Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies

Abstract

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100 -300 m) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.hydrophobicity ͉ self-assembled monolayers ͉ serum-free differentiation T he potential of embryonic stem (ES) cells to differentiate into all specialized cell types has made them attractive models for studying the mechanisms of lineage commitment and has opened pathways for regenerative medicine (1). Various in vitro strategies have been developed for differentiation of ES cells into populations of specific cell types (2). Of these strategies, the formation of three-dimensional cell aggregates known as embryoid bodies (EBs) is a common and critical intermediate to the induction of lineage-specific differentiation (3, 4). In addition, lineage differentiation programs within the EB closely resemble lineage commitment in vivo in the developing embryo, further highlighting the importance of the ES cell-EB culture system (5-8).Although EBs can be generated through several methodologies, the suspension culture technique allows for easy access to the cultured EBs and can be scaled for expansion (9). In this method, EBs are formed when ES cells are removed from feeder contact and dispersed on low-attachment tissue culture plates, supplemented by culture medium absent of key factors necessary for the maintenance of undifferentiated ES cell growth. Lowattachment tissue culture plates typically use neutral, hydrophilic hydrogels to prevent protein adsorption and subsequent cell attachment, facilitating the initial aggregation of ES cells that is critical to EB formation (10). The cellular aggregates formed by this procedure will develop simple EBs that consist of an outer layer of endoderm cells within 2-4 days (3). At this point, two differentiation strategies can be applied. If suspension culture is continued, simple EBs will differentiate further to form cystic EBs that typically contain an inner layer of columnar ectodermlike cells and that accumulate fluid in the interior of the struct...

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

“…3D), while at high cell densities the EB size is primarily between 100 and 300 mm, which has been previously reported to have increased differentiation into various lineages. 53 Additionally, higher rotation speeds demonstrate a trend of an increased number of smaller EBs compared to lower rotation speeds (Fig. 3E).…”

“…3E). Although EB size has been shown to a significant factor in stem cell fate, 53 the flow cytometry data were collected with the same variations in cell seeding density and rotation speeds so that the trends in differentiation are regardless of EB size. We have demonstrated that EB size alone is not the defining factor in ES cell hematopoiesis; even if size differences are not significant, parameters like rotation speed and cell seeding density can have significant influence on differentiation.…”

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

“…Nonetheless, ROCKi is a xeno-factor and has been shown to bias cell fate toward residual pluripotency and inhibit differentiation in neural differentiation studies, reducing the utility of the derived cells for potential clinical applications [30]. To facilitate cell aggregation in suspension for EB formation, nonadhesive culture surfaces were developed [12,31], and soluble factors that promote cell-cell interactions were administered in the culture media [32]. Recently, a serum-free, animal product-free defined medium, mTeSR TM 1, has been developed to support the feeder-independent culture and EB formation from dissociated hESCs [33,34], making it possible to derive clinically relevant human cell lineages from hPSCs in a reproducible manner.…”

Surface Engineering to Control Embryonic Stem Cell Fate

“…Elsewhere, plasma-deposited gradients of octadiene to acrylic acid were fabricated to investigate the effect of carboxylic group (COOH) concentration on mouse ES cell adhesivity and differentiation status (Wells et al, 2009). In addition, by altering the hydrophobicity of a surface, the formation and differentiation potential of ES cells within embryoid bodies (EBs) can be tuned to promote a desirable EB size and composition (Valamehr et al, 2008).…”