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Derivation of naive human embryonic stem cells.

Ware CB, Nelson AM, Mecham B, Hesson J, Zhou W, Jonlin EC, Jimenez-Caliani AJ, Deng X, Cavanaugh C, Cook S, Tesar PJ, Okada J, Margaretha L,Sperber H, Choi M, Blau CA, Treuting PM, Hawkins RD, Cirulli V, Ruohola-Baker H.

Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):4484-9.

Institute for Stem Cell and Regenerative Medicine, Departments of Comparative Medicine, Biology, and Biochemistry, and Divisions of Hematology, Metabolism, Endocrinology and Nutrition, and Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195.




The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve humanES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

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Significance Statement

The authors developed a method to generate a stable, naïve hESC state without the need for transgenes and can be maintained indefinitely through simple trypsin passage. The cells were validated and shown to be karyotypically stable with hallmarks consistent with a mouse naïve state, including gene expression, teratoma formation, and immature mitochondrial and overall morphology. The developed Naïve hESCs are capable of robust differentiation to all three germ lineages, with a heightened capability of endoderm formation not previously seen in hESCs.


Figure: Credit Proc Natl Acad Sci U S A. 2014 Mar 25;111(12):4484-9.