A Chemically Defined PEG Hydrogels for Human Cardiac Organoids Development

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, which often leads to high batch-to-batch variations, poor stability, cumbersome handling, and the relatively high costs. Synthetic poly (ethylene glycol; PEG) hydrogels have emerged as a Matrigel alternative, as they provide control over mechanical stiffness, biochemical composition, and degradability. As a result, PEG hydrogels have been widely used as a synthetic matrix for organoids culture, such as brain and intestinal organoids. However, few studies have been reported on the development of cardiac organoids within the defined hydrogel systems. In this study, we use synthetic PEG-based hydrogels to develop functional cardiac organoids from hiPSCs 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 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 encapsulated cardiac organoids were cultured in the RPMI-B27 completed media till day 21. We varied the PEG hydrogels 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 hiPSC line was used to evaluate the contractile functions in cardiac organoids (contractile motion and calcium transient). The expression of cardiac specific markers was evaluated via cryosection and immunostaining. We also performed bulk RNA sequencing (RNAseq) to elucidate the transcriptomics difference between the organoids generated from different culture conditions (PEG-encapsulated organoids, Matrigel-encapsulated organoids, and suspension-cultured organoids).

Results, Conclusions, and Discussions: The seeded hiPSCs self-assembled to form aggregates within 12 hours and exhibited high viability (>99%) after encapsulation in the hydrogels for 2 days, as measured by Live/Dead staining. Incorporation of integrin binding peptide RGD didn’t affect the viability of the encapsulated aggregates but induced the morphological changes of the organoids (Figure 1A). Cells migrated out from the organoids in the RGD incorporated hydrogel group, similar to those encapsulated in the Matrigel (Figure 1B). We have been able to obtain robust contracting cardiac organoids from the PEG hydrogels. The 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. 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 (suspension) 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 1C). According to the bulk RNA sequencing results, principal component analysis (PCA) (Figure 1D) and hierarchical clustering (Figure 1E) showed significant difference in gene expression patterns between the cardiac organoids encapsulated within 3wt% PEG hydrogels with/out 2 mM RGD, Matrigel and suspension. In all these 4 groups, organoids in 3wt% PEG hydrogels with 2 mM RGD showed a close correlation with those encapsulated in Matrigel, indicating that our synthetic PEG hydrogels can be used as suitable alternatives to Matrigel for organoids culture.

In this work, we developed a PEG-based synthetic hydrogel for cardiac organoids development, as an effective alternative to the current gold standard Matrigel. We also found that cell adhesion motif and hydrogel stiffness had a significant impact on cardiac organoid transcriptomics and functions.

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