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Induction of Human Naïve Pluripotency Using 5i/L/A Medium

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Human Naïve Pluripotent Stem Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2416))

Abstract

Prior to implantation, the cells in the mammalian epiblast constitute a naïve pluripotent state, which is distinguished by absence of lineage priming, freedom from epigenetic restriction, and expression of a unique set of transcription factors. However, human embryonic stem cells (hESCs) derived under conventional conditions have exited this naïve state and acquired a more advanced “primed” pluripotent state that corresponds to the post-implantation epiblast. We have developed a cocktail comprising five kinase inhibitors and two growth factors (5i/L/A) that enables induction of defining features of naïve pluripotency in primed hESCs. These conditions can also be applied to induce naïve pluripotency in patient-specific induced pluripotent stem cells (iPSCs). Here, we provide a detailed protocol for inducing naïve pluripotency in primed hESCs and iPSCs and methods for the routine validation of naïve identity. We also outline the use of two fluorescent reporter systems to track acquisition of naïve identity in live cells: (a) a GFP reporter linked to an endogenous OCT4 allele in which the primed-specific proximal enhancer has been deleted (OCT4-ΔPE-GFP); and (b) a dual-color reporter system targeted to both alleles of an X-linked gene that reports on the status of the X chromosome in female cells (MECP2-GFP/tdTomato). The conditions described herein have given insight into various aspects of naïve human pluripotent stem cells (hPSCs), including their unique transposon transcription profile, X chromosome status, and extraembryonic potential.

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Acknowledgments

Work in our laboratory is supported by the NIH Director’s New Innovator Award (DP2 GM137418) and grants from the Shipley Foundation Program for Innovation in Stem Cell Science, the Edward Mallinckrodt, Jr. Foundation, and the Washington University Children’s Discovery Institute. No federal NIH/NIGMS funds are used to develop 3D models of early human development. We thank Kyoung-mi Park for comments on the manuscript.

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Authors and Affiliations

  1. Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA

    Laura A. Fischer, Shafqat A. Khan & Thorold W. Theunissen

  2. Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Laura A. Fischer, Shafqat A. Khan & Thorold W. Theunissen

Authors
  1. Laura A. Fischer
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  2. Shafqat A. Khan
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  3. Thorold W. Theunissen
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Correspondence to Thorold W. Theunissen .

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Editors and Affiliations

  1. Babraham Institute, Cambridge, UK

    Peter Rugg-Gunn

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Fischer, L.A., Khan, S.A., Theunissen, T.W. (2022). Induction of Human Naïve Pluripotency Using 5i/L/A Medium. In: Rugg-Gunn, P. (eds) Human Naïve Pluripotent Stem Cells. Methods in Molecular Biology, vol 2416. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1908-7_2

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  • DOI: https://doi.org/10.1007/978-1-0716-1908-7_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1907-0

  • Online ISBN: 978-1-0716-1908-7

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