title: Colony size effect on neural differentiation of embryonic stem cells microprinted on stromal cells.
abstract: Controlling cellular microenvironment to induce neural differentiation of embryonic stem cells (ESCs) remains a major challenge. We address this need by introducing a micro-engineered co-culture system that resembles embryonic development in terms of direct intercellular interactions and induces neural differentiation of ESCs. A polymeric aqueous two-phase system (ATPS)-mediated robotic microprinting technology allows precise localization of mouse ESCs (mESCs) over a layer of supporting stromal cells. mESCs proliferate over a 2-week culture period into a single colony of defined size. Physical and chemical cues from the stromal cells guide mESCs to differentiate toward specific neural lineages. We generated mESC colonies of three different sizes from 100, 250 and 500 single cells and showed that size of mESC colonies is an important factor determining the yield of neural cells. Expression of early neural cell markers nestin denoting neural stem cells, NCAM specifying neural progenitors, and β-III tubulin (TuJ) indicating post mitotic neurons escalated from day 4. Differentiation into specific neural cells astrocytes marked by GFAP, oligodendrocytes indicated by CNPase, and TH-positive dopaminergic neurons was observed during the second week of culture. Unexpectedly, analysis of protein expression revealed a disproportionate increase in neural differentiation of mESCs by increase in the colony size. For the first time, our study establishes colony size as an important regulator of fate of ESCs in this heterocellular niche. This approach of deriving neural cells may make a major impact on stem cell research for treating neurodegenerative diseases.
authors.forename: Ramila
authors.initials: R
authors.lastname: Joshi
authors.forename: James
authors.initials: J
authors.lastname: Buchanan
authors.forename: Hossein
authors.initials: H
authors.lastname: Tavana
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D051379:Mice; D008856:Microscopy, Fluorescence; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D064231:Nestin / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
chemical_list: D064231:Nestin; D014404:Tubulin; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases