The ECM evolves during development, aging, injury and disease. Changes to the content, quantity, and organization of the ECM can often direct stem cell fate and cell function. To dissect out the critical parameters that influence cell behavior in 3D environments, biomaterials with independently tunable properties are needed. Here we demonstrate the potential of elastin-like protein biomaterials to be used as an in vitro platform to isolate the effects of crosslinking density on encapsulated hESC-CMs.
Using recombinant protein engineering, we produced clinically relevant biomaterials with repeatable molecular precision and complete bioresorbability. The elastin-like protein provides both elasticity and the stable presentation of cell-adhesion ligands derived from fibronectin while allowing for the independent tuning of crosslinking density by varying crosslinker to protein stoichiometry (). These unique biomaterials were found to support the viability and metabolism (,) of encapsulated human embryoid bodies, enabling sustained expression of a mature cardiomyocyte marker ().
While most studies to date have focused on effects of substrate properties on cardiomyocyte function in 2D, less is known about matrix effects on hESC-CMs in 3D cultures. By changing the crosslinking density, we created a family of 3D matrices with moduli ranging from 0.45 to 2.4 kPa () without altering RGD ligand presentation (1.85 × 10-5 mol/cm3). Despite the small range of elastic moduli explored, we observed differences in transient contractility, suggesting that these cells are sensitive to small changes in matrix stiffness in 3D.
As with all hydrogel materials, changing crosslinking density also alters mesh size and hence the effective diffusion rate of nutrients, waste, and cell-secreted factors whose local accumulation or depletion in the matrix may lead to altered cell metabolism and growth. Confocal microscopic observations of embryoid bodies in hydrogels at all crosslinking densities confirmed the viability of cells throughout the entire gel during 14 days of in vitro
culture (). The isolated, random presence of dead cell clusters and the absence of cell death in the centers of the gels suggest that sufficient diffusion is occurring to support cell metabolism in all matrices. In addition to diffusion effects, changes in mesh size are also known to alter cell morphology and cellular processes affected by cell spreading such as proliferation and migration. For example, the small mesh size of highly crosslinked poly(ethylene glycol) networks physically inhibits cell growth if no proteolytic sites are present in the material 21-22
. Results with other cell-laden 3D hydrogel systems typically observe increased cell proliferation and migration within hydrogels of lower crosslinking densities, although we observed no statistically significant changes in cell metabolism across the range of hydrogels tested here 14, 21, 23
Encapsulated hESC-CMs exhibited bulk, synchronous contractions capable of deforming the bulk hydrogel. Interestingly, we observed a new phenomenon whereby spontaneous cellular contraction was transiently suspended after 3D encapsulation. The duration of this transient suspension was directly correlated with increased crosslinking density (). In a hydrogel network, the work required to deform the material is directly correlated to crosslinking density; therefore, two distinct, yet not mutually exclusive, hypotheses may explain these results. First, the delay in contractility may reflect an adaptation period needed for cardiomyocytes to adjust to a stiffer microenvironment. Over time, the cardiomyocytes may mature and/or adapt to be capable of generating greater contraction forces on the surrounding matrix. Second, the cells may initiate remodeling of their local matrix to yield a more compliant material with lower crosslinking density on which they can then do work. These two hypotheses are not mutually exclusive, and based on previous work by others as described in more detail below, both processes are likely to be occurring simultaneously.
Determining cellular adaptation to the matrix environment is unfortunately harder to elucidate within a 3D matrix as compared to 2D substrates. Evidence of cell interaction and adaptation to the surrounding extracellular matrix has been shown in the form of integrin shedding 24
and cytoskeletal remodeling 25
. Additionally, Jacot et al
. demonstrated an immediate increase in elastic modulus in mouse myocardium after birth 12
. This stiffening is hypothesized to aid in the functional maturation of cardiomyocytes. Uniaxial mechanical stretch has also been shown to increase cardiomyocyte alignment and sarcomeric banding 26
. Thus, cardiomyocytes are capable of sensing and responding to external stimuli by altering their internal contractility machinery. As the field moves in the direction of 3D cultures, new non-destructive assays and technologies will be needed to assess cell adaptation to enable a better understanding of the mechanisms underlying cardiomyocyte maturation and function. Some steps have been made toward this direction with the introduction of 3D particle tracking 27-28
that is capable of visualizing 3D displacement vectors, but data processing can be computationally intensive and interpreting these results still remains a challenge, as matrix modulus is typically assumed to be constant and isotropic.
Rather than adapting to their surroundings, cells are also capable of degrading and remodeling the matrix around them. This ability to alter the surrounding extracellular matrix permits cells to migrate and proliferate, which is critical for developmental and regenerative processes 29
, as well as disease progression 30
. Unfortunately, cell-dictated matrix remodeling often results in local matrix inhomogeneities that cannot be assessed using bulk measurement techniques. Here, we used visual assessments of gels and detection of protease activity by zymography to assess cell-dictated matrix degradation. These results suggest that large-scale matrix remodeling is not occurring; however, this does not preclude local remodeling on the cellular length scale. To visualize matrix remodeling on a cellular length scale, others have utilized specialized techniques involving optical tweezers 31
, microrheology 27, 32
, Förster resonance energy transfer-based protease sensors 33-34
, or non-linear microscopy like second harmonic generation 35-36
. A more rigorous investigation of local cell-remodeling within the elastin-like hydrogels is outside the scope of this study, and left for future exploration.
With a growing interest in the use of embryonic stem cells, tunable systems that mimic nature’s 3D environments are needed to isolate principle parameters that regulate cell-matrix crosstalk. Here we demonstrate that recombinant elastin-like hydrogels are suitable biomaterials for systematic studies of 3D hESC-CM culture. By more fully understanding how hESC-derived cardiomyocytes respond to ECM properties, we may be able to predict how these cells will respond to and integrate with healthy (more compliant) or injured (more rigid) cardiac tissue in the body. In addition, our data demonstrate a new phenomenon of transient suspended contractility upon 3D hESC-CM encapsulation. The encapsulated embryoid bodies were able to overcome this suspended contractility after a time delay that was directly correlated to the matrix crosslinking density. Interestingly, the duration of time spent in the suspended contractility phase did not affect subsequent spontaneous beating frequency or the ability to respond to electrical stimulation. This period of suspended contractility is an intriguing observation, and future investigation into the mechanism of this delay may shed light on hESC-CM adaptability to mechanical properties of their surrounding environment.