Poster AA15 - Hydrogel supported hiPSC differentiation impacts cardiac contractile functionality and cell type composition based on single-cell transcriptomics

Introduction: Scalable production of chamber specific cardiomyocytes (CMs) is necessary for disease modeling and regenerative therapies. Human induced pluripotent stem cells (hiPSCs) have been successfully differentiated to yield cardiomyocytes; however, the information about whether the resulting cardiomyocytes belong to the left or right ventricle remains unknown. The advent of scRNA seq has enabled us to identify cell populations when hiPSCs undergo cardiac differentiation. Building on our previous work of producing 3D engineered cardiac tissues, in this work we investigated the different cell types produced when hiPSCs are differentiated in 3D engineered tissue microspheres (MS) vs scaffold-free tissues (aggregates) as well as assessing and comparing the resulting functionality and CM yield of these two 3D differentiation methods. Robust cardiac differentiation protocols have been around for a decade and 2D cardiac differentiation was found to yield almost exclusively left ventricular (LV) CMs when employing the standard B27 cardiac differentiation protocol ; however, cardiomyocyte sub-type populations generated during 3D differentiation using this protocol have yet to be investigated.

Materials and

Methods: MS hiPSC engineered tissues were produced using a microfluidic encapsulation system which utilizes oil and water emulsion technique. This system consists of a device made up of polydimethylsiloxane (PDMS) containing two inlets, an outlet, and a T-junction. HiPSCs were mixed with poly(ethylene glycol) fibrinogen (PF) precursor solution (40 million cells/mL PF) were pumped into the top inlet of the PDMS device while oil was pumped into the bottom inlet. MS are formed at the T-junction due to emulsification and travel towards the outlet channel where they photocrosslink as a result of exposure to visible light. Self-aggregated hiPSC tissues were made by seeding approximately 3 million cells in a shaker flask placed on a shaker plate.
Following hiPSC encapsulation (MS) or shaker flask initiation of forced aggregation (aggregates), tissues were cultured for 3 days prior to initiation of differentiation. Using flow cytometry, MS and aggregate tissues were analyzed for Ki67 (proliferation marker) and Oct4 (pluripotency marker) on differentiation day (DD) 0 and for cardiac troponin T (cTnT) marker on DD 10. On DD 5 and 15, tissues were dissociated and singularized to be analyzed for single cell RNA sequencing (scRNA seq). Contraction analysis based on video microscopy was performed on DD 10 and 15 . Tissues were also optically mapped on DD45 to measure electrophysiological properties.

Results, Conclusions, and Discussions: Here, we investigated how providing a 3D hydrogel matrix affects cell population, functionality, and cardiomyocyte yield when 3D tissues formed using hiPSCs undergo cardiac differentiation. As seen from flow cytometry analysis, the MS were able to produce a higher percentage of cardiomyocytes as compared to the aggregates; on DD 10, CM yield (average of 3 batches) in MS was 60.7% compared to 47.8% in aggregates observed by flow cytometry analysis for cTnT marker. DD 15 tissue cryosections showed positive staining for cTnT and α-smooth muscle actinin . In addition to CM yield, we also investigated the functional differences in the two tissue types. MS cardiac tissues had higher contraction and relaxation velocities compared to the aggregates; contraction and relaxation velocities for MS cardiac tissues were 88.9±5.8 µm/s and 81.6±4.0 µm/sec respectively, compared to aggregates with 48.0 ±12.9 µm/s and 38.9±22.4 µm/sec respectively. To measure electrophysiological properties, tissues were optically mapped on DD 45; MS cardiac tissues were able to be paced up to 3 Hz. Higher calcium transition velocities and lower calcium transition durations were observed in MS cardiac tissues compared to aggregates. From initial scRNA seq analysis, a higher endodermal population was observed in aggregates compared to MS, whereas MS had a larger endothelial cell population, which has been found to be important in engineered cardiac tissue formation. Aggregates also resulted in higher expression of type III and type V collagen compared to microspheres. Almost all the resulting cardiomyocytes in both the platforms (MS and aggregates) were found to be left ventricular.

Acknowledgements (Optional):