Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells

Background Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands. Methodology/Principal Findings We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs. Conclusions/Significance Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.National Institutes of Health (U.S.) (NIH grant No. EB007278)Swiss National Science FoundationSwiss National Science Foundatio

Spatial organization acts on cell signaling: how physical force contributes to the development of cancer

Cells constantly encounter physical forces and respond to neighbors and circulating factors by triggering intracellular signaling cascades that in turn affect their behavior. The mechanisms by which cells transduce mechanical signals to downstream biochemical changes are not well understood. In their work, Salaita and coworkers show that the spatial organization of cell surface receptors is crucial for mechanotransduction. Consequently, force modulation that disrupts the mechanochemical coupling may represent a critical step in cancerogenesis

How Linear Tension Converts to Curvature: Geometric Control of Bone Tissue Growth

This study investigated how substrate geometry influences in-vitro tissue formation at length scales much larger than a single cell. Two-millimetre thick hydroxyapatite plates containing circular pores and semi-circular channels of 0.5 mm radius, mimicking osteons and hemi-osteons respectively, were incubated with MC3T3-E1 cells for 4 weeks. The amount and shape of the tissue formed in the pores, as measured using phase contrast microscopy, depended on the substrate geometry. It was further demonstrated, using a simple geometric model, that the observed curvature-controlled growth can be derived from the assembly of tensile elements on a curved substrate. These tensile elements are cells anchored on distant points of the curved surface, thus creating an actin “chord” by generating tension between the adhesion sites. Such a chord model was used to link the shape of the substrate to cell organisation and tissue patterning. In a pore with a circular cross-section, tissue growth increases the average curvature of the surface, whereas a semi-circular channel tends to be flattened out. Thereby, a single mechanism could describe new tissue growth in both cortical and trabecular bone after resorption due to remodelling. These similarities between in-vitro and in-vivo patterns suggest geometry as an important signal for bone remodelling

Cellular and Molecular Responses to Gravitational Force-Triggered Stress in Cells of the Immune System

Sensitivity of the human immune system to microgravity has been supposed since the first Apollo missions and was demonstrated during several space missions in the past. In vitro experiments demonstrated that cells of the immune system are exceptionally sensitive to microgravity. Therefore, serious concerns arose whether spaceflight-associated immune system weakening ultimately precludes the expansion of human presence beyond Earth’s orbit. In human cells, gravitational forces may be sensed by an individual cell in the context of altered extracellular matrix mechanics, cell shape, cytoskeletal organization, or internal prestress in the cell–tissue matrix. The development of cellular mechanosensitivity and signal transduction was probably an evolutionary requirement to enable our cells to sense their individual microenvironment. Therefore it is possible that the same mechanisms, which enable human cells to sense and to cope with mechanical stress, are potentially dangerous in microgravity. This chapter reviews the most recent developments in investigation to elucidate the influence of microgravity on immune cell signaling and functions and hereby bridges the phenotypic changes to transcriptome and epigenetic regulators