization assay has been developed (122) and commercialized under the trade name QuantiGene® FlowRNA, which uses a standard flow cytometer to simultaneously detect up to three RNA transcripts per cell. Although this technology has not yet been applied to the study of PACs, it could theoretically be used to characterize levels of intracellular KLF10 RNA and define more accurately the differentiation of hematopoietic stem/progenitor cells into PACs. This would allow an additional advance in that it would not rely on protein detection with antibodies, but could be used to measure RNA transcripts of a variety of proangiogenic genes. SmartFlare^™ RNA probes, which allows RNA detection in live cells, provides the possibility to sort KLF10 expressing cells for further functional studies (123).

Technicalities for Flow Cytometry of Circulating PACs

PACs are infrequent in peripheral blood and therefore strict guidelines for rare event detection must be applied (90–94). Rare events present a unique signal-to-noise ratio problem, necessitating the acquisition of a large number of events combined with the use of creative techniques to limit artifacts in order to obtain appropriate numbers of the desired cell population. Non-specific binding of antibodies through protein-protein interactions is minimized through a blocking step with a blocking serum or Fc-block prior to antibody staining. A nuclear stain is typically used to ensure that the rare events being collected are in fact cells containing a nucleus and not a cell fragment. There are many different nuclear stains and one commonly used dye is DAPI (4′,6-diamidino-2-phenylindole). Staining cellular DNA with DAPI can also be used to identify cells with hypodiploidy. These typically represent dead and dying cells, which can alter marker expression due to nonspecific binding and should be eliminated from the analysis. DRAQ5^™ is one of the several other more recently available nuclear dyes that can be used to stain both live and fixed cells (124). Dead and dying cells can be identified with cell viability assays that stain the nuclei of cells with compromised cellular membranes. Although some can only be used on live cell samples, other stains such as LIVE/DEAD^® have been developed to assess cell viability in samples that have undergone fixation and permeabilization steps.

The number of required events for rare-event detection is typically based on the coefficient of variance (CV) and is determined using Poisson statistics according to the following equation: $C V % = 100 / \sqrt positive events$ . Depending upon the study, patient population, and disease, there are different values for the percentage of PACs in peripheral blood that range from 0.008% to 0.134% of isolated mononuclear cells (MNC) (28,89). Therefore, using a CV of 10% and a 0.02% frequency of PAC in isolated MNC, acquisition of 500,000 events would be required for proper analysis.

During the data analysis, gating strategies can be used to eliminate sources of artifacts such as fluidic disturbances, aggregates, and dead cells (Figure 2). Fluidic disturbances involve turbulent, instead of laminar, flow within the flow cell, which results in increased variability for different measurements. To eliminate this source of interference, time-gating can be done that involves gating out transient fluidic disturbances seen as altered marker expression in a plot of time versus log side scatter. Aggregates, including cell doublets or clusters, can be removed simply by analyzing the triggering parameters of pulse width and height and gating out those events that are too wide for their height. Dead cells and dying cells can be eliminated from the analysis using several different methods depending on the staining technique utilized. Those events with light scatters too low to be intact cells should also be eliminated as they likely represent cellular fragments. Backgating should be performed to check whether the gating strategy contributed to exclusion of subsets of interest.

Various cell surface antigen, probes and intracellular markers are available for the detection of PACs. Although there is no consensus about the exact phenotype of PACs and no single correct protocol exists to analyze for these cells, certain cytometric standards (A–F) apply. In this example human peripheral blood was collected in BD Vacutainer CPT Cell Preparation Tube and mononuclear cell were isolated according to manufactures instructions. Cells were stained for CD34, CD133, CD45 and VEGFR2 after Fc-blocking. DRAQ5 was used as nuclear stain and UV live/dead stain as viability dye. (A). Time gating to control for fluidic disturbances. Any burst, abrupt drop in events or other irregularity should be excluded. (B). Aggregates of cell could give false co-expression results and events with higher FSC-A relative to FSC-H are gated out. (C). Dead cells exhibit increased non-specific affinity for antibodies. Live cells are selected by employing a viablility dye. (D). Cell debris are eliminated by gating for events positive for nuclear stain. (E). Events with FSC/SSC to high or too low for mononuclear cells are rejected. (F). Hematopoietic cells express bright CD45, while hematopoietic stem/progenitor cells have dim CD45 expression. CD45 gating is performed to select for these cells. (G). In this illustration, CD34+CD133+ were gated and (H). VEGFR2+ subset was further analyzed. Backgating (not shown) should be performed to ensure that the gating strategy didn’t exclude any subpopulations of interest.

One example of a flow cytometric protocol for detecting PACs in peripheral blood is illustrated in Figure 2. Although several protocols have been developed for this purpose (reviewed above) (84,87,95–97), this method has been used involving five colors because it is specific enough to isolate the rare proangiogenic progenitor fraction while remaining basic enough to allow for the easy addition of other antibodies/probes against disease-specific pathways to the panel that may be of interest, allowing for the continued study of PACs as a biomarker in various disease processes (20–23,55–57). In this method, DRAQ5 identifies nucleated cells and LIVE/DEAD excludes dead and dying cells with compromised plasma membrane. CD34 and CD133 are essential for isolating immature progenitor cells and CD45 is necessary to isolate the hematopoietic fraction. CD133 is also important for isolating the proangiogenic fraction (28). Quantification of VEGFR2 has been shown as an example for quantifying expression of an additional surface marker, in this case one of proangiogenic differentiation. However, as stressed above, circulating progenitors cells negative for VEGFR2 may still be able to differentiate into PACs after homing into ischemic sites and in situ up-regulation of VEGFR2 (5). In addition to dead/dying cell exclusion, this analysis has incorporated techniques for rare event detection such as time gating and aggregate exclusion that were not mentioned in the previously reviewed protocols (84,87,95–97), but are necessary to increase sensitivity for detection and to eliminate artifacts.

Conclusions

Angiogenesis and hematopoiesis are inter-connected during development as bipotent hemangioblasts that generate both endothelial and hematopoietic cells. This relationship was found to be maintained during postnatal life by Asahara’s original discovery of “endothelial progenitor cells.” Despite the controversy surrounding the exact definition of endothelial progenitor cells, considerable progress has been made in characterizing different subsets of cells that contribute to angiogenesis. One of these types, proangiogenic hematopoietic progenitor cells have been found to contribute greatly to the process of neovascularization and play a critical role in vascular repair in as well as dysfunction in disease. These cells are mobilized from the bone marrow by angiogenic factors, home to sites of vessel formation, and differentiate to become proangiogenic cells. Once present in neovascularizing tissue, they contribute to angiogensis by temporarily incorporating in the vessel wall and elaborating potent paracrine factors. In this way, they represent a true link between the hematopoietic system and angiogenesis.

Detection and isolation of PACs has proven to be important in many different areas of disease research. Although there is no definitive way of characterizing these cells, flow cytometry has been a vital tool for identifying and monitoring these cells in the bone marrow and peripheral blood. Some of the most common protocols involve using cell surface expression of stem/progenitor markers including CD34 and CD133, hematopoietic marker CD45, and angiogenic markers such as VEGFR2, although progenitors lacking expression of angiogenic markers may still have the potential to form PACs. As these cells are infrequent in peripheral blood and bone marrow, strict guidelines for rare event detection must be applied including time gating, aggregate exclusion, nuclear staining, and dead cell exclusion. With an appropriate protocol, these cells can be identified and further evaluated for expression of disease-specific markers. Studying these cells with flow cytometry will help expand our understanding of their biology and role in the pathophysiology of human disease.

Acknowledgments

Support:

Kewal Asosingh is a Scholar of the International Society for Advancement of Cytometry (ISAC). This work was supported by grants HL081064, HL60917, HL115008 and M01 RR018390 from the National Institutes of Health, American Heart Association grant 11SDG4990003, American Thoracic Society/Pulmonary Association Research grant (PH-07-003), and the Hematopoietic Stem Cell Core Facility of the Case Comprehensive Cancer Center (P30 CA43703).

Abbreviations

EPC: endothelial progenitor cell
BM: Bone marrow
PAC: proangiogenic hematopoietic progenitor cell
PHC: proangiogenic hematopoietic cell
CFU-Hill: colony forming unit-Hill
UEA-1: Ulex europaeus agglutinin 1
DiI-acLDL: 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein

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PERMALINK

Biology and Flow Cytometry of Proangiogenic Hematopoietic Progenitors Cells

Jonathan A Rose

Serpil Erzurum

Kewal Asosingh

Abstract

Introduction

Defining Proangiogenic Hematopoietic Progenitor Cells (PAC)

Table 1.

Table 2.

Origin of Proangiogenic Hematopoietic Progenitor Cells

Mobilization from the Bone Marrow

Recruitment to Sites of Neovascularization

Differentiation

Mechanisms of Proangiogenic Activity

Figure 1. Differentiation and angiogenic activity of proangiogenic hematopoietic progenitor cells.

Monitoring PAC in Human Disease

Flow Cytometric Methods to Detect PACs in Peripheral Blood and Bone Marrow

Cell Surface Markers

Functional Probes

Aldehyde Dehydrogenase Activity

Hoechst Uptake

Intracellular Detection of Transcription Factor KLF10

Technicalities for Flow Cytometry of Circulating PACs

Figure 2. Illustration of a gating strategy for enumeration of circulating PACs.

Conclusions

Acknowledgments

Abbreviations

Literature Cited

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

Biology and Flow Cytometry of Proangiogenic Hematopoietic Progenitors Cells

Jonathan A Rose

Serpil Erzurum

Kewal Asosingh

Abstract

Introduction

Defining Proangiogenic Hematopoietic Progenitor Cells (PAC)

Table 1.

Table 2.

Origin of Proangiogenic Hematopoietic Progenitor Cells

Mobilization from the Bone Marrow

Recruitment to Sites of Neovascularization

Differentiation

Mechanisms of Proangiogenic Activity

Figure 1. Differentiation and angiogenic activity of proangiogenic hematopoietic progenitor cells.

Monitoring PAC in Human Disease

Flow Cytometric Methods to Detect PACs in Peripheral Blood and Bone Marrow

Cell Surface Markers

Functional Probes

Aldehyde Dehydrogenase Activity

Hoechst Uptake

Intracellular Detection of Transcription Factor KLF10

Technicalities for Flow Cytometry of Circulating PACs

Figure 2. Illustration of a gating strategy for enumeration of circulating PACs.

Conclusions

Acknowledgments

Abbreviations

Literature Cited

ACTIONS

PERMALINK

RESOURCES

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