(M-488) High Throughput Micropatterning of Chamber-specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Cardiac Fibroblasts Within Multiwell Plates

Introduction:: Given shortages of primary human cardiac tissue and the inadequacy of animal models for modeling human responses, human induced pluripotent stem cell (iPSC) derived cardiac tissues are critical for drug screening, disease modeling, and regenerative medicine. Several protocols have been established to differentiate iPSCs into ventricular or atrial cardiomyocytes (CM); additionally, protocols have been established to differentiate iPSCs into cardiac fibroblast (CF) protocols via different cardiac progenitor pathways including the epicardium, shown to produce a large portion of fibroblast in the heart, and the second heart field (SHF), shown to contribute to atria formation. Advanced culture techniques can improve the functional maturation of iPSC-CMs but often these platforms are low throughput, require expensive equipment, and/or are incompatible with established analysis techniques; furthermore, these techniques are not optimized for CM sub-types and supporting cell types. Here, we demonstrate a high throughput micropatterning technique for the co-culture of iPSC-derived CMs and CFs in a standard well plate format for screening applications.

Materials and Methods:: iPSCs were differentiated into atrial or ventricular CMs via published protocols. Next, these CMs were dissociated and replated onto standard tissue culture plates with micropatterned fibronectin and bovine serum albumin (BSA) in alternating 70-75µm wide parallel lines, generated using a custom soft-lithographic process. The CMs preferentially attached to fibronectin patterns and were subsequently co-cultured with adult atrial or ventricular fibroblasts or iPSC derived CFs. The CFs preferentially attached to the BSA regions. The iPSC-derived CFs were differentiated using two different published protocols that use different progenitor cell pathways: the epicardium and second heart field progenitors. The cell phenotype was assessed using immunostaining analysis, contractility via pixel motion tracking, and optical voltage mapping. The surface of microelectrode arrays (MEA) was micropatterned with fibronectin and CM field potentials were measured.


Results, Conclusions, and Discussions:: Results and Discussion: iPSC derived CMs and CF expressed prototypical markers confirming their identity (Figure 1A). The BSA based micropatterning technique shown here allows for the micropatterned plates to be created faster and easier than published techniques (Figure 1B). Co-cultures CMs and CF showed specific binding of CMs on the fibronectin lines and CF on the BSA lines (Figure 1C). These micropatterned co-cultures (PC-N) show improved contractility over published micropatterned protocols (PC-S), random monocultures (RM) and random co-cultures (RC) as approximated by the mean magnitude of the velocity vector of pixel movements (Figure 1D, E). Lastly, cryopreserved CMs preferentially attached to fibronectin patterns on MEA (Fig. 1F). Electrophysiological data was obtained from the micropatterned CMs with waveforms similar to fresh matured cells (Figure 1G).



Conclusions: Here we show a novel technique for micropatterning iPSC-CMs and CFs in a high-throughput manner within industry-standard multiwell plates. We show enhanced iPSC-CM function in these micropatterned co-cultures. Future efforts are focused on a) utilizing these micropatterned co-cultures for modeling diseases such as fibrosis and atrial fibrillation by utilizing iPSC lines generated from patients with disease causing mutations, and b) incorporating iPSC-derived endothelial cells into the platform to elucidate multicellular crosstalk.


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