(G-271) Hyaluronan Improves the Functional Properties of iPS-derived Cardiac Microtissues in a 3D Heterotypic Environment

Introduction:: The cardiac microenvironment is tightly regulated by spatiotemporally controlled biomechanical factors, which include fluid shear forces, cellular responses, and surrounding extracellular matrix (ECM) components. These finely tuned components play a significant role in cardiac development and disease progression. Biomechanical alterations lead to morphological and functional malfunctions such as those seen in congenital heart defects (CHD). Here we explore the role of wall shear stress on myocardial functional development or disease initiation in iPSC-derived cardiomyocytes by utilizing microfluidic devices containing three-dimensional (3D) ECM hydrogels and stromal cell populations (fibroblasts and endothelial cells).


Materials and Methods:: Human iPSCs (WTC11 line with encoded Ca⁺² GCaMP reporter, Bruce Conklin, Gladstone institute) were differentiated into cardiomyocytes by modulating the Wnt pathway using small molecules (CHIR, IWP2). Progenitor iPSC-CMs at day 7 were dissociated and combined with cardiac fibroblasts at a 3:1 ratio and the cell suspension was seeded on agarose microwell molds (400x400μm), to form spherical aggregates (150μm diameter, 3500 cells/aggregate). Aggregates were transferred to a rotary shaker (65rpm) overnight, where they resumed beating. The myocardial aggregates were suspended in 3D ECM hydrogel containing 1.5mg/ml collagen I and various concentrations of hyaluronic acid (0.5, 2 and 10 mg/ml) and a stiffness of 15-20kpa. For dynamic conditions, a multilayer microfluidic device was fabricated and assembled with a well to house the ECM hydrogel in the middle PDMS layer with a microchannel layer on the top. The hydrogel-aggregate suspension was injected into the ECM well in a microfluidic device, which was then incubated at 37°C for 60 mins to allow ECM polymerization and beating resumption in aggregates. Devices were subjected to shear stress forces of 0.5,1, and 2 dyne/cm² by controlling medium flow rates with a peristaltic pump. Static cultures in the 24 well plates were maintained as a control. Calcium handling and contractility analysis using MUSCLEMOTION was performed on videos obtained at 100 fps at 24, 48, and 72 hr. Immunofluorescence imaging (cTnT) (Fig1, bottom panel) helped to visualize the structural changes in the myocardial cytoskeleton after shear exposure. Flow cytometry was used to establish the purity of CM differentiations.

Results, Conclusions, and Discussions:: Dynamic conditions led to contractility arrest without hyaluronan in the ECM hydrogels (Fig 1, top panel (green represents GCaMP autofluorescence). Static, 3D culture conditions were found to be conducive in maintaining contractile function and structural integrity in cardiac microtissue. A shear stress rate of 0.5 dynes/cm², when applied for 24-48hr, led to an increase in contraction amplitude, peak-to-peak interval, and time to peak in calcium handling properties (Fig 2). With hyaluronan in ECM hydrogels, exposure to 0.5 dyne/cm² shear stress for 48hrs. preserved contractility but made calcium handling properties more fetal-like. Different concentrations of Hyaluronan in ECM hydrogel also affected the calcium handling properties and morphologically sarcomere structures quantified through confocal microscopy of cTnT. Hyaluronan concentration temporarily spikes during earlier stage of heart development (heart tube stage) to sustain hydrostatic homeostasis. In static culture experiments, relation between higher HA concentration in our ECM hydrogels and observation of increasing fetal-like calcium handling properties shows the recapitulation of physiologic phenomenon. In dynamic cultures, loss of contractility can be linked with the activation of disease pathways in cardiac development. This is being further investigated by probing expression of flow-sensitive genes and fibrosis markers. Microtissue structural disintegration with reduction in cell volume and outward cell migration under dynamic conditions reiterate the potential role of fluid-generated forces with morphological rearrangement in the process of tissue specification.

Our model embodies key factors in cardiac microenvironment imperative to understand cardiac development. It offers an excellent platform for testing cardiovascular development, disease progression and new therapeutics.




Acknowledgements (Optional): : This work was supported by Fulbright PhD grant, NSF CMMI 1919438 and NIH-R15HL150745.


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