Three-Dimensional Extracellular Matrix Stimulates Gastrulation-like Events in Human Embryoid Bodies

Abstract

Human embryonic stem (hES) cells cultured in suspension form aggregates called embryoid bodies (EBs), which structurally resemble the pregastrulation-stage embryo. We show that these EBs express genes characteristic of the visceral endoderm (VE) and form an outer boundary of VE-like cells, but only a minority expresses markers of the primitive streak and the definitive endoderm in patterns suggestive of gastrulation-like events. EBs embedded in a Matrigel matrix, however, lack the presumptive VE but up-regulate expression of gastrulation-related genes, which is correlated with the expression of these genes in a greater proportion of individual EBs. Over time, Matrigelembedded EBs form internally organized structures and exhibit higher expression of markers of the definitive endoderm and mesoderm. We hypothesize that a three-dimensional adhesive support provides an organizing influence analogous to the early basal lamina surrounding the epiblast of the pregastrulation embryo. Furthermore, we identify a synthetic three-dimensional scaffold capable of supporting EB formation that partially mimics the effects of Matrigel.

Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells

“…Development of the three primary germ layers occurs in the epiblast by a process called gastrulation (Figure 1.1B). Gastrulation is initiated in the posterior epiblast by movement of cells through the primitive streak (PS) where cells undergo an epithelial to mesenchymal transition, exiting the PS as mesoderm in the proximal-anterior region of the epiblast and as definitive endoderm in the distal-anterior region (Gadue, Huber et al 2005;Rust, Sadasivam et al 2006;Murry and Keller 2008 In the morula, the inner (blue) cells will form ICM and the outer (pink) cells will form trophoblast. In the early blastocyst, a cavity (the blastocoele, B) forms between the inner cell mass and the trophoblast; the embryo is still enclosed in the zona pellucida (ZP).…”

“…However, while gastrulation occurs in a precise, spatially organized manner during embryogenesis, differentiation of PS-like cells in the EB is spatially chaotic. It is believed that in the embryo distinct signaling environments exist that are defined by location in relation to extraembryonic and embryonic tissues which secrete signals that direct lineage commitment (Rust, Sadasivam et al 2006;Murry and Keller 2008). However, in contrast to the epiblast, EBs lack polarity and as a result spatially disorganized germ layer induction may be due to the lack of position-specific cues.…”

Activin A-Induced Differentiation of Embryonic Stem Cells into Endoderm and Pancreatic Progenitors—The Influence of Differentiation Factors and Culture Conditions

“…However, when BMP4 was applied together with activin A at equal concentrations (50 ng/ml) in the presence of Matrigel, the transcript level of Pdx1 was 10 times higher as with activin A alone. The synergistic effect of activin A and BMP4 disappeared, when EBs formed in the absence of Matrigel resulting in suppression of visceral endoderm [100]. Philips and coworkers speculated that the loss of visceral endoderm formation might provide an environment necessary to realize BMP4 effects on PS patterning and endoderm specification [66].…”

Human pluripotent stem cells: an emerging model in developmental biology

“…For example, forced aggregation of defined numbers of hPSCs or the use of 3D microwell cultures tends to generate EBs that are more uniform in size and morphology (Mohr et al, 2010a;Ng et al, 2005). Another study employed a semi-solid 3D extracellular matrix to support the formation of EBs with more organized germ layer structures (Rust et al, 2006). These technological improvements may lead to the wider use of EBs for studies of early embryogenesis.…”