Preparation of Defined Human Embryonic Stem Cell Populations for Transcriptional Profiling

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a useful approach to preparing highly reproducible samples of human embryonic stem cell (hESC) total RNA suitable for transcriptional profiling from heterogeneous mixtures of cells containing undifferentiated hESC and differentiated cell types. In this unit, fluorescence?activated cell sorting (FACS) is used to sub?fractionate hESC populations on the basis of their levels of co?expression of two previously published hESC surface markers, CD9(TG30) and GCTM?2. This sub?fractionation allows for the separation of undifferentiated hESC (CD9hi, GCTM?2hi) from the early stages in hESC differentiation (CD9neg or low, GCTM?2neg or low). Curr. Protoc. Stem Cell Biol. 14:1B.7.1?1B.7.15. © 2010 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; cell surface markers; fluorescence?activated cell sorting

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Introduction
Basic Protocol 1: Enzymatic Passaging of Cultures of hESCs
Alternate Protocol 1: Starting hESC Bulk Culture from Maintenance Culture
Basic Protocol 2: Harvesting hESCs from Bulk Cultures
Basic Protocol 3: Immunofluorescent Staining of Surface Antigens on Live hESCs for FACS
Alternate Protocol 2: Combined Detection of OCT‐4 Intracellular Expression and Other Cell Surface Markers on Fixed hESC by Flow Cytometry
Basic Protocol 4: RNA Isolation
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Enzymatic Passaging of Cultures of hESCs

Materials

hESCs in culture
Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
Collagenase I solution (4 mg/ml in DMEM/F‐12)
Knock‐out serum replacement (KSR) medium (see recipe )
Mouse embryonic fibroblast (MEF) feeder cells (Pera et al., ; unit 1.3 ) plated on gelatinized flasks at 1.2 × 10⁴ cells/cm²

Inverted microscope
Dissecting microscope
37°C incubator
10‐ml pipet
Cell scraper, optional
15‐ml tubes
Centrifuge

Alternate Protocol 1: Starting hESC Bulk Culture from Maintenance Culture

Materials

Mechanically passaged hESCs in organ culture dishes
PBS with Ca⁺⁺ and Mg⁺⁺ (PBS⁺ )
Dispase solution (10 mg/ml in hESC medium; see recipe ), ice cold
Gelatinized 25‐cm² flasks seeded with 1.2 × 10⁴ cells/cm² inactivated MEFs (unit 1.3 )
Knock‐out serum replacement (KSR) medium (see recipe )

Dissecting microscope with warm stage
4‐well culture dish (Nunc, cat. no. 176740)
Pulled glass capillary cutter or a 26‐G needle attached to a 3‐ml syringe or a sterilized ultra sharp splitting blade
Micropipet tips
20‐µl or 200‐µl micropipet tip
1.5‐ml microcentrifuge tubes

Basic Protocol 2: Harvesting hESCs from Bulk Cultures

Materials

hESCs in bulk cultures ( protocol 1 )
Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS)
TrypLE Express (Invitrogen, cat. no. 12604)
20% (v/v) fetal bovine serum (FBS; Invitrogen, cat. no. 16000‐044) in DMEM/F12 medium (chilled to 4°C)

37°C incubator
35‐µm cell strainer (BD Biosciences, cat. no. 352235)
Centrifuge

Additional reagents and equipment for counting cells using a hemacytometer (unit 1.3 )

Basic Protocol 3: Immunofluorescent Staining of Surface Antigens on Live hESCs for FACS

Materials

Harvested bulk hESCs ( protocol 3 )
Mouse anti‐CD9 (TG30) monoclonal antibody (Millipore, cat. no. MAB4427)
Mouse anti‐GCTM2 monoclonal antibody (0.4 mg/ml; kind gift from Martin Pera)—a commercially available alternative, which detects the same antigen is the antibody TG343 (Millipore, cat. no. MAB4346)
PE rat anti‐mouse CD 90.2 (0.2 mg/ml; BD Pharmingen, cat. no. 553014)
Mouse IgG2a isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 554121)
Mouse IgM isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 553472)
20% (v/v) fetal bovine serum (FBS) in DMEM/F12 medium (chilled to 4°C)
Alexa Fluor 488 goat anti–mouse IgG2a (2 mg/ml; Invitrogen, cat. no. A21131)
Alexa Fluor 647 goat anti–mouse IgM (2 mg/ml; Invitrogen, cat. no. A21238)
R‐phycoerythrin goat anti–mouse IgG2a conjugate (1.0 mg/ml; Invitrogen, cat. no. P21139)
Propidium iodide solution (1.0 mg/ml; Sigma, cat. no. P4864)
Spherotech 8 peak Ultra Rainbow beads

Centrifuge
35‐µm cell strainer (BD Biosciences, cat. no. 352235)
FACSVantage DiVa (Becton Dickinson) or equivalent

NOTE: Both primary and secondary antibody staining and washing are carried out in 20% FBS medium prechilled to 4°C. For 1.5 × 10⁷ cells (recommended cell number for test stainings; fewer cells can be used in control tubes, e.g., 50,000 cells/tube); 10 ml and 2 ml medium are used for primary and secondary antibody staining respectively.

Alternate Protocol 2: Combined Detection of OCT‐4 Intracellular Expression and Other Cell Surface Markers on Fixed hESC by Flow Cytometry

Materials

Harvested hESCs ( protocol 3 )
2% (w/v) paraformaldehyde (PFA) solution in CMF‐PBS, pH 7.4
10% (v/v) goat serum in CMF‐PBS
Phosphate‐buffered serum, calcium‐ and magnesium‐free (CMF‐PBS)
0.1% (v/v) Triton X‐100 solution in CMF‐PBS
Mouse anti‐Oct4 monoclonal antibody(1 mg/ml; Chemicon, cat. no. MAB4401)
R‐phycoerythrin goat anti‐mouse IgG1 conjugate (1 mg/ml; Invitrogen, cat. no. P21129)
Mouse IgG1 isotype control (0.5 mg/ml; BD Pharmingen, cat. no. 553447)

Centrifuge
35‐µm cell strainer (BD Biosciences, cat. no. 352235)

Basic Protocol 4: RNA Isolation

Materials

hESC bulk cultures ( protocol 3 )
RNeasy Mini kit (Qiagen, cat. no. 74104)
RNase‐free H₂ O

Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific)
RNase‐free tubes (PCR tubes)

NOTE: Keep all reagents, working solutions, and master mixes on ice during these procedures.

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Figures

Figure 1.B0.1 Bright‐field image of a day 7 hESC colony cultured on mouse embryonic fibroblasts that is ready to be passaged by treatment with collagenase (50×). Note the compact appearance of undifferentiated hESC in the center of the colony, surrounded by a ring of differentiated cells.

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Figure 1.B0.2 Dark‐field image showing a day 7 colony prior to being transferred into bulk passage. The colony has been cut into six pieces. The largest piece has differentiated and the other five pieces are “good” pieces, with minimal visible differentiation, to be transferred into bulk culture.

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Figure 1.B0.3 Typical sorting strategy for hESCs. Sorted cells were initially gated using forward and side scatter, followed by the removal of clumps and doublets by gating on single cells (FSC‐A vs. FSC‐H), nonviable cells were excluded based on propidium iodide (PI) fluorescence and, penultimately, MEF feeder cells were removed using negative selection for Thy1.2‐PE. hESC were then separated by FACS on the basis of cell surface intensity of GCTM‐2 and TG30 (CD9) into four populations: P4 (GCTM‐2^neg CD9^neg ), P5 (GCTM‐2^low CD9^low ), P6 (GCTM‐2^mid CD9^mid ), and P7 (GCTM‐2^high CD9^high ). The P4 gate is set relative to isotype controls and the P5‐P7 gates are reproducibly set based on initial experiments, using the strategy outlined in Figure .

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Figure 1.B0.4 The template used to set up the FACS DIVA prior to each FACS experiment. Spherotech beads were initially gated using forward and side scatter to exclude debris. The FACS DIVA was then calibrated so that beads of known fluorescence were always gated into the same preset gates (P2 for FL1, P3 for FL2, and P4 for FL8). This ensures that the FACS DIVA is identically calibrated for each separate experiment.

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Figure 1.B0.5 Data from a representative flow cytometry analysis of hESC using TG343, CD9, and OCT4. hESC were initially gated using forward and side scatter, followed by the removal of clumps and doublets by gating on single cells (FSC‐A vs. FSC‐H), nonviable cells were excluded based on propidium iodide (PI) fluorescence and, penultimately, MEF feeder cells were removed using negative selection for Thy1.2‐PE (not shown). hESC were then analyzed using the FACS DIVA for immunoreactivity to TG343, CD9, and OCT4. TG343hi TG30hi cells were 90% positive for OCT‐4, TG343medium TG30medium cells were 70% positive for OCT‐4, and TG343negative/low TG30negative/low cells were 22% positive for OCT4.

View Image

Videos

Literature Cited

Literature Cited
	Boyer, L.A., Lee, T.I., Cole, M.F., Johnstone, S.E., Levine, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., Gifford, D.K., Melton, D.A., Jaenisch, R., and Young, R.A. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947‐956.
	Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., and Smith, A. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643‐655.
	Filipczyk, A.A., Laslett, A.L., Mummery, C., and Pera, M.F. 2007. Differentiation is coupled to changes in the cell cycle regulatory apparatus of human embryonic stem cells. Stem Cell Res. 1:45‐60.
	Hough, S.R., Laslett, A.L., Grimmond, S.B., Kolle, G., and Pera, M.F. 2009. Metastable states of pluripotency in human embryonic stem cells. PLOS ONE 4:e7708.
	Kolle, G., Ho, M., Zhou, Q., Chy, H.S., Krishnan, K., Cloonan, N., Bertoncello, I., Laslett, A.L., and Grimmond, S.M. 2009. Identification of human embryonic stem cell surface markers by combined membrane‐polysome translation state array analysis and immunotranscriptional profiling. Stem Cells 27:2446‐2456.
	Laslett, A.L., Filipczyk, A., and Pera, M.F. 2003. Characterization and culture of human embryonic stem cells. Trends Cardiovasc. Med. 13:295‐301.
	Laslett, A.L., Grimmond, S., Gardiner, B., Stamp, L., Lin, A., Hawes, S.M., Wormald, S., Nikolic‐Paterson, D., Haylock, D., and Pera, M.F. 2007. Transcriptional analysis of early lineage commitment in human embryonic stem cells. BMC Dev. Biol. 7:12.
	Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M., and Yamanaka, S. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631‐642.
	Pera, M.F., Filipczyk, A., Hawes, S.M., and Laslett, A.L. 2003. Isolation, characterization, and differentiation of human embryonic stem cells. Methods Enzymol. 365:429‐446.

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Preparation of Defined Human Embryonic Stem Cell Populations for Transcriptional Profiling

Abstract

Table of Contents

Materials

Basic Protocol 1: Enzymatic Passaging of Cultures of hESCs

Alternate Protocol 1: Starting hESC Bulk Culture from Maintenance Culture

Basic Protocol 2: Harvesting hESCs from Bulk Cultures

Basic Protocol 3: Immunofluorescent Staining of Surface Antigens on Live hESCs for FACS

Alternate Protocol 2: Combined Detection of OCT‐4 Intracellular Expression and Other Cell Surface Markers on Fixed hESC by Flow Cytometry

Basic Protocol 4: RNA Isolation

Figures

Videos

Literature Cited