Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells 英文参考文献.docVIP

MicrofluidicPerfusionforRegulatingDiffusibleSignaling inStemCells KatarinaBlagovic1,LilyY.Kim1,2¤,JoelVoldman1,3 * 1Research Laboratory of Electronics, Massachusetts Institute ofTechnology, Cambridge, Massachusetts, United States of America, 2Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3Department of Electrical Engineering and Computer Science, MassachusettsInstituteofTechnology,Cambridge,Massachusetts,UnitedStatesofAmerica Abstract Background: Autocrine paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonicstemcell(ESC)pluripotencyandlineagecommitment.Althoughautocrinesignalingviafibroblastgrowthfactor- 4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrinesignalingissufficient,orwhetherothersolubleligandsarealsoinvolvedinfatespecification,isunknown.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 approachexploredinthisworkistoleveragetransportphenomenaatcellularresolutiontodownregulateoveralldiffusible signalingthroughthephysicalremovalofcell-secretedligands. Methodology/Principal Findings: We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine)factorstodownregulatediffusiblesignaling.Bycomparingcellgrowthanddifferentiationinside-by- sidechamberswithorwithoutaddedcell-secretedfactors,weisolatetheeffectsofdiffusiblesignalingfromartifactssuchas shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermalspecification.ThenweinduceFGF4signalinginminimalchemicallydefinedmedium(N2B27)andinhibit FGFsignalinginfullysupplementeddifferentiationmediumwithcell-secretedfact