Marita L. Rodriguez

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells

BACKGROUND Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. METHODS AND RESULTS A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. CONCLUSIONS Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens.

Decoupling Substrate Stiffness, Spread Area, and Micropost Density: A Close Spatial Relationship between Traction Forces and Focal Adhesions

Mechanical cues can influence the manner in which cells generate traction forces and form focal adhesions. The stiffness of a cells substrate and the available area on which it can spread can influence its generation of traction forces, but to what extent these factors are intertwined is unclear. In this study, we used microcontact printing and micropost arrays to control cell spreading, substrate stiffness, and post density to assess their effect on traction forces and focal adhesions. We find that both the spread area and the substrate stiffness influence traction forces in an independent manner, but these factors have opposite effects: cells on stiffer substrates produce higher average forces, whereas cells with larger spread areas generate lower average forces. We show that post density influences the generation of traction forces in a manner that is more dominant than the effect of spread area. Additionally, we observe that focal adhesions respond to spread area, substrate stiffness, and post density in a manner that closely matches the trends seen for traction forces. This work supports the notion that traction forces and focal adhesions have a close relationship in their response to mechanical cues.

Let-7 family of microRNA is required for maturation and adult-like metabolism in stem cell-derived cardiomyocytes.

Significance The adult human heart is incapable of significant regeneration after injury. Human embryonic stem cells (hESCs) have the capacity to generate an unlimited number of cardiomyocytes (CMs). However, hESC-derived CMs (hESC-CMs) are at a fetal state with respect to their functional and physiological characteristics, diminishing their utility for modeling adult-related heart disease and therapeutic screening. Thus, the potential for hESC-CMs may improve immensely in cardiac-related therapeutic applications if factors that drive their maturation are uncovered. In this study, we show that members of let-7 miRNA family control CM metabolism, cell size, and force contractility, making them one of the best factors identified to date in promoting maturity of stem cell derivatives. In metazoans, transition from fetal to adult heart is accompanied by a switch in energy metabolism-glycolysis to fatty acid oxidation. The molecular factors regulating this metabolic switch remain largely unexplored. We first demonstrate that the molecular signatures in 1-year (y) matured human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are similar to those seen in in vivo-derived mature cardiac tissues, thus making them an excellent model to study human cardiac maturation. We further show that let-7 is the most highly up-regulated microRNA (miRNA) family during in vitro human cardiac maturation. Gain- and loss-of-function analyses of let-7g in hESC-CMs demonstrate it is both required and sufficient for maturation, but not for early differentiation of CMs. Overexpression of let-7 family members in hESC-CMs enhances cell size, sarcomere length, force of contraction, and respiratory capacity. Interestingly, large-scale expression data, target analysis, and metabolic flux assays suggest this let-7–driven CM maturation could be a result of down-regulation of the phosphoinositide 3 kinase (PI3K)/AKT protein kinase/insulin pathway and an up-regulation of fatty acid metabolism. These results indicate let-7 is an important mediator in augmenting metabolic energetics in maturing CMs. Promoting maturation of hESC-CMs with let-7 overexpression will be highly significant for basic and applied research.

Review on Cell Mechanics: Experimental and Modeling Approaches

The interplay between the mechanical properties of cells and the forces that they produce internally or that are externally applied to them play an important role in maintaining the normal function of cells. These forces also have a significant effect on the progression of mechanically related diseases. To study the mechanics of cells, a wide variety of tools have been adapted from the physical sciences. These tools have helped to elucidate the mechanical properties of cells, the nature of cellular forces, and mechanoresponses that cells have to external forces, i.e., mechanotransduction. Information gained from these studies has been utilized in computational models that address cell mechanics as a collection of biomechanical and biochemical processes. These models have been advantageous in explaining experimental observations by providing a framework of underlying cellular mechanisms. They have also enabled predictive, in silico studies, which would otherwise be difficult or impossible to perform with current experimental approaches. In this review, we discuss these novel, experimental approaches and accompanying computational models. We also outline future directions to advance the field of cell mechanics. In particular, we devote our attention to the use of microposts for experiments with cells and a bio-chemical-mechanical model for capturing their unique mechanobiological properties. [DOI: 10.1115/1.4025355]

Measuring the Contractile Forces of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes With Arrays of Microposts

Human stem cell-derived cardiomyocytes hold promise for heart repair, disease modeling, drug screening, and for studies of developmental biology. All of these applications can be improved by assessing the contractility of cardiomyocytes at the single cell level. We have developed an in vitro platform for assessing the contractile performance of stem cell-derived cardiomyocytes that is compatible with other common endpoints such as microscopy and molecular biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded onto elastomeric micropost arrays in order to characterize the contractile force, velocity, and power produced by these cells. We assessed contractile function by tracking the deflection of microposts beneath an individual hiPSC-CM with optical microscopy. Immunofluorescent staining of these cells was employed to assess their spread area, nucleation, and sarcomeric structure on the microposts. Following seeding of hiPSC-CMs onto microposts coated with fibronectin, laminin, and collagen IV, we found that hiPSC-CMs on laminin coatings demonstrated higher attachment, spread area, and contractile velocity than those seeded on fibronectin or collagen IV coatings. Under optimized conditions, hiPSC-CMs spread to an area of approximately 420 μm2, generated systolic forces of approximately 15 nN/cell, showed contraction and relaxation rates of 1.74 μm/s and 1.46 μm/s, respectively, and had a peak contraction power of 29 fW. Thus, elastomeric micropost arrays can be used to study the contractile strength and kinetics of hiPSC-CMs. This system should facilitate studies of hiPSC-CM maturation, disease modeling, and drug screens as well as fundamental studies of human cardiac contraction.

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.

Micropost arrays for measuring stem cell-derived cardiomyocyte contractility

Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes.

Enhanced contractility with 2-deoxy-ATP and EMD 57033 is correlated with reduced myofibril structure and twitch power in neonatal cardiomyocytes

As cardiomyocytes mature, their sarcomeres and Z-band widths increase in length in order for their myofibrils to produce stronger twitch forces during a contraction. In this study, we tested the hypothesis that tensional homeostasis is affected by altering myofibril forces. To assess this hypothesis, neonatal rat cardiomyocytes were cultured on arrays of microposts to measure cellular contractility. An optical line scanning technique was used to measure the deflections in the microposts with high temporal resolution, enabling the analysis of twitch force, twitch velocity, and twitch power. Myofibril force production was elevated by vector-mediated overexpression of ribonucleotide reductase (RR) to increase cellular dATP content or by adding the inotropic agent EMD 57033 (EMD). We found that RR and EMD treatment did not affect cardiomyocyte twitch force, but it did lead to reduced twitch velocity and twitch power. Immunofluorescent analysis of α-actinin revealed that RR-over-expressing cardiomyocytes and EMD-treated cardiomyocytes had lower spread area, sarcomere length, and Z-band width as compared to control cells. These results indicate a correlation between myofibril structure and cardiac power. This correlation was confirmed by exposing the cells to the myosin II inhibitor blebbistatin, and then subsequently washing it out. After wash-out, cardiomyocytes exhibited a reduction in twitch force, velocity, and power due to shorter sarcomere length and Z-band widths. Our results suggest that cardiac myofibril structure is regulated by tensional homeostasis. If myofibril-generated forces in cardiomyocytes are elevated, a state of tensional homeostasis is maintained by producing sufficient twitch forces with a lower degree myofibril structure.

Effect of Silanization Film Thickness in Soft Lithography of Nanoscale Features

Soft lithography was used to replicate nanoscale features made using electron beam lithography on a polymethylmethacrylate (PMMA) master. The PMMA masters were exposed to fluorinated silane vapors to passivate its surfaces so that polydimethylsiloxane (PDMS) did not permanently bond to the master. From scanning electron microscopy, the silanization process was found to deposit a coating on the master that was a few hundreds of nanometers thick. These silane films partially concealed the nanoscale holes on the PMMA master, causing the soft lithography process to produce PDMS features with dimensions that were significantly reduced. The thickness of the silane films was directly measured on silicon or PMMA masters and was found to increase with exposure time to silane vapors. These findings indicate that the thickness of the silane coatings is a critical parameter when using soft lithography to replicate nanoscale features, and caution should be taken on how long a master is exposed to silane vapors. [DOI: 10.1115/1.4005665]

Spatial and temporal coordination of traction forces in one-dimensional cell migration

ABSTRACT Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.