Myocardial Matrix Hydrogel Enhances Cardiac Spheroid Generation and Cardiogenic Commitment

Cardiac progenitor cells (CPCs), which have been examined for the treatment of myocardial infarction, have been shown to have a superior immunomodulatory and paracrine effect when in the form of spheroids compared to single cells. We previously showed that a myocardial matrix hydrogel enhanced cardiogenic properties of disassociated CPCs grown in 2D culture or when encapsulated in the hydrogel in 3D. The aim of this study was to evaluate the effects of the myocardial matrix when incorporated into cardiac spheroids. Sca-1+ CPCs were cultured in poly-D-lysine coated wells with or without the addition of myocardial matrix or collagen type I at 0.06 mg/ml. To form the myocardial matrix hydrogel, porcine left ventricular tissue was decellularized, digested in pepsin, and then diluted in media 1 day after cell seeding. Spheroid number and size were measured after 5 days in culture. A metabolic assay was performed at day 1, 3 and 5. Gene expression analysis of cardiac and vascular markers was evaluated after 4 days.

Myocardial matrix enhanced the generation and diameter of cardiac spheroids in culture compared to media only (113.2±25.9 and 60.0±14.8 µm respectively). Interestingly, no spheroids were observed when collagen was supplemented, but rather the cells clumped together forming a non-homogeneous large cell aggregate. An Alamar Blue assay showed a significant increase in CPC metabolic activity after 5 days in culture when cells were cultured in the presence of matrix. Similarly, gene expression analysis showed a significant increase of the cardiac transcription factor Nkx2.5 with trends towards an increase in GATA-4, Mef2c, and the sarcomeric markers Troponin I and cardiac actin. No differences in the expression of the vascular markers VEGF-R2 and CD31 were observed. Our study demonstrated that the incorporation of myocardial matrix into cardiac spheroids enhances their formation, size, metabolic activity, and cardiac commitment. Our approach represents a promising alternative and advantageous culture condition for cell-based cardiac regeneration or for tissue engineering self-assembly strategies.

Acknowledgments: This work was supported by the NHLBI (1R01HL113468).

Disclosures: KLC is co-founder, board member, consultant, and holds equity interest in Ventrix, Inc.