title: Microengineered embryonic stem cells niche to induce neural differentiation.
abstract: A major challenge in therapeutic use of embryonic stem cells (ESCs) for treating neurodegenerative diseases is creating a niche in vitro for controlled neural-specific differentiation of ESCs. We employ a niche microengineering approach to derive neural cells from ESCs by mimicking embryonic development in terms of direct intercellular interactions. Using a polymeric aqueous two-phase system (ATPS) microprinting technology, murine ESCs (mESCs) are precisely localized over a monolayer of supporting stromal cells to allow formation of individual mESC colonies. Polyethylene glycol (PEG) and dextran (DEX) are dissolved in culture media to form two immiscible aqueous solutions. A robotic liquid handler is used to print a nanoliter-volume drop of the denser DEX phase solution containing mESCs onto a confluent layer of supporting PA6 stromal cells submerged in the aqueous PEG phase. mESCs proliferate into isolated colonies of uniform size. For the first time, a comprehensive protein expression analysis of individual mESC colonies is performed over a two-week culture period to track temporal progression of cells from a pluripotent stage to specific neural cells. Starting from day 4, the expression of nestin, neural cell adhesion molecule (NCAM), and beta-III tubulin shows a significant increase but then levels off after the first week of culture. The expression of specific neural cell markers glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and tyrosine hydroxylase (TH) is elevated during the second week of culture. This microengineering approach to control ESCs differentiation niche combined with the time-course protein expression analysis of individual differentiating colonies facilitates understanding of evolution of specific neural cells from ESCs and identifying underlying molecular markers.
authors.forename: Ramila
authors.initials: R
authors.lastname: Joshi
authors.forename: Hossein
authors.initials: H
authors.lastname: Tavana
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000166:cytology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D051379:Mice; D064231:Nestin / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D017154:Stromal Cells; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media; D005904:Glial Fibrillary Acidic Protein; C577485:Nes protein, mouse; D064231:Nestin; C528900:glial fibrillary astrocytic protein, mouse; D014446:Tyrosine 3-Monooxygenase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases