Isolation and Characterization of Endothelial Progenitor Cells from Human Blood

Laura E. Mead1, Daniel Prater1, Mervin C. Yoder2, David A. Ingram2

1 Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, 2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2C.1
DOI:  10.1002/9780470151808.sc02c01s6
Online Posting Date:  July, 2008
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Abstract

Circulating endothelial progenitor cells (EPCs) in adult human peripheral blood were originally identified in 1997 by Asahara et al., which challenged the paradigm that vasculogenesis is a process restricted to embryonic development. 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. Endothelial colony‐forming cells (ECFCs), which are a subtype of EPCs, were recently identified from circulating adult and human umbilical cord blood. In contrast to other types of EPCs, which display various monocyte/macrophage phenotypes and functions, ECFCs are characterized by robust proliferative potential, secondary and tertiary colony formation upon replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In this unit, we describe detailed methodologies for isolation and characterization of ECFCs from both human peripheral and umbilical cord blood. Curr. Protoc. Stem Cell Biol. 6:2C.1.1‐2C.1.27. © 2008 by John Wiley & Sons, Inc.

Keywords: endothelial progenitor cell (EPC); adult progenitor cells; endothelial cell transplantation; endothelial colony‐forming cells (ECFC)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation, Cloning, and Propagation of Endothelial Colony‐Forming Cells from Human Umbilical Cord Blood
  • Alternate Protocol 1: Isolation, Cloning, and Propagation of Endothelial Colony‐Forming Cells from Human Peripheral Blood
  • Basic Protocol 2: Phenotypic Characterization of ECFCs: Endothelial‐Specific Cell Surface Antigen Expression
  • Alternate Protocol 2: Phenotypic Characterization of ECFCs: Uptake of AcLDL
  • Alternate Protocol 3: Phenotypic Characterization of ECFCs: Matrigel Lattice Formation
  • Basic Protocol 3: Transplantation of ECFCs into Mice
  • Alternate Protocol 4: Transplant of Mixed Cell Implants into Mice
  • Support Protocol 1: Preparation of Collagen‐Coated Tissue Culture Surfaces
  • Support Protocol 2: Preparation of Cloning Cylinders
  • Support Protocol 3: Cryopreservation of ECFCs
  • Support Protocol 4: Thawing Cryopreserved ECFCs
  • Support Protocol 5: Coating 96‐Well Plates with Matrigel
  • Support Protocol 6: ADSC Culture
  • Support Protocol 7: CD31 Immunohistochemical Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation, Cloning, and Propagation of Endothelial Colony‐Forming Cells from Human Umbilical Cord Blood

  Materials
  • Anticoagulated UCB (e.g., the authors typically use 10 U heparin/ml blood)
  • Phosphate‐buffered saline (PBS), without calcium and magnesium, pH 7.2
  • Ficoll‐Paque PLUS (Ficoll, Amersham Biosciences, cat. no. 17‐1440‐03)
  • EBM‐2 10:1 (see recipe; Lonza)
  • 0.4% (w/v) trypan blue solution (Sigma, cat. no. T8154)
  • cEGM‐2 (see recipe; Lonza)
  • 15‐ml conical centrifuge tubes, sterile
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054), warm
  • 50‐ml conical centrifuge tubes, sterile
  • Assorted pipets, sterile
  • 20‐ml syringe, sterile
  • Mixing cannula (Maersk Medical, cat. no. 500.11.012)
  • Centrifuge
  • Transfer pipet, sterile
  • Hemacytometer
  • Collagen I‐coated 6‐well plates (BD Biosciences Discovery Labware, cat. no. 356400)
  • Inverted microscope
  • Fine‐tipped marker
  • Prepared cloning cylinders (see protocol 9)
  • Forceps, sterile
  • Pasteur pipets (Fisher Scientific, cat. no. 13‐678‐20C), sterilized
  • 1.5‐ and 2.0‐ml microcentrifuge tubes
  • Collagen I coated 24‐well tissue culture plate (see protocol 8)
  • 25‐ and 75‐cm2 vented tissue culture flasks (BD Biosciences Falcon), coated with rat tail collagen I (see protocol 8)
  • Additional reagents and equipment for counting viable cells using a hemacytometer and trypan blue exclusion (unit 1.3)

Alternate Protocol 1: Isolation, Cloning, and Propagation of Endothelial Colony‐Forming Cells from Human Peripheral Blood

  Materials
  • ECFC cultures grown in 25‐ or 75‐cm2 flasks ( protocol 1)
  • Trypsin‐EDTA (Invitrogen, cat. no. 25300‐054)
  • Staining buffer (see recipe), ice cold
  • Phosphate‐buffered saline (PBS), without calcium and magnesium
  • 0.4% (w/v) trypan blue solution
  • Fc Block (Miltenyi, cat. no. 130‐059‐901)
  • hCD31 antibody, FITC conjugated (BD Pharmingen, cat. no. 555445)
  • hCD45 antibody, FITC conjugated (BD Pharmingen, cat. no. 555482)
  • hCD14 antibody, FITC conjugated (BD Pharmingen, cat. no. 555397)
  • hCD144 antibody, PE conjugated (eBioscience, cat. no. 12‐1449‐80)
  • hCD146 antibody, PE conjugated (BD Pharmingen, cat. no. 550315)
  • hCD105 antibody, PE conjugated (Invitrogen, cat. no. MHCD10504)
  • Ms IgG 1,κ antibody, FITC conjugated (BD Pharmingen, cat. no. 555748)
  • Ms IgG 1,κ antibody, PE (BD Pharmingen, cat. no. 559320)
  • Ms IgG 2a,κ FITC conjugated (BD Pharmingen, cat. no. 555573)
  • Fixing buffer (see recipe), ice cold
  • 15‐ or 50‐ml conical centrifuge tubes, sterile
  • Hemacytometer
  • 12 × 75–mm polystyrene round bottom tubes (BD Falcon, cat. no. 352008)
  • FACSCalibur flow cytometer (Becton Dickinson) or equivalent
  • Flow cytometry analysis software (e.g., Cellquest; Becton Dickinson or FlowJo; Tree Star)
  • Additional reagents and equipment for detaching cells with tryspin/EDTA ( protocol 1) and counting viable cells using a hemacytometer and trypan blue exclusion (unit 1.3)

Basic Protocol 2: Phenotypic Characterization of ECFCs: Endothelial‐Specific Cell Surface Antigen Expression

  • Collagen I coated 24‐well tissue culture plate (see protocol 8)
  • DiI‐complexed acetylated low density lipoprotein from plasma (DiI AcLDL; Invitrogen, cat. no. L‐3484)
  • EBM‐2 10:1 (see recipe)
  • Inverted fluorescence microscope with a rhodamine filter
  • Additional reagents and equipment for preparing a culture of ECFCs ( protocol 1)

Alternate Protocol 2: Phenotypic Characterization of ECFCs: Uptake of AcLDL

  • cEGM‐2 (see recipe)
  • Additional reagents and equipment for preparing a Matrigel‐coated tissue culture plate ( protocol 12) and obtaining a viable cell count using a hemacytometer and trypan blue exclusion (unit 1.3)

Alternate Protocol 3: Phenotypic Characterization of ECFCs: Matrigel Lattice Formation

  Materials
  • Fetal bovine serum (FBS; Hyclone, cat. no. SH30070.03)
  • EBM‐2 10:1 (see recipe), ice cold
  • 7.5% (w/v) sodium bicarbonate (Sigma, cat. no. S8761), sterile and ice cold
  • 1 N NaOH, sterile and ice cold
  • 1 M HEPES (Lonza, cat. no. 17‐737E), ice cold
  • 1 mg/ml fibronectin (Millipore, cat. no. FC10‐10MG), ice cold
  • Rat tail collagen type I, (BD Biosciences Discovery Labware, cat. no. 354236), ice cold
  • cEGM‐2 (see recipe), warm
  • ECFC cultures grown in 25‐ or 75‐cm2 flasks ( protocol 1)
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • Phosphate‐buffered saline (PBS), without calcium and magnesium
  • 0.4% (w/v) trypan blue solution (Sigma, cat. no. T8154)
  • Immunodeficient mice, 8‐ to 12‐weeks‐old (see )
  • Isoflurane inhalant
  • Alcohol pads or 70% ethanol
  • Zinc fixative (BD Biosciences, cat no. 550523)
  • 37°C water bath
  • Hemacytometer
  • 15‐ or 50‐ml conical centrifuge tubes, sterile
  • Micropipettor with a 1‐ml tip
  • 12‐well tissue culture plate
  • Thin surgical spatula (e.g., FST, cat. no. 10091‐12), sterile
  • Fine iris scissors, sterile
  • Electric shears
  • Smooth forceps (2), sterile
  • Sharp iris scissors, sterile
  • Blunt‐end iris scissors
  • Light microscope with an eyepiece micrometer
  • 5‐0 polypropylene suture on a cutting needle
  • Glass slides
  • Additional reagents and equipment for obtaining a viable cell count using a hemacytometer and trypan blue exclusion (unit 1.3), isoflurane anesthesia (unit 1.4), euthanizing the mouse (Donovan and Brown, ), paraffin‐embedding the gel (Bancroft and Gamble, ), and staining with hematoxylin and eosin (Bancroft and Gamble, ) or anti–human CD31 or anti–mouse CD31 ( protocol 14) to visualize the vasculature within the gel

Basic Protocol 3: Transplantation of ECFCs into Mice

  • Adipose‐derived stem cells (ADSC; Lonza, cat. no. PT‐5006), grown in 25‐ or 75‐cm2 flasks (see protocol 13)

Alternate Protocol 4: Transplant of Mixed Cell Implants into Mice

  Materials
  • Collagen I solution (see recipe)
  • Phosphate‐buffered saline (PBS), without calcium and magnesium
  • Tissue culture–treated plates or flasks
  • Pipets, sterile
  • Pasteur pipets, sterile
  • 37°C incubator

Support Protocol 1: Preparation of Collagen‐Coated Tissue Culture Surfaces

  Materials
  • Vacuum grease (Dow Corning, cat. no. 1658832)
  • Glass dish
  • Forceps, sterile
  • Cloning cylinders, sterile (Fisher Scientific, cat. no. 07‐907‐10)
  • 10‐cm petri dish, sterile

Support Protocol 2: Preparation of Cloning Cylinders

  Materials
  • ECFC cultures grown in 25‐ or 75‐cm2 flasks ( protocol 1)
  • Trypsin/EDTA (Invitrogen, cat. no. 25300‐054)
  • EBM‐2 10:1 (see recipe)
  • 0.4% (w/v) trypan blue solution (Sigma, cat. no. T8154)
  • Freezing medium (see recipe), ice cold
  • Phosphate‐buffered saline (PBS), without calcium and magnesium
  • 15‐ or 50‐ml conical centrifuge tubes, sterile
  • Hemacytometer
  • Cryovials
  • Cryogenic‐controlled rate freezing container (Nalgene) or insulated polystyrene foam box
  • Liquid nitrogen storage container
  • Additional reagents and equipment for performing a viable cell count using a hemacytometer and trypan blue exclusion (unit 1.3)

Support Protocol 3: Cryopreservation of ECFCs

  Materials
  • Cryopreserved ECFCs in cryovials ( protocol 10)
  • cEGM‐2 (see recipe)
  • 0.4% (w/v) trypan blue solution (Sigma, cat. no. T8154)
  • 37°C water bath
  • 15‐ or 50‐ml conical centrifuge tubes, sterile
  • Hemacytometer
  • 25‐ and 75‐cm2 vented tissue culture flasks (BD Falcon), coated with rat tail collagen I (see protocol 8)
  • Additional reagents and equipment for obtaining a viable cell count using a hemacytometer and trypan blue exclusion (unit 1.3)

Support Protocol 4: Thawing Cryopreserved ECFCs

  Materials
  • Matrigel (BD Biosciences, cat. no. 356234)
  • Pipet tips
  • Pipettor
  • 96‐well, flat‐bottomed tissue culture plate

Support Protocol 5: Coating 96‐Well Plates with Matrigel

  Materials
  • Adipose‐derived stem cells (ADSC; Lonza, cat. no. PT‐5006)
  • ADSC‐GM (see recipe; Lonza)
  • Trypsin/EDTA
  • 0.4% (w/v) trypan blue solution (Sigma, cat. no. T8154)
  • 25‐ or 75‐cm2 tissue culture–treated flasks
  • Hemacytometer
  • Assorted pipets
  • 15‐ or 50‐ml conical centrifuge tubes, sterile
  • Additional reagents and equipment for obtaining a viable cell count using a hemacytometer and trypan blue exclusion (unit 1.3)

Support Protocol 6: ADSC Culture

  Materials
  • Zinc‐fixed, paraffin‐embedded 5‐µm tissue sections on glass slides
  • Xylenes
  • Ethyl alcohol
  • Phosphate‐buffered saline (PBS) with calcium and magnesium
  • Retrieval solution (Dako, cat. no. S236984)
  • Blocking solution/diluent (Vector Labs, cat. no. SP‐5050)
  • Anti–mouse CD31 (clone mec13.3, available from various suppliers)
  • Anti–human CD31 (clone JC70/A, Dako)
  • Universal LSAB2 link‐biotin kit (Dako, cat. no. K0675)
  • DAB solution (Dako, cat. no. K3467)
  • Coplin jars
  • Additional reagents and equipment for deparaffinizing and hydrating tissue sections through a series of xylenes and serial alcohol dilutions (Bancroft and Gamble, )
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Figures

Videos

Literature Cited

Literature Cited
   Asahara, T., Murohara, T., Sullivan A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schattman G., and Isner J.M. 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964‐967.
   Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C., Silver, M., Kearne, M., Magner, M., and Isner, J.M. 1999. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ. Res. 85:221‐228.
   Bancroft, J.D. and Gamble, M. 2002. Theory and Practice of Histological Techniques. 5th Ed.. Churchill Livingstone, New York.
   Baumgarth, N. and Roederer, M. 2000. A practical approach to multicolor flow cytometry for immunophenotyping. J. Immunol. Methods 243:77‐97.
   Bompais, H., Chagraoui, J., Canron, X., Crisen, M., Liu, X.H., Anjo, A., Tolla‐LePort, C., Leboef, M., Charbord, P., Bikfalvi, A., and Uzan, G. 2004. Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells. Blood 103:2577‐2584.
   Dimmeler, S., Aicher, A., Vasa, M., Mildner‐Rihm, C., Adler, K., Tiemann, M., Rutten, H., Fichtlscherer, S., Martin, H., and Zeiher, M. 2001. HMG‐CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3‐kinase/Akt pathway. J. Clin. Invest. 108:391‐397.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Enis, D.R., Shepherd, B.R., Wang, R., Qasim, A., Shanahan, C.M., Weissberg, P.L., Kashgarian, M., Pober, J.S., and Schechner, J.S. 2005. Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl‐2‐transduced endothelial cells. Proc. Natl. Acad. Sci. U.S.A 102:425‐430.
   Gulati, R., Jevremovic, D., Peterson, T.E., Chaterjee, S., Shah, V., Vile, R.G., and Simon, R.D. 2003. Diverse origin and function of cells with endothelial phenotype obtained from adult human blood. Circ. Res. 93:1023‐1025.
   Hassan, N.F., Campbell, D.E., and Douglas, S.D. 1986. Purification of human monocytes on gelatin‐coated surfaces. J. Immunol. Methods 95:273‐276.
   Hill, J.M., Zalos, G., Halcox, J.P., Schenke, W.H., Waclawiw, M.A., Quyyumi, A.A., and Finkel, T. 2003. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 348:593‐600.
   Hur, J., Yoon, C.H., Kim, H.S., Choi, J.H., Kang, H.J., Hwang, K.K., Oh, B.H., Lee, M.M., and Park, Y.B. 2004. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler. Thromb. Vasc. Biol. 24:288‐293.
   Ingram, D.A., Mead, L.E., Tanaka, H., Meade, V., Fenoglio, A., Mortell, K., Pollok, K., Ferkowicz, M.J., Gilley, D., and Yoder, M.C. 2004. Identification of a novel hierarchy of endothelial progenitor cells utilizing human peripheral and umbilical cord blood. Blood 104:2752‐2760.
   Ito, H., Rovira, I.I., Bloom, M.L., Takeda, K., Ferrans, V.J., Quyyumi, A.A., and Finkel, T. 1999. Endothelial progenitor cells as putative targets for angiostatin. Cancer Res. 59:5875‐5877.
   Kalka, C., Masuda, H., Takahashi, T., Kalka‐Moll, W.M., Silver, M., Kearney, M., Li, T., Isner, J.M., and Asahara, T. 2000. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. U.S.A. 97:3422‐3427.
   Lin, Y., Weisdorf, D.J., Solovey, A., and Hebbel, R.P. 2000. Origins of circulating endothelial cells and endothelial outgrowth from blood. J. Clin. Invest. 105:71‐77.
   Melero‐Martin, J.M., Khan, Z.A., Picard, A., Wu, X., Paruchuri, S., and Bischoff, J. 2007. In vivo vasculogenic potential of human blood‐derived endothelial progenitor cells. Blood 109:4761‐4768.
   Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P., and Roederer, M. 2006. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1:1522‐1530.
   Prater, D.N., Case, J., Ingram, D.A., and Yoder, M.C. 2007. Working hypothesis to redefine endothelial progenitor cells. Leukemia 21:1141‐1149.
   Rafii, S. and Lyden, D. 2003. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat. Med. 9:702‐712.
   Rehman, J., Li, J., Orschell, C.M., and March, K.L. 2003. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors.” Circulation 107:1164‐1169.
   Schechner, J.S., Nath, A.K., Zheng, L., Kluger, M.S., Hughes, C.C., Sierra‐Honigmann, M.R., Lorber, M.I., Tellides, G., Kashgarian, M., Bothwell, A.L., and Pober, J.S. 2000. In vivo formation of complex microvessels lined by human endothelial cells in an mmunodeficient mouse. Proc. Natl. Acad. Sci. U.S.A. 97:9191‐9196.
   Schmeisser, A., Graffy, C., Daniel, W.G., and Strasser, R.H. 2003. Phenotypic overlap between monocytes and vascular endothelial cells. Adv. Exp. Med. Biol. 522:59‐74.
   Shapiro, H.M. 2003. Practical Flow Cytometry. 4th Edition. Wiley‐Liss, Wilmington, Del.
   Shepherd, B.R., Enis, D.R., Wang, F., Suarez, Y., Pober, J.S., and Schechner, J.S. 2006. Vascularization and engraftment of a human skin substitute using circulating progenitor cell‐derived endothelial cells. Faseb J. 20:1739‐1741.
   Werner, N., Kosiol, S., Schiegl, T., Ahlers, P., Walenta, K., Link, A., Böhm, M., and Nickenig, G. 2005. Circulating endothelial progenitor cells and cardiovascular outcomes. N. Engl. J. Med. 353:999‐1007.
   Yoder, M.C., Mead, L.E., Prater, D., Krier, T.R., Mroueh, K.N., Li, F., Krasich, R., Temm, C.J., Prchal, J.T., and Ingram, D.A. 2007. Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 109:1801‐1809.
   Ziegelhoeffer, T., Fernandez, B., Kostin, S., Heil, M., Voswinckel, R., Helisch, A., Kostin S, Heil M, Voswinckel R., and Helisch, A. 2004. Bone marrow‐derived cells do not incorporate into the adult growing vasculature. Circ. Res. 94:230‐238.
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