Serum‐Free Generation of Multipotent Mesoderm (Kdr+) Progenitor Cells in Mouse Embryonic Stem Cells for Functional Genomics Screening

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Abstract
Table of Contents
Materials
Literature Cited

Abstract

This unit describes a robust protocol for producing multipotent Kdr?expressing mesoderm progenitor cells in serum?free conditions, and for functional genomics screening using these cells. Kdr?positive cells are able to differentiate into a wide array of mesodermal derivatives, including vascular endothelial cells, cardiomyocytes, hematopoietic progenitors, and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. In addition, the oligonucleotide transfection protocol used to functionally screen siRNA and miRNA libraries is a powerful tool to reveal networks of genes, signaling proteins, and miRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. Technical limitations, troubleshooting, and potential applications of these methods are discussed. Curr. Protoc. Stem Cell Biol. 23:1F.13.1?1F.13.13. © 2012 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cells; mesendoderm; mesoderm; endoderm; siRNA; transfection; Kdr; Foxa2

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Introduction
Basic Protocol 1: Culture and Freezing of Mouse Embryonic Stem Cells
Basic Protocol 2: Differentiation of Mouse Embryonic Stem Cells into Multipotent Mesoderm Progenitor (Kdr+) Cells
Alternate Protocol 1: Differentiation of Mouse Embryonic Stem Cells for High‐Throughput Functional Screening of siRNA/miRNA Libraries
Basic Protocol 3: Paraformaldehyde Fixation and Immunostaining of Differentiated mESCs
Basic Protocol 4: High‐Throughput Imaging and Quantification of Kdr and Foxa2 Expression
Reagents and Solutions
Commentary
Literature Cited
Tables

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Materials

Basic Protocol 1: Culture and Freezing of Mouse Embryonic Stem Cells

Materials

0.1% gelatin solution (Stem Cell Technologies, cat. no. 07903)
Kdr‐eGFP mouse embryonic stem cells (Ema et al., ), frozen
mESC growth medium (see recipe )
0.25% trypsin EDTA (Gibco, cat. no. 25200)
Freezing medium (see recipe )

10‐cm tissue culture plates (Corning, cat. no. 430167)
37°C water bath
15‐ml centrifuge tubes
Inverted bright‐field microscope
5‐ and 10‐ml serological pipets
1‐ml cryotubes
Cryo 1°C freezing container (Nalgene, cat. no 5100‐0001) or equivalent

Basic Protocol 2: Differentiation of Mouse Embryonic Stem Cells into Multipotent Mesoderm Progenitor (Kdr+) Cells

Materials

1× phosphate‐buffered saline (PBS, Mediatech, cat. no. 21‐040‐CV)
Chemically defined medium (CDM, see recipe )
0.25% trypsin EDTA (Gibco, cat. no. 25200)
mESC growth medium (see recipe )
Activin A (R&D Systems, cat. no. 338‐AC‐050)
0.1% gelatin solution (Stem Cell Technologies, cat. no. 07903)
siRNA against Acvr1b (Ambion, cat. no. s61928)
Lipofectamine RNAiMAX (Invitrogen, cat. no. 13778‐100)
Opti‐MEM reduced‐serum medium (Invitrogen, cat. no. 31985‐070)

40‐µm nylon cell strainer (BD Falcon, cat. no. 352340)
15‐ and 50‐ml centrifuge tubes
Hemacytometer
Inverted bright‐field microscope
10‐cm low‐attachment tissue culture plates (Fisherbrand Petri dishes, cat. no. 08‐757‐13)
10‐ml serological pipets
384‐well optical tissue culture plates, sterile (Greiner Bio‐One, cat. no. EK‐30091)
16‐channel electronic pipettor, 2.0‐125 µl (Thermo Scientific, cat. no. 2061)
384‐well aerosol‐free pipet tips (Thermo Scientific Cat, cat. no. 7445)
Centrifuge for 384‐well plates

Additional reagents and equipment for culturing Kdr‐eGFP mouse ES cells (see protocol 1 ) and counting cells (unit 1.3 )

Alternate Protocol 1: Differentiation of Mouse Embryonic Stem Cells for High‐Throughput Functional Screening of siRNA/miRNA Libraries

Fluid‐handling robot (e.g., Star Line workstations, Hamilton)
siRNA or miRNA library (e.g., Ambion/Life Technologies or Dharmacon/Thermo Scientific)

Basic Protocol 3: Paraformaldehyde Fixation and Immunostaining of Differentiated mESCs

Materials

Differentiated mESCs (see protocol 2 or protocol 3 )
8% (w/v) paraformaldehyde (PFA) in 1× PBS
Blocking buffer (see recipe )
Primary antibodies (Santa Cruz Biotechnology):
- Anti‐Foxa2 (M‐20, goat polyclonal, cat. no. SC‐6554)
- Anti‐Pecam1 (V‐16, goat polyclonal, cat. no. SC‐31045)
- Anti‐smooth muscle actin (B4, mouse monoclonal, cat. no. SC‐53142)
1× PBS without calcium and magnesium (Mediatech, cat. no. 21‐040‐CV)
Secondary antibodies (Molecular Probes):
- Donkey anti‐goat Alexa Fluor 568 (cat. no. A‐11057)
- Donkey anti‐goat Alexa Fluor 647 (cat. no. A‐21447)
- Donkey anti‐mouse Alexa Fluor 568 (cat. no. A‐10037)
DAPI solution (Invitrogen, cat. no. D3571)
Storage solution (see recipe )

16‐channel electronic pipettor, 2.0‐125 µl (Thermo Scientific, cat. no. 2061)
384‐well aerosol‐free pipet tips (Thermo Scientific, cat. no. 7445)
Shaker appropriate for microtiter plates
5‐liter plastic beaker

NOTE: For all steps, use a 16‐channel pipettor or fluid‐handling robot.

Basic Protocol 4: High‐Throughput Imaging and Quantification of Kdr and Foxa2 Expression

Materials

Differentiated and immunostained cells (see protocol 4 )
Automated inverted fluorescence microscopy workstation (GE Healthcare, IN Cell Analyzer 1000)
Nikon Plan Apo VC objective (10×, 0.45 N.A.)
Filter sets (Table 1.13.1 )

CyteSeer automated image analysis program (Vala Biosciences)

Table 1.1.1 MaterialsFilter Sets and Exposure Settings for Imaging Mesodermal and Endodermal Differentiation

	Mesoderm	Endoderm
Color	Green	Red
Dye	eGFP	Alexa Fluor 568
Excitation λ (nm)	488	578
Excitation filter	480/20	535/25
Emission λ (nm)	509	603
Emission filter	535/25	620/30
Exposure time	60 msec	300 msec
HWAF offset	−6 µm	−6 µm

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Figures

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Literature Cited

Literature Cited
	Bushway, P.J. and Mercola, M. 2006. High‐throughput screening for modulators of stem cell differentiation. Methods Enzymol. 414:300‐316.
	Cheung, C., Bernardo, A.S., Trotter, M.W., Pedersen, R.A., and Sinha, S. 2012. Generation of human vascular smooth muscle subtypes provides insight into embryological origin‐dependent disease susceptibility. Nat. Biotechnol. 30:165‐173.
	Ema, M., Takahashi, S., and Rossant, J. 2006. Deletion of selection cassette but not cis‐acting elements in targeted Flk1‐lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood 107:111‐117.
	Kane, N.M., Meloni, M., Spencer, H.L., Craig, M.A., Strehl, R., Milligan, G., Houslay, M.D., Mountford, J.C., Emanueli, C., and Baker, A.H. 2010. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: Analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscl. Thromb. Vasc. Biol. 30:1389‐1397.
	Kattman, S.J., Witty, A.D., Gagliardi, M., Dubois, N.C., Niapour, M., Hotta, A., Ellis, J., and Keller, G. 2011. Stage‐specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8:228‐240.
	Mercola, M., Colas, A., and Willems, E. 2012. iPSCs in cardiovascular drug discovery. Circ. Res. In press.
	Savitzky, A. and Golay, B. 1964. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36:1627‐1639.
	Vazao, H., das Neves, R.P., Graos, M., and Ferreira, L. 2011. Towards the maturation and characterization of smooth muscle cells derived from human embryonic stem cells. PLoS One 6:e17771.