Molecular Criteria for Defining the Naive Human Pluripotent State

Thorold W Theunissen ,Haoyi Wang,Rosa Castanon,Julien Duc,Evarist Planet,Zhuzhu Zhang,Marc Friedli,Rudolf Jaenisch,Jesse Drotar,Yanmei Huang,Didier Trono,Styliani Markoulaki,Julien Pontis,Michaël Imbeault,Alexandra Iouranova,Tenzin Lungjangwa,Joseph R Ecker ,Ryan C O’neil ,Yiping He ,Joseph R Nery ,Malkiel A Cohen ,Katherine J Wert

doi:10.1016/j.stem.2016.06.011

Abstract

SummaryRecent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.

Highlights

Pluripotent stem cells from mouse and human have distinct morphologies, signaling requirements and epigenetic configurations
It has been proposed that mouse embryonic stem cells (ESCs) and induced pluripotent stem cells represent a naive state of pluripotency corresponding to the inner cell mass (ICM), whereas human ESCs/iPSCs correspond to a more advanced, or ‘‘primed,’’ state of pluripotency found in the postimplantation epiblast (Nichols and Smith, 2009)
Naive Human ESCs Display a Transposon Transcription Signature of Cleavage-Stage Embryos Transposable elements (TEs) are mobile genetic entities that constitute over half the human genome, and whose sequential expression during embryonic development is tightly regulated by species-specific trans-acting factors (Friedli and Trono, 2015)

Summary

Introduction

Pluripotent stem cells from mouse and human have distinct morphologies, signaling requirements and epigenetic configurations. A number of protocols have been described for inducing naive features in human ESCs, mostly testing candidates (Chan et al, 2013; Gafni et al, 2013; Takashima et al, 2014; Ware et al, 2014). We identified a combination of five kinase inhibitors that, together with LIF and activin A (5i/L/A), enabled the conversion of pre-existing human ESCs to the naive state in the absence of transgenes. An independent analysis concluded that naive human cells generated with this method or in titrated 2i/L medium supplemented with a protein kinase C (PKC) inhibitor (Takashima et al, 2014) displayed the closest transcriptional similarity to both the human blastocyst and mouse ESCs in 2i/L (Huang et al, 2014)

Results

Discussion

Conclusion