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.

Signals from the surface modulate differentiation of human pluripotent stem cells through glycosaminoglycans and integrins

“…Several surfaces have been used for differentiation to specific cell types, such as cardiomyocytes (22,33,41), endothelial and bone cells (36), neurons (38,42), or definitive endoderm (38,43). Although polymers can be produced inexpensively, it can be difficult to characterize or control how these surfaces interact with cells.…”

Organizational metrics of interchromatin speckle factor domains: integrative classifier for stem cell adhesion & lineage signaling

“…Samples were then counterstained with Hoescht to identify osteogenic- and adipogenic-positive cells. Lysed samples were either used to determine ALP concentrations using an ALP activity assay per manufacturer’s instructions (QuantiChromTM), or the relative gene expression of ALP (Qiagen, ALP) or lipoprotein lipase (Qiagen, LPL), as previously reported 62 .…”

Feeder Cell Sources and Feeder-Free Methods for Human iPS Cell Culture

“…For example, a 3-D porous natural polymer scaffold prepared from a chitosan and alginate complex was reported to sustain the self-renewal of human ESCs without the support of feeder cells or conditioned medium [82]. Similarly, Carlson et al [83] reported the combination of poly-D-lysine, which is a synthetic positively charged amino acid chain commonly used as a coating to enhance cell adhesion, with synthetic polymer scaffolds with a 3-D fibrous architecture promote the adhesion, proliferation, and self-renewal of human ESCs. Additionally, microcarrier particles have also been used as substrates to amplify various types of adherent cells [84].…”