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Naïve Pluripotency - New Direction, Same Destination

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Review of “Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells” from Stem Cells by Stuart P. Atkinson

Mouse embryonic stem cells (mESCs) reside in a “naïve” pluripotent state, in that that they display unbiased lineage-specific differentiation. However, human ESCs (hESCs) reside in a “primed” state and exhibit lineage-specific biases which may create problems for their clinical application. To solve this problem, many groups have attempted to derive or convert hESCs or human induced pluripotent stem cells (hiPSCs) to the naïve state, meeting with differing success (see original study for extensive references). 

In a new study published in Stem Cells, researchers from the laboratory of Björn Heindryckx (Ghent University Hospital, Belgium) have now taken conversion to the naïve state in a new direction. They describe a novel combination of small molecules and growth factors which can rapidly and efficiently induce naïve pluripotency in the absence of ectopic gene expression [1].

The group’s study used various hESC lines cultured on mouse embryonic fibroblasts (MEFs) in low oxygen concentrations to test various media formulations for their ability to establish naïve pluripotency. The optimal conversion media they found contained: 

  • Basic fibroblast growth factor (bFGF) - essential to promote pluripotency acting through PI3K signaling
  • Human leukemia inhibitory factor (LIF) - upregulates JAK/STAT signaling
  • Forskolin (FK) - protein kinase A agonist which blocks the MAPK and RAF signaling pathways
  • Ascorbic acid (AA) – Promotes DNA demethylation and enhances cell reprogramming
  • 2i small molecule combination - PD0325901 (PD) which inhibits MAPK/ERK1/2 and MEK and CHIR99021 (CH) which inhibits GSK3.

This “simple” combination efficiently converted primed hESCs to a naïve state where they displayed similarities to mouse embryonic stem cells. This included the ability of naïve hESCs to be passaged as single cells, the formation of dome-shaped colonies in culture (See figure), and similarities in gene expression patterns, differentiation capacity, and the use of the same signaling pathways to maintain this cellular state. Of note, the authors demonstrated that the combination of factors likely induced naïve pluripotency through PI3K/AKT/mTORC signaling, and not, as many others studies had found, through the potent inhibition of the MEK pathway.

These new findings represent a quick and easy way to convert hESCs into their more useful naïve state without the need for transgene expression. The production of the naïve hESCs removes heterogeneity from cell cultures and removes any lineage-barriers that may exist in the primed state. In this way, this study represents an important step forward for the application of cells in regenerative cell applications such as genome engineering and gene correction. 

References

  1. Duggal G, Warrier S, Ghimire S, et al. Alternative Routes to Induce Naive Pluripotency in Human Embryonic Stem Cells. Stem Cells 2015;33:2686-2698.