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-β 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-β activity on signalling dynamics. Finally, we show that our microfluidic system, unlike conventional culture systems, can detect TGF-β ligands secreted in the conditioned medium from hESCs.

Development of a microfluidic approach for the real-time analysis of extrinsic TGF-β signalling

Vetralla Massimo;Bolego Chiara;Gagliano Onelia;Montagner Marco;Elvassore Nicola
2020

Abstract

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-β 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-β activity on signalling dynamics. Finally, we show that our microfluidic system, unlike conventional culture systems, can detect TGF-β ligands secreted in the conditioned medium from hESCs.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3353815
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