Molecular mechanisms involved in the transition between the primed and naïve state of embryonic stem cells

Author(s)

Amanda Cristina Corveloni

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-08-17

Examining board members

Rodrigo Alexandre Panepucci; Virgínia Picanço e Castro; Aparecida Maria Fontes; Lílian Figueiredo Moreira

• Advisor: Rodrigo Alexandre Panepucci

Abstract

Pluripotent embryonic stem cells (ESCs) are defined as cells with differentiation potential for the three embryonic leaflets (ectoderm, mesoderm and endoderm). Mouse pluripotent ESCs (mESCs) are considered to have a \"naïve\" pluripotent state, defined by the ability to self-renew, maintaining the potential for differentiation (without tendencies), while the epiblast cells (EpiSCs) formed after implantation undergo marked changes, becoming \"primed\" to respond to differentiating inducing signals. Human ESCs (hESCs) are considered, based on transcriptional, morphological and signaling-dependent characteristics, closer to mouse EpiSCs than to mESCs, understood as in the primed state. Our group recently showed that miR-363 promotes pluripotency in hESCs by post-transcriptional inhibition of Notch signaling components. Thus, the objective of this work was to evaluate the role of the Notch signaling pathway in the context of the naïve and primed states of pluripotency hESCs. For this, hESCs cell lines H1 and H9 were used, and several methods of conversion from primed to naïve state were explored, with inhibitors (LIF-5i/3i) with two different culture mediums and with commercial medium RSeT (TM) in hypoxia and normoxic conditions, in addition to inhibiting Notch signaling with a gamma secretase inhibitor (DAPT). Characteristic parameters of each state (surface markers and genes associated with primed and naïve states) were evaluated by quantitative immunofluorescence microscopy (High Content Analysis/Microscopy), flow cytometry and gene expression. The changes on these markers varied according to the conversion method used, but the most striking changes were the increase in DPPA3 and KLF4 transcripts, important genes associated with naïve status, an effect also caused by the inhibition of Notch signaling in different evaluated approaches. Furthermore, an increase in the DNMT3L transcript (also an important naïve marker) and a decrease in the H3K27me3 labeling under naïve culture conditions were observed, as well as with the inhibition of Notch signaling. Our data demonstrate that inhibition of Notch signaling positively regulates the transcriptional network related to the naïve state, in addition to possibly being related to an epigenetic modulation, supporting our hypothesis that Notch signaling is involved in the primed / naïve transition process, however the mechanism by which it acts has not yet been fully elucidated. We also demonstrate that the association of a drug that inhibits Notch signaling can be explored as a more effective method for the primed/naive transition process in vitro. (AU)

• FAPESP's process: 17/15929-6 - Molecular mechanisms involved in the transition between the Primed into Naive state of embryonic stem cells
• Grantee: Amanda Cristina Corveloni
• Support Opportunities: Scholarships in Brazil - Master