Live‐Cell Immunofluorescence Staining of Human Pluripotent Stem Cells

Philip D. Manos1, Sutheera Ratanasirintrawoot2, Sabine Loewer2, George Q. Daley3, Thorsten M. Schlaeger4

1 Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, 2 Children's Hospital Boston, Boston, Massachusetts, 3 Harvard Medical School, Boston, Massachusetts, 4 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.12
DOI:  10.1002/9780470151808.sc01c12s19
Online Posting Date:  December, 2011
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Abstract

Antibodies are instrumental tools in stem cell identification, purification, and analysis. Most commonly, cell samples are either dissociated to obtain a single‐cell suspension suitable for FACS analysis or cell sorting, or fixed in situ for immunostaining and fluorescence microscopy imaging. This unit describes an alternative method in which live adherent cells are stained and imaged in situ without the need for cell dissociation, fixation, or fluorescent reporter genes. This minimally invasive method is particularly useful for identification and distinction of fully and partially reprogrammed induced pluripotent stem cells (iPSCs). The unit also describes the use of mCD49e and hCD29 antibodies in live‐cell (vital) imaging. mCD49e strongly stains mouse embryonic fibroblast (MEF) feeder cells in human pluripotent stem cell cultures, whereas hCD29 recognizes an antigen expressed on undifferentiated and many differentiated cells. A distinguishing feature of hCD29 in live‐cell staining is that its antigen is precluded from detection wherever cells have formed tight epithelial junctions (e.g., in the center but not the periphery of pluripotent stem cell colonies) due to basolateral location. A non‐fluorescent fixed‐cell staining protocol is also provided for medium‐ to high‐throughput quantification of stem cell experiments without an automated microscope. The discussion addresses technical limitations, pitfalls, troubleshooting, and potential applications, such as identification of emerging bona fide human iPSC colonies in reprogramming experiments. Curr. Protoc. Stem Cell Biol. 19:1C.12.1‐1C.12.14. © 2011 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; human pluripotent stem cells; human induced pluripotent stem cells; reprogramming; imaging; immunofluorescence staining; vital staining

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Live Immunofluorescence Staining of Adherent Pluripotent Stem Cells
  • Basic Protocol 2: Automated Fluorescence Microscopy of Live‐Stained Stem Cells
  • Basic Protocol 3: Image Processing and Analysis
  • Alternate Protocol 1: Non‐Fluorescent Chromogenic Development of Live and Fixed Cell Antibody Stainings
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Live Immunofluorescence Staining of Adherent Pluripotent Stem Cells

  Materials
  • Live hESC/hiPSC/reprogramming culture in 96‐ or 6‐well plate (e.g., Matrix, cat. no. 4940, or Nunc, cat. no. 140675)
  • hESC growth medium without phenol red, 37°C
  • DMEM/F12 without phenol red (Invitrogen, cat. no. 11039‐047), 37°C
  • Antibodies (Table 1.12.1)
  • 10× Hoechst solution (5 µg/ml; optional; see recipe)
    Table 1.1.1   Materials   Antibodies for Staining Pluripotent Stem Cells a   Antibodies for Staining Pluripotent Stem Cells   Fluorescent Dyes and Filter Sets b   Fluorescent Dyes and Filter Sets

    Agent Vendor Conjugate (cat. no.) Dilution Comment
    TRA‐1‐60 BD Alexa Fluor 555 (560121); Alexa Fluor 647 (560122) 1:100 Stringent human pluripotent stem cell marker; Alexa Fluor 647 requires suitable CCD camera (signal is poorly visible to human eye)
    β‐Tubulin BD Alexa Fluor 555 (558608); Alexa Fluor 647 (558606) 1:100 Isotype control for TRA‐1‐60 (Ms IgM, κ)
    mCD49e BD PE (557447) 1:100 Stains live MEFs; PE bleaches rapidly
    hCD13 Biolegend PE (301703) 1:100 Stains live human fibroblasts, lost during reprogramming; PE bleaches rapidly
    hCD29 Biolegend Alexa Fluor 488 (303015) 1:100 Basolaterally expressed antigen on human pluripotent stem cells (epitope not accessible to live‐cell staining in tight epithelia)
    hCD29 Biolegend Unconjugated (303001) 1:100 Sample primary antibody for use in immunostaining protocol
    hTRA‐1‐60 eBioscience Biotin (13‐8863‐82) 1:200 Sample primary antibody for use in immunostaining protocol
    Anti‐mouse IgG Santa Cruz HRP (sc‐2005) 1:500 Use as secondary reagent for detection of unconjugated primary mouse IgG antibodies (e.g., hCD29)
    Streptavidin‐HRP Biolegend HRP (405210) 1:200 Use as secondary reagent for detection of biotinylated primary antibodies
    Color Blue Green Red Far‐Red
    Dye Hoechst Alexa Fluor 488 FITC GFP Alexa Fluor 555 Cy3 PE Alexa Fluor 647 Cy5
    Excitation (nm) 377/50 ND 482/35 543/22 628/40
    Dichroic (nm) 409 506 562 660
    Emission (nm) 435/20 536/40 593/40 692/40
    Comment Hoechst and UV light are cytotoxic High autofluorescence from medium, plastic, cells PE photobleaches extremely quickly Poorly visible to the eye but detectable by most CCD cameras

     aHRP, horseradish peroxidase; MEF, mouse embryonic fibroblast; PE, phycoerythrin.
  • 1.5‐ml microcentrifuge tubes (e.g., Eppendorf Safe‐Lock, cat. no. 0030 121.589)
  • Automated inverted fluorescence microscope equipped with 10× objective and filters for excitation/detection of DAPI/Hoechst, FITC/GFP/Alexa Fluor 488, Cy3/Alexa Fluor 555, and Cy5/Alexa Fluor 647 (Table 1.12.2)
    Table 1.1.2   Materials   Antibodies for Staining Pluripotent Stem Cells a   Antibodies for Staining Pluripotent Stem Cells   Fluorescent Dyes and Filter Sets b   Fluorescent Dyes and Filter Sets

    Agent Vendor Conjugate (cat. no.) Dilution Comment
    TRA‐1‐60 BD Alexa Fluor 555 (560121); Alexa Fluor 647 (560122) 1:100 Stringent human pluripotent stem cell marker; Alexa Fluor 647 requires suitable CCD camera (signal is poorly visible to human eye)
    β‐Tubulin BD Alexa Fluor 555 (558608); Alexa Fluor 647 (558606) 1:100 Isotype control for TRA‐1‐60 (Ms IgM, κ)
    mCD49e BD PE (557447) 1:100 Stains live MEFs; PE bleaches rapidly
    hCD13 Biolegend PE (301703) 1:100 Stains live human fibroblasts, lost during reprogramming; PE bleaches rapidly
    hCD29 Biolegend Alexa Fluor 488 (303015) 1:100 Basolaterally expressed antigen on human pluripotent stem cells (epitope not accessible to live‐cell staining in tight epithelia)
    hCD29 Biolegend Unconjugated (303001) 1:100 Sample primary antibody for use in immunostaining protocol
    hTRA‐1‐60 eBioscience Biotin (13‐8863‐82) 1:200 Sample primary antibody for use in immunostaining protocol
    Anti‐mouse IgG Santa Cruz HRP (sc‐2005) 1:500 Use as secondary reagent for detection of unconjugated primary mouse IgG antibodies (e.g., hCD29)
    Streptavidin‐HRP Biolegend HRP (405210) 1:200 Use as secondary reagent for detection of biotinylated primary antibodies
    Color Blue Green Red Far‐Red
    Dye Hoechst Alexa Fluor 488 FITC GFP Alexa Fluor 555 Cy3 PE Alexa Fluor 647 Cy5
    Excitation (nm) 377/50 ND 482/35 543/22 628/40
    Dichroic (nm) 409 506 562 660
    Emission (nm) 435/20 536/40 593/40 692/40
    Comment Hoechst and UV light are cytotoxic High autofluorescence from medium, plastic, cells PE photobleaches extremely quickly Poorly visible to the eye but detectable by most CCD cameras

     bFITC, fluorescein isothiocyanate; GFP, green fluorescent protein; PE, phycoerythrin.
NOTE: All steps in which liquids are transferred or tubes or plates are opened must be performed in a suitable a Class II Biological biosafety cabinet under sterile conditions.

Basic Protocol 2: Automated Fluorescence Microscopy of Live‐Stained Stem Cells

  Materials
  • Live hESC/hiPSC culture for staining
  • Primary antibody (Table 1.12.1)
  • 16% paraformaldehyde solution (e.g., Electron Microscopy Sciences, cat. no. 15710)
  • Dulbecco's phosphate‐buffered saline with calcium and magnesium (DPBS+/+; e.g., Invitrogen, cat. no. 14040‐133)
  • Staining buffer (see recipe)
  • Appropriate secondary reagent (HRP‐labeled secondary antibody or HRP‐streptavidin, Table 1.12.1)
  • DAB peroxidase substrate kit (Vector Labs, cat. no. SK‐4100)
  • Half and half (“half cream” light cream from a grocery store)
  • Parafilm (VWR)
  • Computer‐operated flatbed scanner (e.g., Epson Perfection V500 Photo, 6400 × 9600 dpi)
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Figures

Videos

Literature Cited

Literature Cited
   Chan, E.M., Ratanasirintrawoot, S., Park, I.H., Manos, P.D., Loh, Y.H., Huo, H., Miller, J.D., Hartung, O., Rho, J., Ince, T.A., Daley, G.Q., and Schlaeger, T.M. 2009. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27:1033‐1037.
   Loewer, S., Cabili, M.N., Guttman, M., Loh, Y.H., Thomas, K., Park, I.H., Garber, M., Curran, M., Onder, T., Agarwal, S., Manos, P.D., Datta, S., Lander, E.S., Schlaeger, T.M., Daley, G.Q., and Rinn, J.L. 2010. Large intergenic non‐coding RNA‐RoR modulates reprogramming of human induced pluripotent stem cells. Nat. Genet. 42:1113‐1117.
   Lowry, W.E., Richter, L., Yachechko, R., Pyle, A.D., Tchieu, J., Sridharan, R., Clark, A.T., and Plath, K. 2008. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 105:2883‐2888.
   Park, I.H., Lerou, P.H., Zhao, R., Huo, H., and Daley, G.Q. 2008. Generation of human‐induced pluripotent stem cells. Nat. Protoc. 3:1180‐1186.
   Warren, L., Manos, P.D., Ahfeldt, T., Loh, Y.H., Li, H., Lau, F., Ebina, W., Mandal, P.K., Smith, Z.D., Meissner, A., Daley, G.Q., Brack, A.S., Collins, J.J., Cowan, C., Schlaeger, T.M., and Rossi, D.J. 2010. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7:618‐630.
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