Differentiation of Multipotent Mesenchymal Precursors and Skeletal Myoblasts from Human Embryonic Stem Cells

Maria Elena Stavropoulos1, Isabella Mengarelli1, Tiziano Barberi1

1 Beckman Research Institute of City of Hope, Duarte, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.8
DOI:  10.1002/9780470151808.sc01f08s9
Online Posting Date:  June, 2009
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Abstract

This unit describes a protocol for the derivation of multipotent mesenchymal precursors from human embryonic stem cells (hESCs). hESCs cultured at low density in the presence of a chemically defined serum‐free medium are induced to adopt an endomesodermal fate and later a mesenchymal phenotype. FACS sorting for the surface antigen CD73 is used to purify mesenchymal precursors able to differentiate into fat, bone, cartilage, and skeletal muscle cells. Enrichment in mesenchymal precursors with a myogenic potential is achieved via an additional FACS sorting for the embryonic skeletal muscle surface marker N‐CAM. Curr. Protoc. Stem Cell Biol. 9:1F.8.1‐1F.8.10. © 2009 by John Wiley & Sons, Inc.

Keywords: hESC; FACS; hESC‐MP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differentiation of hESCs into Mesenchymal Progenitors: Endomesodermal Induction
  • Support Protocol 1: Maintenance of hESC Cultures
  • Alternate Protocol 1: Enrichment for hESC‐MP with Myogenic Potential by FACS Sorting
  • Basic Protocol 2: Terminal Skeletal Muscle Differentiation of Myogenic hESC‐MP
  • Alternate Protocol 2: Terminal Differentiation of Mesenchymal Derivatives
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differentiation of hESCs into Mesenchymal Progenitors: Endomesodermal Induction

  Materials
  • hESCs
  • hESC medium (see recipe)
  • ITS medium (see recipe)
  • 60‐mm 1 µg/ml fibronectin‐coated culture dishes (Biocoat; BD Falcon)
  • Phosphate‐buffered saline, Ca2+/Mg2+‐free (CMF‐PBS; Cellgro, Mediatech)
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • MEM medium (see recipe)
  • FACS sorting buffer (see recipe)
  • Anti‐human CD73 monoclonal antibody, PE conjugated (BD Pharmingen) or PE‐conjugated IgG isotype control (BD Pharmingen)
  • Gelatin (0.1% in water; Specialty Media, Millipore)
  • Aspirator
  • 37°C incubator
  • 15‐ml centrifuge tubes
  • Tissue culture grade dishes (60‐mm; BD Falcon)
  • Hemacytometer
  • MoFlo FACS sorter (Dako Cytomation)

Support Protocol 1: Maintenance of hESC Cultures

  Materials
  • hESC cultures, undifferentiated and grown on MEFs
  • Phosphate buffered saline (PBS; Cellgro, Mediatech)
  • Collagenase IV/dispase (see recipe)
  • hESC medium (see recipe)
  • Mitotically inactivated MEFs (Specialty Media, Millipore)
  • MEF medium (see recipe)
  • Gelatin (0.1% in water; Specialty Media, Millipore)
  • 37°C incubator
  • 15‐ml tubes
  • Aspirator
  • Tissue culture grade dishes (60‐mm; BD Falcon)

Alternate Protocol 1: Enrichment for hESC‐MP with Myogenic Potential by FACS Sorting

  Materials
  • hESC‐MP (<70% confluent dish)
  • Phosphate buffered saline (PBS; Cellgro, Mediatech)
  • 0.05% (w/v) trypsin/EDTA (Invitrogen)
  • MEM medium (see recipe)
  • FACS sorting buffer (see recipe)
  • Anti‐N‐CAM monoclonal antibody (clone 5.1H11; DSHB)
  • Fluorochrome‐conjugated secondary antibody (chosen by investigator)
  • 0.1% (w/v) gelatin (Embryomax, Millipore)
  • Aspirator
  • 37°C incubator
  • 15‐ml tubes
  • Hemacytometer
  • MoFlo FACS sorter (Dako Cytomation)
  • Tissue culture dishes (various sizes)

Basic Protocol 2: Terminal Skeletal Muscle Differentiation of Myogenic hESC‐MP

  Materials
  • Myogenic hESC‐MP cultures (see protocol 3)
  • MEM medium (see recipe)
  • N2 medium (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Barberi, T., Willis, L.M., Socci, N.D., and Studer, L. 2005. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med. 2:e161.
   Barberi, T., Bradbury, M., Dincer, Z., Panagiotakos, G., Socci, N.D., and Studer, L. 2007. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat. Med. 13:642‐648.
   Lian, Q., Lye, E., Yeo, K.S., Tan, E.K.W., Salto‐Tellez, M., Liu, T.M., Palanisamy, N., Oakley, R.M.E., Lee, E.H., Lim, B., and Lim, S.K. 2007. Derivation of clinically compliant MSCs from CD105+, CD24‐differentiated human ESCs. Stem Cells 25:425‐436.
   Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284:143‐147.
   Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O., and Thomson, J.A. 2001. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat. Biotech. 19:1129‐1133.
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