Induction of ES Cell–Derived Cartilage Formation

Jan Kramer1, Peter Schlenke1, Jürgen Rohwedel1

1 University of Lübeck, Lübeck
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.5
DOI:  10.1002/0471143030.cb2305s34
Online Posting Date:  March, 2007
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Abstract

This unit describes the protocols used for cultivation of murine embryonic stem (ES) cells and their differentiation into chondrogenic cell types in vitro. ES cells cultivated as cellular aggregates, so‐called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro‐ and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, marker molecules indicating bone formation can be detected in the nodules. This unit also contains protocols for characterization of the differentiated cells by immunostaining, mRNA‐in situ hybridization, electron microscopy, and RT‐PCR analysis.

Keywords: ES cells; chondrogenesis; osteogenesis; collagen II; proteoglycans; Sox5; Sox6

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: In Vitro Differentiation of Embryonic Stem Cells from Embryoid Bodies
  • Alternate Protocol 1: Alternative Plating Method for Generating EBs of the Same Size
  • Basic Protocol 2: Microdissection and Characterization of Mesenchymal Condensations and Cartilage Nodules from EBs
  • Basic Protocol 3: Isolation of Cells from Selected Cartilage Nodules by Collagenase Treatment
  • Histochemical Staining of EB
  • Support Protocol 1: Fixation for Histochemical Staining
  • Support Protocol 2: Alcian Blue Staining
  • Support Protocol 3: Alizarin Red Staining
  • Support Protocol 4: Von Kossa Staining
  • Support Protocol 5: Sudan III Staining
  • Support Protocol 6: Alkaline Phosphatase (AP) Staining
  • Support Protocol 7: Indirect Immunostaining of EBs
  • Support Protocol 8: Whole‐Mount Fluorescence in situ Hybridization for mRNA of a Gene of Interest Coupled with Immunostaining for Collagen II
  • Support Protocol 9: RT‐PCR Analysis to Detect Cartilage‐Specific Gene Expression in EBs
  • Support Protocol 10: Ultractructural Analysis of ES Cell‐Derived Mesenchymal Condensations and Cartilage Nodules by Electron Microscopy
  • Support Protocol 11: Derivation, Cultivation, and Inactivation of Embryonic Fibroblasts
  • Support Protocol 12: Proliferation and Subcultivation of ES Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vitro Differentiation of Embryonic Stem Cells from Embryoid Bodies

  Materials
  • ES cell lines BLC6 (Wobus et al., ), D3 (Doetschman et al., ), E14 (Hooper et al., ), and/or R1 (Nagy et al., ) growing ( protocol 16) on feeder layers ( protocol 15) in Medium 2 (see recipe) and Medium 4 (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Trypsin/EDTA solution (see recipe)
  • Medium 2 (see recipe) and Medium 4 (see recipe)
  • Low‐FBS medium (see recipe)
  • 100‐mm bacteriological petri dishes (Greiner), uncoated
  • 100‐µl sterile pipet tips with aerosol‐filter barriers (Eppendorf)
  • 5‐ml glass pipets
  • Gelatin‐coated (see recipe) 60‐mm tissue culture dishes (Nunc), chamber slides (Falcon CultureSlides from BD Biosciences), and 24‐well tissue culture plates
  • Additional reagents and equipment for basic cell culture techniques including counting cells (unit 1.1)

Alternate Protocol 1: Alternative Plating Method for Generating EBs of the Same Size

  Materials
  • EB cultures, (day 5 plus 10 days or day 5 plus 20 days; see protocol 1 or protocol 2)
  • Tissue‐Tek OCT compound (Sakura Finetek)
  • Acetone
  • Phosphate‐buffered saline (PBS; see recipe)
  • Microscalpel or microdissector (Eppendorf)
  • Tissue‐Tek Cryomold 10 × 10 × 5–mm (Sakura Finetek; http://www.sakuraus.com/)
  • Cryostat (Leica)
  • Vectabond‐coated glass microscope slides (Vector)
  • Additional reagents and equipment for indirect immunostaining ( protocol 11)

Basic Protocol 2: Microdissection and Characterization of Mesenchymal Condensations and Cartilage Nodules from EBs

  Materials
  • Microdissected nodules from EB cultures ( protocol 3, steps 1 to 2)
  • 0.1% (w/v) collagenase in Medium 4 (see recipe for Medium 4)
  • Medium 4 (see recipe)
  • Gelatin‐coated (see recipe) or collagen II‐coated 60‐mm tissue culture dishes or chamber slides
  • Shaking water bath or incubator
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Additional reagents and equipment for basic cell culture techniques including counting cells (unit 1.1)

Basic Protocol 3: Isolation of Cells from Selected Cartilage Nodules by Collagenase Treatment

  Materials
  • Cultures with differentiating EBs ( protocol 1), day 5 plus 2 days to day 5 plus 35 days, in chamber slides or tissue culture dishes depending on staining technique to be used (see Support Protocols below)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 3.7% (v/v) formaldehyde in distilled H 2O

Support Protocol 1: Fixation for Histochemical Staining

  Materials
  • Fixed EB cultures at the desired interval after plating on day 5 ( protocol 5) in 60‐mm culture dishes
  • Alcian blue working solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 5% (v/v) acetic acid solution

Support Protocol 2: Alcian Blue Staining

  Materials
  • Fixed EB cultures at the desired interval after day 5 ( protocol 5) in chamber slides
  • 100 mM Tris⋅Cl, pH 9 ( appendix 2A)
  • Alizarin red staining solution 1: 5% (w/v) Alizarin red S (Sigma) in H 2O; adjust to pH 9 with sodium hydroxide
  • Alizarin red staining solution 2: 0.5% (w/v) Alizarin red S (Sigma) in H 2O; adjust to pH 9 with sodium hydroxide
  • Vectashield mounting medium (Vector)
  • Clear nail polish
  • Coverslips

Support Protocol 3: Alizarin Red Staining

  Materials
  • Fixed EB cultures at the desired interval after plating on day 5 ( protocol 5) in chamber slides
  • Von Kossa silver stain: 500 mg silver nitrate (Fluka) in 10 ml distilled water (5% w/v AgNO 3)
  • Von Kossa fixation solution: 1 g anhydrous sodium thiosulfate (Merck) in 20 ml distilled water (5% w/v sodium thiosulfate)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Vectashield mounting medium (Vector)
  • Clear nail polish
  • Coverslips

Support Protocol 4: Von Kossa Staining

  Materials
  • Unfixed EBs cultured in chamber slides for the desired interval after plating on day 5 ( protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Sudan III staining solution (see recipe)
  • Vectashield mounting medium (Vector)
  • Clear nail polish
  • Coverslips

Support Protocol 5: Sudan III Staining

  Materials
  • Unfixed EBs cultured in chamber slides for the desired interval after plating on day 5 ( protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Leukocyte Alkaline Phosphatase staining kit (Sigma, cat. no. 86‐R) containing:
    • Citrate solution
    • FRV‐alkaline solution
    • Hematoxylin solution, Gill No. 3
    • Naphthol AS‐BI alkaline solution
    • Sodium nitrite solution
  • Acetone
  • 37% (w/v) formaldehyde
  • Vectashield mounting medium (Vector)
  • Clear nail polish
  • 15‐ml conical polypropylene centrifuge tubes
  • Coverslips

Support Protocol 6: Alkaline Phosphatase (AP) Staining

  Materials
  • Unfixed EBs cultured on chamber slides for the desired interval after plating on day 5 ( protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 7:3 (v/v) methanol/acetone (e.g., 42 ml methanol/18 ml acetone), −20°C
  • 10% (v/v) goat serum (Dianova, http://www.dianova.de) in PBS (see recipe)
  • Antibodies for immunostaining, primary and fluorophore‐labeled secondary, appropriately diluted (see recipe)
  • Vectashield mounting medium (Vector)
  • Clear nail polish
  • Humidified chamber (15‐cm covered glass dish with wet piece of filter paper)
  • Coverslips
  • Fluorescence microscope (e.g., AXIOSKOP; Zeiss)

Support Protocol 7: Indirect Immunostaining of EBs

  Materials
  • 16‐day post‐coitum (p.c.) mouse embryo limb buds
  • TOPO II cloning kit (Invitrogen,)
  • pCR‐BluntII‐TOPO vector (Invitrogen)
  • DIG RNA labeling mix kit including Polymerases SP6 and T7 (Boehringer Mannheim; cat. no. 1175025).
  • EB cultures plated on chamber slides at day 5 ( protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% (w/v) paraformaldehyde/4% (w/v) sucrose in PBS
  • 2× SSC, 0.2× SSC, and 0.1× SSC (see recipe for 20× SSC)
  • 50%, 70%, 95%, and 100% ethanol series
  • Prehybridization buffer (see recipe) with and without salmon sperm DNA
  • Monoclonal antibody II‐II6B3 against collagen II (diluted 1:20 in PBS; see recipe for Antibodies for immunostaining)
  • FITC‐conjugated sheep F(ab) fragments against digoxigenin (Roche, cat. no. 1207741).
  • Cy3‐conjugated goat anti‐mouse secondary antibodies (Dianova; cat. no. 115‐165‐062; http://www.dianova.de) for indirect detection of collagen II, both diluted 1:800 in PBS.
  • Vectashield mounting medium (Vector; cat. no. H‐1000).
  • Clear nail polish
  • Humidified chamber (15‐cm covered glass dish with wet piece of filter paper)
  • 45° and 70°C incubators
  • Coverslips
  • Fluorescence microscope with FITC and Cy3 filters
  • Additional reagents and equipment for RNA isolation and cDNA synthesis via RT‐PCR ( protocol 13), and DNA sequencing (Chapter 7 in Ausubel et al., )

Support Protocol 8: Whole‐Mount Fluorescence in situ Hybridization for mRNA of a Gene of Interest Coupled with Immunostaining for Collagen II

  Materials
  • Cultures of EBs at the desired time points ( protocol 1 or protocol 2)
  • Phosphate‐buffered saline (PBS; see recipe)
  • RNeasy Mini Kit (Qiagen)
  • RNase‐free DNase set (Qiagen)
  • Oligo‐dT primer (Life Technologies)
  • Superscript II reverse transcriptase (Life Technologies)
  • Taq DNA polymerase (Roche)
  • Primer (see Table 23.5.3)
  • 2% (w/v) agarose gel (Voytas, )
  • Additional reagents and equipment for quantification of nucleic acids ( appendix 3D), isolation of total RNA (Ausubel et al. , Chapter 4), RT‐PCR (Beverley, ), agarose gel electrophoresis (Voytas, ), and densitometry (unit 6.3)

Support Protocol 9: RT‐PCR Analysis to Detect Cartilage‐Specific Gene Expression in EBs

  Materials
  • ES cell–derived mesenchymal condensations and cartilage nodules (see protocol 3)
  • 5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4
  • 1% (w/v) OsO 4
  • 50%, 70%, 95% and 100% ethanol
  • Araldite embedding kit (e.g., Fluka)
  • Ultracut E (Leica Microsystems Nussloch, http://www.leica‐microsystems.com/)
  • LKB Bromma Ultrostainer Carlsberg System for uranyl acetate and lead citrate staining
CAUTION: Glutaraldehyde and OsO 4 are potentially hazardous reagents. Work should be performed under a hood and protective clothing and gloves must be used during the procedure.

Support Protocol 10: Ultractructural Analysis of ES Cell‐Derived Mesenchymal Condensations and Cartilage Nodules by Electron Microscopy

  Materials
  • Day 14 p.c. pregnant mice
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA solution (see recipe)
  • Medium 1 (see recipe) for cultivation of embryonic fibroblasts
  • 1 µg/ml MMC working solution (see recipe)
  • Medium 3 (see recipe)
  • 100 mm‐bacteriological petri dishes (Greiner), uncoated
  • Sterile dissection instruments: scissors, forceps
  • 100‐ml Erlenmeyer flask with a stir bar, sterile
  • ∼4‐mm‐diameter glass beads
  • Sieve (e.g., autoclavable metal tea filter) with pore diameter of ∼0.5 mm
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Gelatin‐coated (see recipe) 100‐mm and 60‐mm dishes
  • 2 ml‐cryopreservation vials (Nunc)
  • Dewar flasks with liquid nitrogen
  • Additional reagents and equipment for counting cells (unit 1.1)

Support Protocol 11: Derivation, Cultivation, and Inactivation of Embryonic Fibroblasts

  Materials
  • Feeder layer cells: 60‐mm dishes of MMC‐treated embryo fibroblasts ( protocol 15)
  • Medium 2 (see recipe)
  • Frozen vial of ES cells
  • Phosphate‐buffered saline (PBS; see recipe)
  • Trypsin/EDTA solution (see recipe)
  • Medium 3 for freezing the ES cells
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • 2‐ml glass pipet
  • 2‐ml cryopreservation vials (Nunc)
  • Dewar flasks with liquid nitrogen
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Figures

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Literature Cited

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