Live‐Cell Immunofluorescence Staining of Human Pluripotent Stem Cells

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

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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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

^a HRP, 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

^b FITC, 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

Figure 1.C1.1 (A ) A grayscale flatfield image (single 256 × 256–pixel frame). (B ) 2 × 2 flatfield correction frames. (C ) A human pluripotent stem cell colony (2 × 2 montage). (D ) The same montage in (C) after flatfield correction using the flatfield correction image in (B). (E ) Pseudocolored multi‐frame montage of live‐stained and imaged human embryonic stem cells grown on mouse embryonic fibroblasts. Red, mCD49e; green, hCD29 (stains differentiated, mesenchymal human cells and incompletely epithelialized periphery of stem cell colonies); blue, TRA‐1‐60 (only expressed by undifferentiated stem cells). (F ) A 10 × 1 montage flatfield correction image (pseudocolored 3D plot; purple = brightest, yellow = darkest). Note that each frame has a unique shape and height due to positional effects (e.g., different optical properties across the culture dish, well walls, etc.).

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Figure 1.C1.2 (A ) Brightfield image of two human pluripotent stem cell colonies grown on mouse embryonic fibroblast feeder cells. (B ‐C ) The same colonies are shown after live‐cell staining with hCD29 (green; B) and post‐fixation staining with SSEA4 (C). Arrowhead in (A, B) denotes human mesenchymal cells that differentiated and migrated away from the nearby human pluripotent stem cell colony. Note that these CD29+ cells (B) cannot be easily identified using phase contrast alone (A). (D ) In the merge of (B) and (C), live‐cell staining allows human cells to be detected among mouse embryonic fibroblasts, which would be difficult, if not impossible, using standard brightfield microscopy. (E ) HRP‐based chromogenic stain (hCD29, dark‐purple precipitate; stained post‐fixation) of human pluripotent stem cell colonies. (F ) HRP‐based stain (hCD29, dark purple precipitate; live‐staining followed by fixation) of human pluripotent stem cell colonies. (E) and (F) were counterstained using a standard alkaline phosphatase staining kit to reveal expression of the pluripotent stem cell marker APLP. Note that while hCD29 is expressed by all human pluripotent stem cells throughout each colony (E), this antibody fails to stain live cells in the epithelialized centers of undifferentiated human pluripotent stem cell colonies (F).

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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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Live‐Cell Immunofluorescence Staining of Human Pluripotent Stem Cells

Abstract

Table of Contents

Materials

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

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

Figures

Videos

Literature Cited