Directed Differentiation of Size‐Controlled Embryoid Bodies Towards Endothelial and Cardiac Lineages in RGD‐Modified Poly(Ethylene Glycol) Hydrogels

Abstract

Recent advances in stem cell research have demonstrated the importance of microenvironmental cues in directing stem cell fate towards specific cell lineages. For instance, the size of the embryoid body (EB) was shown to play a role in stem cell differentiation. Other studies have used cell adhesive RGD peptides to direct stem cell fate towards endothelial cells. In this study, materials and cell-based approaches are combined by using microwell arrays to produce size-controlled EBs and encapsulating the resulting aggregates in high molecular weight PEG-4 arm acrylate with and without conjugated RGD to study their effect on stem cell differentiation in a 3D microenvironment. Increasing EB size is observed along with a decrease in the total number of EBs in pristine PEG hydrogel, regardless of the initial EB size. In correlation with this aggregation, EBs in PEG show enhanced cardiogenic differentiation compared to RGD-PEG hydrogel. Both aggregation and cardiogenic differentiation are significantly reduced when RGD peptides are introduced to the microenvironment, while endothelial cell differentiation is accelerated by 3 to 5 days, depending on the EB size, and doubled over the course of cell culture for both EB sizes. Presented results indicate that RGD sequence has a dominant effect in driving endothelial cell differentiation in size-controlled EBs, while pristine multi-arm, high molecular weight PEG can induce cardiogenic differentiation, possibly through EB aggregation. The photopatternable nature of the hydrogel used in this study enabled patterning of such domains devoid or abundant of cell attachment sequences. Therefore, these hydrogels can potentially be used for spatially patterned embryonic stem cell differentiation, which may be beneficial for tissue engineering and regenerative medicine applications.

Direct 3D bioprinting of perfusable vascular constructs using a blend bioink

“…For example, a tri-layered coaxial nozzle is the key to successful deposition of perfusable structures, and the PEGTA component used in this study is also superior to commonly used linear PEG derivatives through enhanced crosslinking density and thus increased mechanical strength while maintaining the beneficial porous structure, due to its branched tetravalent chemical structure and multiple active crosslinking sites. This porous structure could induce better cell growth and spreading compared to hydrogels based on conventional PEG-diacrylate, which was proved in our previous study [45]. By combining benefits of both natural and synthetic biomaterials, this bioink formulation displayed favorable biochemical characteristics for survival and proliferation of encapsulated vascular cells and tunable mechanical properties for bioprinting complex perfusable vascular constructs, inducing the formation of biologically relevant, highly organized, intact vessels.…”

Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering

“…Bioactive moieties in the scaffold play a key role in signal transduction between matrix and cells. A variety of peptides have been incorporated into the bioinert PEG network in order to elicit specific responses of resident or recruited cells by mimicking their microenvironment [43, 53, 54]. In particular, RGDS, which serves as a ligand for several integrins [55], has been widely used to support adhesion of a variety of cells to different substrates [56-59].…”

Cell-laden microfluidic microgels for tissue regeneration

“…135 Furthermore, the monodispersity of microfluidic droplets is particularly important in microscale tissue engineering, in which the spheroid size influences stem cell behavior and differentiation potential. 143,144 …”