title: Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.
abstract: Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.
authors.affiliation: Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Carlson
authors.forename: Charles A
authors.initials: CA
authors.lastname: Florek
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Kim
authors.forename: Thomas
authors.initials: T
authors.lastname: Neubauer
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Moore
authors.forename: Rick I
authors.initials: RI
authors.lastname: Cohen
authors.forename: Joachim
authors.initials: J
authors.lastname: Kohn
authors.forename: Martin
authors.initials: M
authors.lastname: Grumet
authors.forename: Prabhas V
authors.initials: PV
authors.lastname: Moghe
mesh_terms: D001704:Biopolymers; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D003094:Collagen; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007797:Laminin / Q000096:biosynthesis; D011107:Polylysine / Q000494:pharmacology; D011509:Proteoglycans; D013237:Stereoisomerism; D054457:Tissue Scaffolds; D014443:Tyrosine / Q000031:analogs & derivatives
chemical_list: D001704:Biopolymers; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C505373:poly(desaminotyrosyl-tyrosine-ethyl ester carbonate); C059485:matrigel; D011107:Polylysine; D014443:Tyrosine; D003094:Collagen