Cermola, Federica (2020) Characterization of an early-primed state of pluripotency competent to gastruloid formation and primordial germ cells induction. [Tesi di dottorato]

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Mouse embryonic stem cells (mESCs) exposed to a high L-Proline regimen undergo a phenotypic transition named embryonic stem-to-mesenchymal transition (esMT). The resulting cells, named L-Proline-induced (PiCs), maintain some features of naïve pluripotent cells (LIF reliance, teratoma and chimera formation), and acquire other (morphology, energetic metabolism, and epigenetic profile) specific of primed pluripotent cells. These data suggest that PiCs represent an early-primed pluripotency state [1-3]. To further characterize the pluripotency state of PiCs, we set up a high-performing assay to measure the gastruloids formation efficiency (GFE) of mESCs, i.e. the ability of spherical aggregates of mESCs to develop into elongated/polarized embryonic organoids [4]. By exploiting this innovative experimental approach we provide morphological and molecular evidences that gastruloid development relies on Cripto gene, a key regulator of stem cells pluripotency [5, 6]. We also found that GFE assay discriminate different phenotypic/functional states of pluripotency. Indeed, while naïve cells efficiently give rise to aggregates, most of which elongate (GFE ≥ 95%), primed Epiblast stem cells (EpiSCs) fail to aggregate, and consequently to generate gastruloids (GFE = 0%). Moreover, although early-primed Epiblast-like cells (EpiLCs) efficiently aggregate, the resulting aggregates remain as undeveloped organoids (GFE = 0%). PiCs generate cell aggregates that elongate earlier and develop into smaller gastruloids, exhibiting highly differentiated areas (GFE ≥ 50%). Moreover, like EpiLCs, PiCs are competent to differentiate into primordial germ cell-like cells (PGCLCs). These results characterize PiCs as a unique pluripotency state, with competence for both gastruloid formation and PGCLCs differentiation. How L-Pro impact ESC identity is not yet fully understood. It is known that L-Pro supplementation increases the biosynthesis of L-Pro-rich protein such as collagen [2, 7], and that Collagen prolyl hydroxylation (CPH), catalysed by prolyl 4-hydroxylase (P4H), requires Vitamin C (VitC). Here, we identify CPH as an epigenetic modulator. The induction of collagen synthesis, and thus of VitC-dependent CPH, increases global DNA/histone methylation level and promote cell state transition. Interfering with CPH, by either genetic ablation of P4H alpha subunits or pharmacologic treatment, avoid both epigenetic changes and cell state transition. Mechanistically, these results suggest that a sudden increment in CPH can modify the epigenetic landscape by reducing VitC availability for DNA and histone demethylases enzymes. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control cell state transition.

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