RESUMO
The circadian clock has paramount implications in physiology and pathology. Although the circadian clock has been widely investigated in adults, up to now very little is known about how circadian rhythms emerge during embryonic development. Some studies about the ontology of the circadian system are focused on the development of the central pacemaker, whereas there is still no agreement about the development of the circadian clock in peripheral tissues. Our work represents the first attempt at investigating the onset of peripheral circadian clocks in the liver, which has a central role in controlling several aspects of human physiology. We profile the emergence of the circadian genes during the transition from the initial state of human pluripotency to the final state of hepatic maturation. We demonstrate that circadian rhythmicity is absent in human pluripotent stem cells, and it arises gradually during the process of hepatic commitment. The clock genes expression reaches a peak at the hepatic progenitor stage. At this point o hiPSC-derived f differentiation the gene oscillations start to be observed with a period of 13 h and approaches 24 h in a later stage when the clock primary feedback loop starts working properly. At the end of differentiation, circadian rhythmicity appears, with genes of primary and secondary feedback loops in antiphase (CLOCK, BMAL1 and REV-ERBα) a sign that the system becomes to be functional.
Assuntos
Relógios Circadianos , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relógios Circadianos/genética , Ritmo Circadiano/genética , Feminino , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/metabolismo , GravidezRESUMO
Autocrine and paracrine signalling are traditionally difficult to study due to the sub-micromolar concentrations involved. This has proven to be especially limiting in the study of embryonic stem cells that rely on such signalling for viability, self-renewal, and proliferation. Microfluidics allows to achieve local concentrations of ligands representative of the in vivo stem cell niche, gaining more precise control over the cell microenvironment, as well as to manipulate ligands availability with high temporal resolution and minimal amount of reagents. Here we developed a microfluidics-based system for monitoring the dynamics of TGF-ß pathway activity by means of a SMAD2/3-dependent luciferase reporter. We first validated our system by showing dose-dependent transcriptional activation. We then tested the effects of pulsatile stimulation and delayed inhibition of TGF-ß activity on signalling dynamics. Finally, we show that our microfluidic system, unlike conventional culture systems, can detect TGF-ß ligands secreted in the conditioned medium from hESCs.