Hydrogel crosslinking density regulates temporal contractility of human embryonic stem cell-derived cardiomyocytes in 3D cultures
ABSTRACT Systematically tunable in vitro platforms are invaluable in gaining insight to stem cell-microenvironment interactions in three-dimensional cultures. Utilizing recombinant protein technology, we independently tune hydrogel properties to systematically isolate the effects of matrix crosslinking density on cardiomyocyte differentiation, maturation, and function. We show that contracting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) remain viable within four engineered elastin-like hydrogels of varying crosslinking densities with elastic moduli ranging from 0.45 to 2.4 kPa. Cardiomyocyte phenotype and function was maintained within hESC embryoid bodies for up to 2 weeks. Interestingly, increased crosslinking density was shown to transiently suspend spontaneous contractility. While encapsulated cells began spontaneous contractions at day 1 in hydrogels of the lowest crosslinking density, onset of contraction was increasingly delayed at higher crosslinking densities up to 6 days. However, once spontaneous contraction was restored, the rate of contraction was similar within all materials (71 ± 8 beats/min). Additionally, all groups successfully responded to electrical pacing at both 1 and 2 Hz. This study demonstrates that encapsulated hESC-CMs respond to 3D matrix crosslinking density within elastin-like hydrogels and stresses the importance of investigating temporal cellular responses in 3D cultures.
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ABSTRACT: Hydrogels have been developed as extracellular matrix (ECM) mimics both for therapeutic applications and basic biological studies. In particular, elastin-like polypeptide (ELP) hydrogels, which can be tuned to mimic several biochemical and physical characteristics of native ECM, have been constructed to encapsulate various types of cells to create in vitro mimics of in vivo tissues. However, ELP hydrogels become opaque at body temperature because of ELP's lower critical solution temperature behavior. This opacity obstructs light-based observation of the morphology and behavior of encapsulated cells. In order to improve the transparency of ELP hydrogels for better imaging, we have designed a hybrid ELP-polyethylene glycol (PEG) hydrogel system that rapidly crosslinks with tris(hydroxymethyl) phosphine (THP) in aqueous solution via Mannich-type condensation. As expected, addition of the hydrophilic PEG component significantly improves the light transmittance. Coherent Anti-Stokes Raman Scattering (CARS) microscopy reveals that the hybrid ELP-PEG hydrogels have smaller hydrophobic ELP aggregates at 37 °C. Importantly, this hydrogel platform enables independent tuning of adhesion ligand density and matrix stiffness, which is desirable for studies of cell-matrix interactions. Human fibroblasts encapsulated in these hydrogels show high viability (> 98%) after 7 days of culture. High-resolution confocal microscopy of encapsulated fibroblasts reveals that the cells adopt a more spread morphology in response to higher RGD ligand concentrations and softer gel mechanics.Biomacromolecules 08/2014; 15(9). DOI:10.1021/bm500969d · 5.79 Impact Factor
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ABSTRACT: The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell-material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed.Interface focus: a theme supplement of Journal of the Royal Society interface 04/2014; 4(2):20130038. DOI:10.1098/rsfs.2013.0038 · 3.12 Impact Factor
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ABSTRACT: Cellular therapies have great potential to provide alternative treatment options for those suffering from heart disease. In order to optimize cell delivery for therapeutic efficacy, a greater understanding of parameters that impact stem cell differentiation, survival, growth, and development are needed. In this study, we examine the role of hydrogel crosslink density on spontaneous cardiomyocyte (CM) differentiation of murine embryoid bodies (EBs). CM differentiation was accelerated in hydrogels of low crosslink density, where 100% of the hydrogels were positive for CM differentiation compared to only 53% in the high crosslink density group after 8 days of culture. DNA microarray data suggests that enhanced CM differentiation in the low crosslink density hydrogels was not tissue specific but rather a result of favoured EB development and cell proliferation. Additionally, enhanced EB growth and differentiation in low crosslink density hydrogels was independent of RGD ligand density and not a consequence of enhanced diffusion. We also demonstrate that matrix metalloproteinase activity is required for spontaneous CM differentiation in 3D hydrogels. Low hydrogel crosslink density regulates spontaneous EB differentiation by promoting EB growth and development. Elucidating the effects of microenvironmental cues on cell differentiation can aid in the optimization of stem cell-based therapies for tissue regeneration.10/2013; 1(10):1082-1090. DOI:10.1039/C3BM60139K