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

Myka L. Estes^1,2, Julie A. Mund^1,2, David A. Ingram^3,1,2, Jamie Case^1,2

¹Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana
²Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana
³Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana

Publication Name:

Current Protocols in Cytometry

Unit Number:

Unit 9.33

DOI:

10.1002/0471142956.cy0933s52

Online Posting Date:

April, 2010

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Abstract

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. © 2010 by John Wiley & Sons, Inc.

Keywords: endothelial cells; circulating endothelial cells; endothelial progenitor cells; circulating endothelial progenitors; human peripheral blood

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Introduction
Strategic Planning
Basic Protocol: Identification of Endothelial Colony Forming Cells and Circulating Progenitor Cell Subsets in Human Peripheral Blood by Polychromatic Flow Cytometry
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Identification of Endothelial Colony Forming Cells and Circulating Progenitor Cell Subsets in Human Peripheral Blood by Polychromatic Flow Cytometry

Materials

Peripheral blood (PB)
EDTA collection tubes (Vacutainer; BD, cat. no. 366450)
Phosphate buffered saline (PBS; Invitrogen, cat. no. 20012-027)
Ficoll-Paque Plus (GE Healthcare, cat. no. 17-1440-03)
Fetal bovine serum (FBS; see recipe)
FcR-blocking agent (Miltenyi Biotec, cat. no. 130-059-901)
CD34-PE (BD Biosciences, cat. no. 550761)
AC133-APC (Miltenyi Biotec, cat. no. 130-090-826)
CD14-PE-Cy5.5 (abCAM, cat. no. ab25395)
CD45-APC-AF750 (Invitrogen, cat. no. MHCD4527)
CD31-FITC (BD Biosciences, cat. no. 555445)
CD235a (glyA; R&D Systems, cat. no. MAB1228)
Pacific blue monoclonal antibody labeling kit (Invitrogen, cat. no. P30013)
CD41a (Invitrogen, cat. no. MHCD4100)
LIVE/DEAD fixable violet dead cell stain kit (Invitrogen, cat. no. L34955)
FACS formaldehyde (see recipe), optional
BD CompBeads (BD Biosciences, cat. no. 552843)
Amine polymer microspheres (Bangs Lab, cat. no. PA04N)

Quantum simply cellular beads (Bangs Lab, cat. no. 816) or equivalent
15- and 50-ml conical centrifuge tubes (BD Falcon, cat. no. 352097 and 352098, respectively)
10- and 30-ml disposable syringes (BD Falcon, cat. no. 309604 and 309650, respectively)
Mixing cannulas (UNO Medical, cat. no. 500.11.012)
14 × 5–ml round-bottom polystyrene tubes (BD Falcon, cat. no. 352008)
Aluminum foil
40-µm cell strainers (BD Falcon, cat. no. 352340)
LSRII flow cytometer with 405-nm, 488-nm, and 633-nm laser configurations (BD)
Flowjo software (Treestar)

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Figures

Figure 9.33.1

FMO gating controls for analysis of circulating progenitor cell populations. Shown are the corresponding FMO gating controls necessary for the frequency analysis of CD34^brightCD45^–CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– cells in PB (ECFCs, b and e), CD31⁺CD34^brightCD45^dimAC133⁺CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– (pro-angiogenic progenitor, (A-E) and CD31⁺CD34^brightCD45^dimAC133^–CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– (non-angiogenic progenitors, (A-E). Each panel shows MNCs stained with all reagents in the eight-antibody/viability marker panel except for one: (A) lacks AC133 APC, (B) lacks CD45 APC-AF750, (C) lacks CD31 FITC, and (D-E) lacks CD34 PE. Black gates in A, B, and D-E delineate the positive staining threshold for APC, APC-AF750, and PE, respectively. The green gates show the ECFC FMO gating strategy. The pink marker gates in B and E are the parent gates for the hematopoietic progenitor cell populations. Blue gates in A and D are the actual gates used for frequency analysis of the two hematopoietic progenitor cell populations (proangiogenic and nonangiogenic progenitors) when MNCs are stained with the full eight-antibody/viability marker panel (as seen in Fig. 9.33.3A-F). The black marker in (C) delineates events that are positive for FITC staining.

View Image
Figure 9.33.2

Logarithmic scaling does not allow the visualization of CD45 negative populations. Representative PFC analysis of a MNC preparation stained with the eight-antibody/viability marker panel. Logarithmic scaling (A) prevents the visualization of populations with negative or dim fluorescence values, which are compressed against the axes (as indicated by the arrows). Conversion to bi-exponential (B) scaling allows for the visualization of all events and reveals the previously undetectable ECFC population (gated in green).

View Image
Figure 9.33.3

Representative PFC analysis of PB MNCs co-stained with the eight-antibody/viability marker panel. In (A), CD14⁺ monocytes (yellow bar), contained within the initial MNC gate, were excluded. All CD14^– cells are then assessed for viability, CD235a, and CD41a expression (excluded in purple gate) (B). CD14^–CD235a^–CD41a^–LIVE/DEAD Violet^– cells (black events in B and C) are sub-gated onto a bi-variant antigen plot to identify CD34^brightCD45^–CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– cells (ECFCs, green gate in C) and CD34^brightCD45^dimCD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– (pink gate in C). CD34^brightCD45^–CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– events are further assessed as CD31⁺ (green histogram in E). CD34^brightCD45^dimCD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– events are further discriminated based upon AC133 expression to identify CD31⁺CD34^brightCD45^dimAC133⁺CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– (pro-angiogenic progenitors, blue gate in D) and CD31⁺CD34^brightCD45^dimAC133^–CD14^–CD41a^–CD235a^– LIVE/DEAD Violet^– (non-angiogenic progenitors, red gate in D). Both proangiogenic and nonangiogenic progenitors express CD31 uniformly (green histogram in F). FMO gating controls are used to set gate boundaries (Fig. 9.33.1).

View Image

Literature Cited

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