title: A biomimetic synthetic feeder layer supports the proliferation and self-renewal of mouse embryonic stem cells.
abstract: Successful realization of the enormous potential of pluripotent stem cells in regenerative medicine demands the development of well-defined culture conditions. Maintenance of embryonic stem cells (ESCs) typically requires co-culture with feeder layer cells, generally mouse embryonic fibroblasts (MEFs). Concerns about xenogeneic pathogen contamination and immune reaction to feeder cells underlie the need for ensuring the safety and efficacy of future stem cell-based products through the development of a controlled culture environment. To gain insight into the effectiveness of MEF layers, here we have developed a biomimetic synthetic feeder layer (BSFL) that is acellular and replicates the stiffness and topography of MEFs. The mechanical properties of MEFs were measured using atomic force microscopy. The average Young's modulus of the MEF monolayers was replicated using tunable polyacrylamide (PA) gels. BSFLs replicated topographical features of the MEFs, including cellular, subcellular, and cytoskeletal features. On BSFLs, mouse ESCs adhered and formed compact round colonies; similar to on MEF controls but not on Flat PA. ESCs on BSFLs maintained their pluripotency and self-renewal across passages, formed embryoid bodies and differentiated into progenitors of the three germ layers. This acellular biomimetic synthetic feeder layer supports stem cell culture without requiring co-culture of live xenogeneic feeder cells, and provides a versatile, tailorable platform for investigating stem cell growth.
  
  
  
  Embryonic stem cells have enormous potential to aid therapeutics, because they can renew themselves and become different cell types. This study addresses a key challenge for ESC use - growing them safely for human patients. ESCs typically grow with a feeder layer of mouse fibroblasts. Since patients have a risk of immune response to feeder layer cells, we have developed a material to mimic the feeder layer and eliminate this risk. We investigated the influence of feeder layer topography and stiffness on mouse ESCs. While the biomimetic synthetic feeder layer contains no live cells, it replicates the stiffness and topography of feeder layer cells. Significantly, ESCs grown on BSFLs retain their abilities to grow and become multiple cell types.
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA; Center for Biomedical Engineering, Brown University, Providence, RI 02912, USA. Electronic address: clopezfagundo@gmail.com.
authors.forename: Cristina
authors.initials: C
authors.lastname: López-Fagundo
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA. Electronic address: liane_livi@brown.edu.
authors.forename: Liane L
authors.initials: LL
authors.lastname: Livi
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA. Electronic address: talisha.ramchal@duke.edu.
authors.forename: Talisha
authors.initials: T
authors.lastname: Ramchal
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA; Center for Biomedical Engineering, Brown University, Providence, RI 02912, USA; School of Engineering, Brown University, Providence, RI 02912, USA; Department of Orthopaedics, Brown University, Providence, RI 02912, USA. Electronic address: Eric_Darling@brown.edu.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Darling
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA; Center for Biomedical Engineering, Brown University, Providence, RI 02912, USA; School of Engineering, Brown University, Providence, RI 02912, USA; Brown Institute for Brain Science, Brown University, Providence, RI 02912, USA. Electronic address: dhk@brown.edu.
authors.forename: Diane
authors.initials: D
authors.lastname: Hoffman-Kim
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D040761:Biomimetic Materials / Q000737:chemistry*; D018929:Cell Culture Techniques / Q000379:methods*; D055119:Elastic Modulus; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D000099166:Polyacrylamides
keywords: Embryonic stem cells; Feeder layer; Mouse embryonic fibroblast; Polyacrylamide