title: Microfluidic generated EGF-gradients induce chemokinesis of transplantable retinal progenitor cells via the JAK/STAT and PI3kinase signaling pathways.
abstract: A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400 ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.
authors.affiliation: Biochemistry Doctoral Program, The Graduate School and University Center, City University of New York, New York, New York, United States of America.
authors.forename: Uchenna J
authors.initials: UJ
authors.lastname: Unachukwu
authors.affiliation: Department of Biological Sciences, Herbert Lehman College, City University of New York, Bronx, New York, United States of America.
authors.forename: Moira
authors.initials: M
authors.lastname: Sauane
authors.affiliation: Department of Biomedical Engineering, City College of New York, City University of New York, New York, New York, United States of America.
authors.forename: Maribel
authors.initials: M
authors.lastname: Vazquez
authors.affiliation: Biochemistry Doctoral Program, The Graduate School and University Center, City University of New York, Department of Biological Sciences, Herbert Lehman College, City University of New York, Bronx, New York, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Redenti
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D046210:Microfluidic Analytical Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012160:Retina / Q000166:cytology*; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*
chemical_list: D050791:STAT Transcription Factors; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D048031:JNK Mitogen-Activated Protein Kinases