Associate Professor Syracuse University, United States
Introduction:: Organoids are multicellular structures that can be derived from primary cells or human induced pluripotent stem cells (hiPSCs), which can be used to model human organ development and various human pathologies ‘in a dish’. Currently, most organoid culture systems rely on Matrigel, a commercialized matrix composed of various elements including laminin, collagen type IV, and growth factors. However, high batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs strictly limit their use in organoids culture. In this regard, we use synthetic chemically-defined poly(ethylene glycol) (PEG)-based hydrogels to develop functional cardiac organoids and to investigate the role of matrix cues (RGD) and hydrogel stiffness on cardiac organoids functions.
Materials and Methods:: hiPSCs was seeded into ultra-low attachment 96 well plate at the density of 3000, 5000 and 10000 cells/well (day -2) and cultured for 48 h to form aggregates respectively. Cardiac differentiation was initiated by the treatment of 10μM GSK3 inhibitor CHIR99021 in the 96 well plate (day 0), and then the aggregates were encapsulated in the matrix metalloproteinase (MMP)-degradable PEG hydrogels after treatment of 5μM WNT pathway inhibitor IWP4 (day 2). The cardiac organoids were cultured in the RPMI-B27 completed media to day 21. We varied the PEG hydrogel with different stiffness (3%, 5% and 8%) and RGD concentrations (0, 1 and 2 mM). The Live/Dead assay was used to evaluate the viability of the encapsulated aggregates. The GCaMP6f reporter was used to visualize calcium transients in cardiac organoids. The expression of cardiac specific markers was evaluated cryosection and immunostaining.
Results, Conclusions, and Discussions:: The seeded hiPSCs self-assembled to form aggregates within 12 hours, and initial hiPSCs seeding density showed great impact on aggregates dimensions (Figure 1a), but no significant difference in the beating behaviors was detected after differentiation for 21 days. Aggregates also exhibited high viability ( >99%) after encapsulation in the hydrogels for 2 days, as measured by Live/Dead staining (Figure 1f). Incorporation of integrin binding peptide RGD didn’t affect the viability of the encapsulated aggregates but induced the morphological changes of the organoids. Cells migrated out from the organoids in the RGD incorporated hydrogel group, similar to those encapsulated in the Matrigel (Figure 1 c-e). We have been able to obtain robust contracting cardiac organoids from the PEG hydrogels. We observed that beat rate decreased with the increase of hydrogel concentration from 3wt% to 8wt%, but no significant difference on beat rate of the organoids in 3wt% PEG hydrogels with different RGD concentrations (Figure 1b). Intracellular calcium transient measured from GCaMP6f-hiPSCs-derived cardiac organoids further demonstrated that 3wt% PEG hydrogel resulted in higher calcium fluorescent intensity and faster calcium decay than the controls (no hydrogel and Matrigel encapsulation) and other hydrogel concentrations. Positive immunofluorescent staining of cardiomyocyte-specific markers (cardiac troponin T (cTnT), sarcomeric α-actinin, and cardiac troponin I (cTnI)) and smooth muscle specific markers (α-smooth muscle actin (SMA)) indicated the formation of functional cardiac organoids in the PEG hydrogels (Figure 1g, h). In this work, we demonstrated successful development of functional cardiac organoids in the synthetic RGD-incorporated PEG hydrogels with different mechanical properties. We also investigated the effect of hydrogel encapsulation on cardiac organoid formation, viability, and contractile functions.
