High‐Throughput Screening Assay for Embryoid Body Differentiation of Human Embryonic Stem Cells

Joel T. Outten1, Paul Gadue2, Deborah L. French2, Scott L. Diamond3

1 Departments of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, 2 Department of Pathology, Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, 3 Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.6
DOI:  10.1002/9780470151808.sc01d06s20
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Serum‐free human pluripotent stem cell media offer the potential to develop reproducible clinically applicable differentiation strategies and protocols. The vast array of possible growth factor and cytokine combinations for media formulations makes differentiation protocol optimization both labor and cost‐intensive. This unit describes a 96‐well plate, 4‐color flow cytometry–based screening assay to optimize pluripotent stem cell differentiation protocols. We provide conditions both to differentiate human embryonic stem cells (hESCs) into the three primary germ layers, ectoderm, endoderm, and mesoderm, and to utilize flow cytometry to distinguish between them. This assay exhibits low inter‐well variability and can be utilized to efficiently screen a variety of media formulations, reducing cost, incubator space, and labor. Protocols can be adapted to a variety of differentiation stages and lineages. Curr. Protoc. Stem Cell Biol. 20:1D.6.1‐1D.6.13. © 2012 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; hESCs; differentiation; high throughput; serum‐free; differentiation; embryoid body; suspension culture

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: 96‐Well Embryoid Body Differentiation
  • Basic Protocol 2: Flow Cytometry Analysis
  • Support Protocol 1: Feeder Depletion of Human Embryonic Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: 96‐Well Embryoid Body Differentiation

  • Collagenase B (see recipe)
  • Cultures of hESCs on Matrigel (approximately 70% confluent is optimal; see protocol 3)
  • 1 mg/ml DNase I (see recipe)
  • Serum‐free differentiation (SFD) medium (see recipe)
  • 0.25% Trypsin‐EDTA (Mediatech, cat. no. 25‐053‐CI)
  • Stop medium (see recipe)
  • Iscove's modified Dulbecco's medium (IMDM; Mediatech, cat. no. 10‐016‐CV)
  • Y26732 ROCK Inhibitor (see recipe)
  • Cell scrapers
  • Serological pipets
  • 50‐ml conical tubes
  • Centrifuge
  • 25‐ml sterile reagent reservoirs
  • 96‐well ultra‐low cluster flat‐bottom plates (Costar, cat. no. 3474)
  • Multichannel pipettor
  • Breathe Easy gas‐permeable membranes (Diversified Biotech)
  • 5‐ml round‐bottom polystyrene tubes, optional
  • 12‐channel sterile reagent reservoir (Costar, cat. no. 4877), optional
  • 8‐channel sterile reagent reservoir (Costar, cat. no. 4878), optional
  • Stainless steel micro plate lids (Wako/Kalypsys)
  • Additional reagents and equipment for trypsinization and counting cells (unit 1.3)

Basic Protocol 2: Flow Cytometry Analysis

  • 96‐well ultra‐low cluster flat‐bottom plates containing differentiated EBs (see protocol 1)
  • 0.25% Trypsin‐EDTA (Mediatech, cat. no. 25‐053‐CI)
  • Stop medium (see recipe)
  • Iscove's modified Dulbecco's medium (IMDM; Mediatech, cat. no. 10‐016‐CV)
  • Cell staining buffer (CSB; Biolegend)
  • 1 mg/ml DNase I (see recipe)
  • Fluorescent‐conjugated antibodies (see recipe)
  • 200‐ to 300‐µl multichannel pipettor
  • 96‐well V‐bottom plates
  • Centrifuge
  • 8‐channel aspirator nozzle
  • Plate agitator
  • C6 Flow Cytometer with C‐sampler (Accuri)

Support Protocol 1: Feeder Depletion of Human Embryonic Stem Cells

  • Matrigel solution (frozen; see recipe)
  • Cultures of hESC cells on MEF feeder cells (70% to 80% confluent is optimal)
  • hESC maintenance medium (see recipe)
  • Y26732 ROCK Inhibitor (see recipe)
  • TrypLE Express cell dissociation enzyme (Invitrogen)
  • Iscove's modified Dulbecco's medium (IMDM; Mediatech, cat. no. 10‐016‐CV)
  • 6‐well tissue culture plates
  • 5‐ml serological pipets
  • Cell scrapers
  • 50‐ml conical tubes
PDF or HTML at Wiley Online Library


  •   FigureFigure 1.D0.1 Endoderm EB differentiation. (A) H9 hESCs on Matrigel prior to harvesting for day 0 suspension seeding. Scale bar = 400 µm. (B) Typical EBs over a 6 day differentiation to endoderm. Scale bar = 200 µm.
  •   FigureFigure 1.D0.2 Representative marker profiles for directed mesoderm differentiation of H9 cells over 6 days. EBs at each time point were dissociated and cells were stained with antibodies corresponding to the indicated surface markers (SSEA‐3, CXCR4, NCAM, KDR). Plots show the transition of SSEA‐3+NCAM pluripotent cells to KDR+SSEA‐3 mesoderm cells. Ectoderm (SSEA‐3+NCAM+) and endoderm (CXCR4+KDR) percentages are given at day 6 as well (Outten et al., ). Stem Cell Research by Elsevier. Reproduced with permission of Elsevier in the format Journal via Copyright Clearance Center


Literature Cited

Literature Cited
   Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M., and Studer, L. 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27:275‐280.
   Chase, L.G. and Firpo, M.T. 2007. Development of serum‐free culture systems for human embryonic stem cells. Curr. Opin. Chem. Biol. 11:367‐372.
   D'Amour, K.A., Bang, A.G., Eliazer, S., Kelly, O.G., Agulnick, A.D., Smart, N.G., Moorman, M.A., Kroon, E., Carpenter, M.K., and Baetge, E.E. 2006. Production of pancreatic hormone‐expressing endocrine cells from human embryonic stem cells. Nat. Biotechnol. 24:1392‐1401.
   Eiselleova, L., Matulka, K., Kriz, V., Kunova, M., Schmidtova, Z., Neradil, J., Tichy, B., Dvorakova, D., Pospisilova, S., Hampl, A., and Dvorak, P. 2009. A complex role for FGF‐2 in self‐renewal, survival, and adhesion of human embryonic stem cells. Stem Cells 27:1847‐1857.
   Enver, T., Soneji, S., Joshi, C., Brown, J., Iborra, F., Orntoft, T., Thykjaer, T., Maltby, E., Smith, K., Dawud, R.A., Jones, M., Matin, M., Gokhale, P., Draper, J., and Andrews, P.W. 2005. Cellular differentiation hierarchies in normal and culture‐adapted human embryonic stem cells. Hum. Mol. Genet. 14:3129‐3140.
   Green, M.D., Chen, A., Nostro, M.C., d'Souza, S.L., Schaniel, C., Lemischka, I.R., Gouon‐Evans, V., Keller, G., and Snoeck, H.W. 2011. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol. 29:267‐272.
   Kennedy, M., D'Souza, S.L., Lynch‐Kattman, M., Schwantz,, S., and Keller, G. 2007. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109:2679‐2687.
   Koike, M., Kurosawa, H., and Amano, Y. 2005. A round‐bottom 96‐well polystyrene plate coated with 2‐methacryloyloxyethyl phosphorylcholine as an effective tool for embryoid body formation. Cytotechnology 47:3‐10.
   McGrath, K.E., Koniski, A.D., Maltby, K.M., McGann, J.K., and Palis, J. 1999. Embryonic expression and function of the chemokine SDF‐1 and its receptor, CXCR4. Devel. Biol. 213:442‐456.
   Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G., and Elefanty, A.G. 2005. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 106:1601‐1603.
   Nostro, M.C., Cheng, X., Keller, G.M., and Gadue, P. 2008. Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell 2:60‐71.
   Outten, J.T., Cheng, X., Gadue, P., French, D.L., and Diamond, S.L. 2011. A high‐throughput multiplexed screening assay for optimizing serum‐free differentiation protocols of human embryonic stem cells. Stem Cell Res. 6:129‐142.
   Park, I.H., Zhao, R., West, J.A., Yabuuchi, A., Huo, H., Ince, T.A., Lerou, P.H., Lensch, M.W., and Daley, G.Q. 2008. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141‐146.
   Pruszak, J., Sonntag, K.C., Aung, M.H., Sanchez‐Pernaute, R., and Isacson, O. 2007. Markers and methods for cell sorting of human embryonic stem cell‐derived neural cell populations. Stem Cells 25:2257‐2268.
   Sakurai, H., Era, T., Jakt, L.M., Okada, M., Nakai, S., Nishikawa, S., and Nishikawa, S. 2006. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 24:575‐586.
   Vallier, L., Touboul, T., Chng, Z., Brimpari, M., Hannan, N., Millan, E., Smithers, L.E., Trotter, M., Rugg‐Gunn, P., Weber, A., and Pedersen, R.A. 2009. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS One 4:e6082.
   Yang, L., Soonpaa, M.H., Adler, E.D., Roepke, T.K., Kattman, S.J., Kennedy, M., Henckaerts, E., Bonham, K., Abbott, G.W., Linden, R.M., Field, L.J., and Keller, G.M. 2008. Human cardiovascular progenitor cells develop from a KDR+ embryonic‐stem‐cell‐derived population. Nature 453:524‐528.
   Yasunaga, M., Tada, S., Torikai‐Nishikawa, S., Nakano, Y., Okada, M., Jakt, L.M., Nishikawa, S., Chiba, T., Era, T., and Nishikawa, S. 2005. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat. Biotechnol. 23:1542‐1550.
PDF or HTML at Wiley Online Library