Poster T11 - Fibrin-Enriched Cardiac Extracellular Matrix Hydrogel for Cardiac Cell Therapy

Introduction: Cardiac cell therapy is a promising therapeutic approach for myocardial infarction treatment. Human mesenchymal stem cells (hMSCs) and human -induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are the two main cell types currently under investigation in cardiac repair. Our lab has previously developed a fibrin enriched cardiac extracellular matrix (cECM) hybrid hydrogel (Fn-cECM) derived from porcine heart and demonstrated their pro-angiogenic capacity in vitro. The goal of this project is to explore the feasibility of using our Fn-cECM hydrogel as a cell delivery platform for cardiac repair. In this study, we investigated the effects of Fn-cECM on hMSCs and hiPSC-CMs in both 2D and 3D settings.

Materials and

Methods: cECM and Fn-cECM hybrid hydrogels were fabricated following our previously established protocol. Briefly, Fn-cECM hybrid hydrogels were prepared by blending fibrin matrix into cECM during hydrogel formation. hMSCs (Lonza, Walkersville, MD) and hiPSC-CMs (FUJIFILM cellular Dynamics Inc.) were cultured separately following manufacturer’s protocol. When confluent, both cell types were harvested and seeded separately onto the wells coated with either cECM hydrogel or Fn-cECM hybrid hydrogels and were cultured inside a cell culture incubator at 37ºC with 5% CO2. For hMSCs, angiogenesis analysis was performed by staining the cells using Calcein AM, and images were analyzed using ImageJ software with angiogenesis analyzer plugin. hiPSC-CMs were cultured on the hydrogels for 7 days, after which cell phenotypes were assessed by using immunofluorescence staining with cardiac cell markers, Cx43, Myh6, cTnT and Myh7 and imaged using confocal microscopy. Nuclei were stained with DAPI.

Results, Conclusions, and Discussions:

Results:
Fn-cECM hydrogels induced more robust vascular network formation with greater number of nodes and tubes on seeded hMSCs compared with cECM hydrogel (Figure 1). Similarly, we observed that VEGF secretion from these cells was also significantly increased. hiPSC-CMs seeded on both cECM and Fn-cECM hydrogels clearly expressed both the early cardiac markers, Cx43 and Myh6. hiPSC-CMs seeded on cECM hydrogel showed the clear nuclei staining and no expression for late cardiac marker, cTnT. However, distinct expression of this marker is shown by hiPSC-CMs seeded on Fn-cECM hydrogel. Development of prominent striations was also observed on these cells. Cells on both the hydrogels expressed the late marker Myh7, demonstrating the presence of beta-myosin heavy chain protein that interacts with actin protein which is necessary to generate the contractile force that is indicative of mature hiPSC-CMs (Figure 2).

Conclusions:
Our obtained results from hMSCs culture demonstrated that Fn-cECM can cause morphological alteration of hMSCs toward vascular tube formation and VEGF secretion. We also observed that Fn-cECM hybrid hydrogel accelerates the maturation of hiPSC-CMs. Within 7 days of culture on our fabricated hybrid hydrogel, the cells showed distinct striations which is one of the important features of mature cardiomyocytes. In our future work, we aim to optimize the hydrogel cell seeding parameters to encourage further hMSC vascularization and cardiomyocyte maturation as well as exploring the potential of these hydrogels in 3D formats for application in cardiac therapy.

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