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Higher Developmental State Reached in Human Pluripotent Cells

Review of “Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human” from Cell by Stuart P. Atkinson

Mouse pluripotent stem cells exist in two related forms; (i) embryonic stem cells (ESCs), described as being ‘naïve’ (or in the ground state) in that they describe early epiblast with the greatest developmental potential and (ii) epiblast stem cells (EpiSCs), described as being ‘primed’, in that they describe late epiblast and have a lower developmental potential. Currently derived human pluripotent stem cells (hPSCs) resemble mouse EpiSCs and, until now, naïve human PSCs have not been described. Now, in a recent article in the journal Cell, researchers from the groups of Paul Bertone (EMBL-EBI) and  Austin Smith (Cambridge Stem Cell Institute, Cambridge, UK) have demonstrated the conversion of hPSCs to the naïve state, and present their findings [1].

Expression of Nanog or Klf2 with two inhibitors (2i) of the Erk pathway and of glycogen synthase kinase-3 with LIF (2iL) converts mouse EpiSCs are to the naïve state [2, 3]. Similar experiments using hESCs and DOX-inducible NANOG/KLF4 transgenes generated expandable colonies of mESC-like cells, which could propagated as single cells without the requirement for Rho-associated kinase inhibitor (ROCKi). The authors suggest that these derived cells were naïve hPSCs. After removal of DOX, maintenance of naïve hPSCs required the presence of a protein kinase C (PKC) inhibitor (Gö6983), a factor known to suppress mESC differentiation [4] In this state, mRNA/protein expression of factors associated with the naïve state in mice were all upregulated, except for the estrogen-related receptor beta gene (ESRRB).

Embryoid body (EB)-mediated differentiation of naïve hPSCs demonstrated mRNA expression from all three germ layers and grafting into NOD/SCID mice generated teratomas that contained well-differentiated regions of neuroepithelium, cartilage and digestive tract. Mitochondrial/metabolic assessment also demonstrated profound alterations; naïve hPSCs displayed a higher basal oxygen consumption rate (OCR) and high levels of mitochondrial membrane depolarization as compared to hPSCs, akin to mESCs. Naïve hPSCs also had the ability to grow in the presence of 2-deoxyglucose and under low glucose concentration, while hPSCs could not. The group also observed epigenetic alterations; naïve hPSCs displayed lower 5-methylcytosine (5mC) and trimethylation of histone 3 lysine 9 (H3K9me3) levels over most genomic contexts, as observed in mouse ground-state ESC and cells of the human inner cell mass (ICM) [5, 6].  As expected, the transcriptional state also changed, with naïve hPSCs being similar to human blastocyst ICM [7]. Naïve hPSCs expressed, as expected, robust levels of ground-state pluripotency regulators, with a reduction in lineage-specific gene expression, and also lower levels of DNMT3B, in line with the reduction in DNA methylation. Short hairpin (sh) RNA studies found that, unlike hPSCs, naïve hPSCs relied on the continued expression of TFCP2L1 and KLF4, similar to ground state pluripotency in mESCs. The researchers further examined naïve identity through morula aggregation and monitoring of subsequent embryo development. While hPSCs did not contribute to cells of the blastocyst, naïve hPSCs did (6 of 42 blastocysts) and, upon blastocyst injection, the group observed naïve hPSCs-derived cells in the mature ICM/epiblast.

These findings suggest that human pluripotent stem cells can convert to a naïve ground state akin to that of ground state mouse ESCs. Indeed, this study also demonstrates a conserved functionality of ground state transcription factors, supporting the idea of a generic naïve state of pluripotency in mammals. The confirmation of ground-state pluripotency in human cells will hopefully soon lead to in depth studies of early human development, which have remained impossible to study to this point, and furthermore, may represent a more suitable material for regenerative medicine applications.


  1. Takashima Y, Guo G, Loos R, et al. Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human. Cell 2014;158:1254-1269.
  2. Hall J, Guo G, Wray J, et al. Oct4 and LIF/Stat3 additively induce Kruppel factors to sustain embryonic stem cell self-renewal. Cell Stem Cell 2009;5:597-609.
  3. Silva J, Nichols J, Theunissen TW, et al. Nanog is the gateway to the pluripotent ground state. Cell 2009;138:722-737.
  4. Dutta D, Ray S, Home P, et al. Self-renewal versus lineage commitment of embryonic stem cells: protein kinase C signaling shifts the balance. Stem Cells 2011;29:618-628.
  5. Guo H, Zhu P, Yan L, et al. The DNA methylation landscape of human early embryos. Nature 2014;511:606-610.
  6. Smith ZD, Chan MM, Humm KC, et al. DNA methylation dynamics of the human preimplantation embryo. Nature 2014;511:611-615.
  7. Yan L, Yang M, Guo H, et al. Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 2013;20:1131-1139.