title: Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells.
abstract: Local (cell-level) signaling environments, regulated by autocrine and paracrine signaling, and modulated by cell organization, are hypothesized to be fundamental stem cell fate control mechanisms used during development. It has, however, been challenging to demonstrate the impact of cell-level organization on stem cell fate control and to relate stem cell fate outcomes to autocrine and paracrine signaling. We address this fundamental problem using a combined in silico and experimental approach in which we directly manipulate, using laminar fluid flow, the local impact of endogenously secreted gp130-activating ligands and their activation of signal transducer and activator of transcription3 (STAT3) signaling in mouse embryonic stem cells (mESC). Our model analysis predicted that flow-dependent changes in autocrine and paracrine ligand binding would impact heterogeneity in cell- and colony-level STAT3 signaling activation and cause a gradient of cell fate determination along the direction of flow. Interestingly, analysis also predicted that local cell density would be inversely proportional to the degree to which endogenous secretion contributed to cell fate determination. Experimental validation using functional activation of STAT3 by secreted factors under microfluidic perfusion culture demonstrated that STAT3 activation and consequently mESC fate were manipulable by flow rate, position in the flow field, and local cell organization. As a unique demonstration of how quantitative control of autocrine and paracrine signaling can be integrated with spatial organization to elicit higher order cell fate effects, this work provides a general template to investigate organizing principles due to secreted factors.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada M5S 3G9.
authors.forename: Faisal
authors.initials: F
authors.lastname: Moledina
authors.forename: Geoff
authors.initials: G
authors.lastname: Clarke
authors.forename: Ali
authors.initials: A
authors.lastname: Oskooei
authors.forename: Kento
authors.initials: K
authors.lastname: Onishi
authors.forename: Axel
authors.initials: A
authors.lastname: Günther
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D000818:Animals; D019898:Autocrine Communication; D002454:Cell Differentiation; D002478:Cells, Cultured / Q000187:drug effects; D003198:Computer Simulation; D050822:Cytokine Receptor gp130 / Q000502:physiology; D004058:Diffusion; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015850:Interleukin-6 / Q000502:physiology; D053612:Janus Kinases / Q000502:physiology; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit / Q000502:physiology; D008024:Ligands; D051379:Mice; D044085:Microfluidics; D008954:Models, Biological; D009994:Osmolar Concentration; D019899:Paracrine Communication; D010766:Phosphorylation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D055153:Stem Cell Niche
chemical_list: C494200:Il6st protein, mouse; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; C507841:Lifr protein, mouse; D008024:Ligands; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D050822:Cytokine Receptor gp130; D053612:Janus Kinases