Skip to main content
SpringerLink
Log in

Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells

  • Protocol
  • First Online:
Stem Cell Assays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2429))

  • 1943 Accesses

Abstract

Human pluripotent stem cells have a wide variety of potential applications, ranging from clinical translation to in vitro disease modeling. However, there is significant variation in the potential of individual cell lines to differentiate towards each of the three germ layers as a result of (epi)genetic background, culture conditions, and other factors. We describe here in detail a methodology to evaluate this bias using short directed differentiation towards neuroectoderm, mesendoderm, and definitive endoderm in combination with quantification by RT-qPCR and immunofluorescent stains.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    Article  CAS  Google Scholar 

  2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872

    Article  CAS  Google Scholar 

  3. Keller A, Dziedzicka D, Zambelli F, Markouli C, Sermon K, Spits C, Geens M (2018) Genetic and epigenetic factors which modulate differentiation propensity in human pluripotent stem cells. Hum Reprod 24:162–175

    CAS  Google Scholar 

  4. Avior Y, Biancotti JC, Benvenisty N (2015) TeratoScore: assessing the differentiation potential of human pluripotent stem cells by quantitative expression analysis of Teratomas. Stem cell Rep 4:967–974

    Article  CAS  Google Scholar 

  5. Bock C, Kiskinis E, Verstappen G, Gu H, Boulting G, Smith ZD, Ziller M, Croft GF, Amoroso MW, Oakley (2011) Reference maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell 144:439–452

    Article  CAS  Google Scholar 

  6. Tsankov AM, Akopian V, Pop R, Chetty S, Gifford CA, Daheron L, Tsankova NM, Meissner A (2015) A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat Biotechnol 33:1182–1192

    Article  CAS  Google Scholar 

  7. Markouli C, Couvreu De Deckersberg E, Regin M, Nguyen HT, Zambelli F, Keller A, Dziedzicka D, De Kock J, Tilleman L, Van Nieuwerburgh F, Franceschini L, Sermon K, Geens M, Spits C (2019) Gain of 20q11.21 in human pluripotent stem cells impairs TGF-β-dependent Neuroectodermal commitment. Stem Cell Rep 13:163–176

    Article  CAS  Google Scholar 

  8. Markouli C, De Deckersberg EC, Dziedzicka D, Regin M, Franck S, Keller A, Gheldof A, Geens M, Sermon K, Spits C (2021) Sustained intrinsic WNT and BMP4 activation impairs hESC differentiation to definitive endoderm and drives the cells towards extra-embryonic mesoderm. Sci Rep 11(1):8242. https://doi.org/10.1038/s41598-021-87547-7. PMID: 33859268

  9. Chetty S, Pagliuca FW, Honore C, Kweudjeu A, Rezania A, Melton DA (2013) A simple tool to improve pluripotent stem cell differentiation. Nat Methods 10:553–556

    Article  CAS  Google Scholar 

  10. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280

    Article  CAS  Google Scholar 

  11. Sui L, Mfopou JK, Geens M, Sermon K, Bouwens L (2012) FGF signaling via MAPK is required early and improves Activin A-induced definitive endoderm formation from human embryonic stem cells. Biochem Biophys Res Commun 426:380–385

    Article  CAS  Google Scholar 

  12. Rodin S, Antonsson L, Hovatta O, Tryggvason K (2014) Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions. Nat Protoc 9:2354–2368

    Article  CAS  Google Scholar 

  13. Baker D, Hirst AJ, Gokhale PJ, Juarez MA, Williams S, Wheeler M, Bean K, Allison TF, Moore HD, Andrews PW, Barbaric I (2016) Detecting genetic mosaicism in cultures of human pluripotent stem cells. Stem Cell Rep 7:998–1012

    Article  CAS  Google Scholar 

  14. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Alexander Keller and Nuša Krivec contributed equally to this work.

Authors and Affiliations

  1. Research Group Reproduction and Genetics, Vrije Universiteit Brussel, Jette, Belgium

    Alexander Keller, Nuša Krivec, Christina Markouli & Claudia Spits

Authors
  1. Alexander Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nuša Krivec
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christina Markouli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudia Spits
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Claudia Spits .

Editor information

Editors and Affiliations

  1. Stem Cell and Cancer Biology Laboratory, Division of Experimental Medicine and Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

    Nagarajan Kannan

  2. Hull York Medical School, York, UK

    Philip Beer

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Keller, A., Krivec, N., Markouli, C., Spits, C. (2022). Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells. In: Kannan, N., Beer, P. (eds) Stem Cell Assays. Methods in Molecular Biology, vol 2429. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1979-7_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1979-7_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1978-0

  • Online ISBN: 978-1-0716-1979-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation