Jamie Case

Endothelial progenitor cells: identity defined?

•  Introduction •  The proof‐of‐concept in vivo: the cell, the read‐out and the animal model ‐  The CEPC: Still a putative cell ‐  Do CEPCs play an essential role in vascular (patho)physiology? ‐  The in vivo read‐out and animal model •  EPCs defined in vitro: the achilles heel in EPC biology ‐  EOCs and EC‐like cells ‐  What are potential caveats with in vitro defined cells? ‐  The search for the EOC precursor: lessons from embryonic development ‐  Do EOCs derive from an immature CEPC? ‐  Do EOCs derive from high proliferative vessel wall ECs? ‐  CEPCs and CECs: Different cells having the same identity? •  Summary

Flow Cytometric Identification and Functional Characterization of Immature and Mature Circulating Endothelial Cells

Objective—We sought to identify and characterize 2 distinct populations of bona fide circulating endothelial cells, including the endothelial colony-forming cell (ECFC), by polychromatic flow cytometry (PFC), colony assays, immunomagnetic selection, and electron microscopy. Methods and Results—Mononuclear cells from human umbilical cord blood and peripheral blood were analyzed using our recently published PFC protocol. A population of cells containing both ECFCs and mature circulating endothelial cells was determined by varying expressions of CD34, CD31, and CD146 but not AC133 and CD45. After immunomagnetic separation, these cells failed to form hematopoietic colonies, yet clonogenic endothelial colonies with proliferative potential were obtained, thus verifying their identity as ECFCs. The frequency of ECFCs were increased in cord blood and were extremely rare in the peripheral blood of healthy adults. We also detected another mature endothelial cell population in the circulation that was apoptotic. Finally, when comparing this new protocol with a prior method, we determined that the present protocol identifies circulating endothelial cells, whereas the earlier protocol identified extracellular vesicles. Conclusion—Two populations of circulating endothelial cells, including the functionally characterized ECFC, are now identifiable in human cord blood and peripheral blood by PFC.

Oxidative Stress Impairs Endothelial Progenitor Cell Function

Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were identified in 1997. Since their original identification, EPCs have been extensively studied as biomarkers to assess the risk of cardiovascular disease in human subjects and as a potential cell therapeutic for vascular regeneration. EPCs are exposed to oxidative stress during vascular injury as residents of blood vessel walls or as circulating cells homing to sites of neovascularization. Given the links between oxidative injury, endothelial cell dysfunction, and vascular disease, recent investigation has focused on the responses of EPCs to oxidant stress and the molecular mechanisms that control redox regulation in these specialized cells. In this review, we discuss the various cell and flow-cytometric techniques used to define and isolate EPCs from circulating blood and the current human and mouse genetic data, which offer insights into redox control in EPC biology and angiogenesis. Finally, we review how EPC responses to oxidant stress may be a critical determinant in maintaining the integrity and function of the cardiovascular system and how perturbations of redox control in EPCs may lead to various human diseases.

Endothelial colony forming cells and mesenchymal stem cells are enriched at different gestational ages in human umbilical cord blood.

: Endothelial progenitor cells (EPCs) are used for angiogenic therapies and as biomarkers of cardiovascular disease. Human umbilical cord blood (UCB) is a rich source of endothelial colony forming cells (ECFCs), which are EPCs with robust proliferative potential that may be useful for clinical vascular regeneration. Previous studies show that hematopoietic progenitor cells are increased in premature UCB compared with term controls. Based on this paradigm, we hypothesized that premature UCB would be an enriched source of ECFCs. Thirty-nine UCB samples were obtained from premature infants (24–37 wk gestational age (GA)) and term controls. ECFC colonies were enumerated, clonally isolated, and identified by expression of endothelial cell surface antigens and functional analysis. GA of 33–36 wk UCB yielded predominantly ECFC colonies at equivalent numbers to term infants. UCB from 24 to 28 wk GA infants had significantly fewer ECFCs. Surprisingly, 24–28 wk GA UCB yielded predominantly mesenchymal stem cell (MSC) colonies, capable of differentiating into adipocytes, chondrocytes, and osteocytes. MSCs were rarely identified in 37–40 wk GA UCB. These studies demonstrate that circulating MSCs and ECFCs appear at different GA in the human UCB, and that 24–28 wk GA UCB may be a novel source of MSCs for therapeutic use in human diseases.

Endothelial progenitor cells and cardiovascular cell-based therapies

Since their initial discovery more than a decade ago, bone marrow (BM)-derived circulating endothelial progenitor cells (EPC) have been reported to play a role in postnatal vasculogenesis through vessel regeneration and remodeling. These cells have been reported to mobilize into the blood stream in response to vascular injury, and differentiate into cells expressing a host of endothelial cell (EC) markers in vitro. Because of demonstrable regenerative capacity in animal models of human disease, EPC are thought to represent a novel treatment option for problematic cardiovascular conditions such as myocardial infarction (MI) and peripheral vascular disease (PVD). Various studies have been performed to test the clinical efficacy of EPC in patients with cardiovascular disease (CVD), including the mobilization of EPC with pharmacologic agents in patients with heart disease, and harvesting of cells from the circulation and BM for autologous reinfusion in affected patients. The outcomes of these trials have been mixed and not as robust as predicted from the animal models, partly because of the variation in the definition of human EPC and the resulting heterogeneity in cell populations used in the treatments. This review will decipher a number of published studies that have been conducted to examine cell therapies for treatment of CVD, will attempt to explain why efficacy of treatment with putative EPC has been inconsistent, and predict which aspects of these trials may need to be redesigned for future successful treatment of CVD.

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34+CD45dim cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133+ subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133+ proangiogenic CHSPCs to AC133− nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.

Identification of Endothelial Cells and Progenitor Cell Subsets in Human Peripheral Blood

An assay for circulating cell subsets in human peripheral blood by flow cytometry is used as a biomarker to determine cardiovascular disease risk and tumor responsiveness to chemotherapy since endothelial progenitor cells (EPCs) function in vasculogenesis and angiogenesis. Despite analytical advances in polychromatic flow cytometry (PFC), conventional approaches are routinely utilized to enumerate and isolate EPCs, which has led to varied results in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function. Herein, a reproducible PFC protocol is provided to identify a rare circulating endothelial colony‐forming cell (ECFC) with proliferative potential, along with a population of circulating progenitor cells (CPCs) in which the ratio analysis distinguishes between healthy and disease populations. In sum, a reliable PFC protocol, which can be used to investigate the roles of human hematopoietic and endothelial elements in the growth and maintenance of the vasculature, is described. Curr. Protoc. Cytom. 52:9.33.1‐9.33.11.

Clonal multilineage differentiation of murine common pluripotent stem cells isolated from skeletal muscle and adipose stromal cells.

Abstract: Pluripotent stem cells (PSCs) with transdifferentiation capacity may provide useful therapeutic modalities in the areas of cellular restoration and regenerative medicine. The utility of PSCs depends on their ability to respond to different stimuli and to adapt to tissue‐specific differentiation conditions. Given that a number of cells possessing characteristics of PSCs have been identified and isolated from several adult murine tissues, we hypothesized that a common PSC may exist in multiple murine tissues and that these cells may either reside permanently in specific sites or continue to circulate and colonize tissues as needed. Previous data from our laboratory suggest that PSCs exhibiting an immunophenotype of CD45−Sca‐1+c‐kit−Thy‐1+ can be isolated from multiple murine tissues and may represent putative common PSCs (CoPSCs). To investigate whether the multiple tissue differentiation potential observed with these cells resulted from the presence of different tissue‐restricted progenitors within CD45−Sca‐1+c‐kit−Thy‐1+ cells or was the product of clonal differentiation of CoPSCs, clonality studies were performed. Single skeletal muscle (SM)‐derived CoPSCs were expanded for 10 days, and progeny cells were split into three culture conditions designed to stimulate myogenic, adipogenic, and neurogenic differentiation. Analysis of 600 clones indicated that 2.16%, 0.83%, and 0.33% of the total number of plated single cells were capable of unipotent, bipotent, and tripotent differentiation, respectively, into combinations of myocytes, adipocytes, and neuronal cells. Given that SM‐derived CoPSCs represent 4.78% of the total cells analyzed, tripotent CoPSCs made up 0.016% of the total muscle cells. Similar results were obtained in clonal analyses using adipose stromal cell (ASC)‐derived CoPSCs, suggesting that both SM‐ and ASC‐derived CoPSCs may be phenotypically and functionally identical. Taken together, these data demonstrate that a common PSC can be identified in different murine tissues and suggest that a small fraction of these cells are capable of clonal differentiation into multiple cell types.

Suppressed hindlimb perfusion in Rac2−/− and Nox2−/− mice does not result from impaired collateral growth

While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.

The ontogeny of endothelial progenitor cells through flow cytometry

Purpose of reviewSince the discovery of endothelial progenitor cells (EPCs), there have been conflicting reports as to the precise phenotypic identity, and thus an accurate description of the function of these cells in disease pathology is lacking. This review will detail the protocols that have been published within 2010 to help decipher the true identity of the various cells that have been reported as EPCs in numerous clinical trials. Recent findingsThroughout 2010, three protocols have been published alleging to identify EPCs, yet only one provides a true nonhematopoietic origin for a cell that is classified as an EPC. In addition to the protocols published to try to establish a consensus definition, 10 studies involving EPCs across disease pathologies were published with various degrees of correlation to disease phenotype and cellular level. SummaryA true phenotypic definition of a circulating EPC capable of becoming an endothelial colony forming cell with proliferative potential has been given. It is now time the EPC field drops this ambiguous term (i.e. EPCs), as many studies purporting to measure EPCs are in fact still quantifying cells of a hematopoietic origin. It is necessary for cross study comparisons that a uniform phenotypic definition be adhered to when using the term EPC.