(112b) Direct Production of Human Cardiac Tissues By Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl

The ultimate goal in cardiac tissue engineering is the production of functional and reproducible 3D human cardiac tissues using human induced pluripotent stem cells (hiPSCs). However, the current protocol for the generation of 3D tissues requires multiple steps that will disrupt the developing CMs and negatively impact their viability and function, and while many animal models exist, none of them accurately replicate human systems for investigative uses. Thus, the objective of this study was to create functional 3D cardiac tissues by directly encapsulating and differentiating hiPSCs using a novel photocrosslinkable hydrogel, methacrylated gelatin (GelMA), which is mechanically robust, biologically responsive, and supports hiPSC survival and CM differentiation. As gelatin is a denatured form of collagen, it is believed that this similarity to the native heart will benefit the differentiation process, making GelMA an attractive choice as a biomaterial.

GelMA was synthesized by reacting methacrylic anhydride (MA; 5% w/v) and gelatin for 2 hours, followed by dialysis and freeze-drying. Lyophilized GelMA was re-dissolved into PBS (15% w/v) and combined photoinitiators to form a GelMA precursor solution. Dissociated hiPSCs were combined with the GelMA precursor solution, pipetted into a circular polydimethylsiloxane (PDMS) mold, and photocrosslinked using visible light for 40 seconds, forming circular 3D tissues. Encapsulated hiPSCs were maintained in their pluripotent state for three days followed by the initiation of cardiac differentiation.

Overall, it was seen that the synthesized GelMA successfully photocrosslinked in the presence of hiPSCs and could be used to create 3D cardiac tissues. The first isolated areas of contractions by in the tissues were demonstrated by day 8 of differentiation and these tissues resulted in uniformly contracting tissues by day 20. Confocal microscopy of dissociated tissues showed the presence of large nuclei and well-defined sarcomeres that are indicative of CMs. GelMA is a suitable biomaterial for hiPSC encapsulation and 3D cardiac differentiation. Future work will make use of these cardiac tissues in order to investigate the impacts of drugs on developing CMs in an ontogenic model. This model will allow for in vitro study of a microenvironment very similar to the developing heart.

The results show that GelMA is able to form cell-laden tissues while still maintaining its bioactivity. Using GelMA for both the encapsulation and differentiation, we are able to have only a single cellâ??handling step while still creating functional CMs. As this method requires minimal cell disturbance, it is beneficial for the development and function of 3D human cardiac tissue.