Delineating routes and applications of naive pluripotency

Ohad Gafni

doi:10.34933/wis.000121

Even though it has been almost 40 years since the first mammalian embryo that was made by somatic cell nuclear transfer, fundamental routes and principals of somatic cell reprogramming into the naïve state are still elusive. Pluripotency can also be induced in somatic cells by ectopic expression of a variety of transcription factors, classically Oct4, Sox2, Klf4 and c-Myc (abbreviated as OSKM)[1-4]. This process is accompanied by genome wide epigenetic Histone 3 Lysine 4 tri-methylation (H3K4me3) and Histone 3 Lysine 27 tri-methylation (H3K27me3) correlated with active and repressive transcriptional states, respectively. However how do these critical changes get biochemically dictated and which epigenetic regulators drive the progression of cellular reprogramming, requires further investigation [5, 6]. My work is divided to two major parts; the first part revealed Utx H3K27me3 demethylase as a key component required for reprogramming. We have showed that although Utx deficient embryonic stem cells maintain pluripotent characteristics, somatic cells lacking Utx failed to reprogram back to a pluripotent state mainly due to severe accumulation of H3K27me3 repressive marks on key pluripotency regulating genes. The second part deals with defined growth conditions that facilitate the derivation of naïve human pluripotent stem cells (PSCs; refer to both ESCs and iPSCs). Mouse embryonic stem cells (mESCs) are isolated from the inner cell mass (ICM) of blastocysts, and can be preserved in vitro in a naïve ICM-like configuration by providing exogenous stimulation, typically with leukemia inhibitory factor and small molecule inhibition of ERK1/2 and GSK3β signaling (termed 2i/LIF conditions). Upon withdrawal of 2i/LIF, naïve mESCs epigenetically drift towards a primed pluripotent state resembling that of the post-implantation epiblast. While human embryonic stem cells (hESCs) share several molecular features with naïve mESCs, they also share a variety of epigenetic properties with primed murine Epiblast stem cells (mEpiSCs). A combination of small inhibitors of signaling pathways and cytokines established in our laboratory retained growth characteristics, molecular circuits, chromatin landscape, and signaling pathway dependence that are highly similar to mouse naïve ESCs, and drastically distinct from conventional primed hPSCs. The potential of naïve hiPSCs to faithfully contribute to chimeric animals is not defined yet. To address this issue, I engineered naïve human iPSC knock-in line that constitutively expresses GFP. These cells were injected into mouse E2.5 morulas, implanted into pseudo-pregnant female mice and their progeny was traced during embryonic development, using immuno-histological analysis and in-toto live confocal imaging

Delineating routes and applications of naive pluripotency

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