Adhesive forces in embryonic stem cell cultures

Abstract

Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregatation, rather than "spreading" on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 x 10(5) pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 x 10(4) pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC.

Figure 1

Embyryonic stem cell colonies grow as 3D structures. (A) Micrographs of dome-shaped murine embryonic stem cell (mESC) colonies growing on top of murine embryonic fibroblasts (MEF) and (B) embryonic stem cell (ESC) aggregates in forming embryoid bodies (EB) in suspension culture. Scale bar = 50 µm.

Figure 2

Mouse embryonic fibroblasts synthesize various extracellular matrix proteins. Extracellular matrix (ECM) proteins produced by MEF cells include: (A) fibronectin, (B) laminin, (C) collagen-type I and (D) collagen-type IV. Scale bar = 50 µm.

Figure 3

The antibody binding capacity values correlate with fluorescence intensity values for specific monoclonal antibodies. (A and B) Histograms of the Quantum Simply Cellular (QSC) flow cytometry calibration microbeads stained with (A) PE-conjugated E-cadherin antibodies and (B) FITC-conjugated integrin-β1 antibodies. (C and D) The linear relationship of the measured mean fluorescence intensity (FI) values for a range of antibody binding capacities (ABC) on QSC flow cytometry calibration microbeads were obtained for QSC microbeads stained with (C) PE -conjugated E-cadherin antibodies and (D) FITC-conjugated integrin-β1 antibodies.

Figure 4

ESC express larger numbers of E-cadherin molecules. Histograms of the mESC and MEF stained with (A) PE-conjugated E-cadherin antibodies and (B) FITC-conjugated integrin-β1 antibodies. The MEF do not express either molecule. The mESC express more integrin-β1 when cultured on gelatin (FI = 10) compare with when cultured on MEF (FI = 5, approximately equivalent to autofluorescence for the cells and calibration beads). The ESC express high levels of E-cadherin while cultured on MEF (mean FI = 139) or gelatin (mean FI = 148).

Figure 5

E-cadherin blocking reduces ESC-ESC contacts. Images of ESC colonies cultured (A–C) without E-cadherin blocking antibody and (D and E) with the blocking antibody. Note that the ESC colonies cultured with the blocking antibody are significantly smaller with more cells growing as single cells. Scale bar = 50 µm.