Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, “green” technology for regenerating large volume vascularized tissue mass.

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.

The ability to modulate the mechanical properties, and cell alignment within a cardiac patch without hindering cell functionality may have significant impact on developing therapies for treating myocardial infarctions. We developed fibrin-based composite layers comprising aligned microthreads distributed uniformly throughout a hydrogel. Increasing the microthread volume fraction (∼5%, 11% and 22%) significantly increased the moduli of the scaffolds (20.6 ± 8.1, 46.4 ± 23.0, and 97.5 ± 49.3 kPa, respectively), p < 0.05. Analyses of cell-mediated contractile strains and frequencies showed no significant differences among composite layers and fibrin hydrogel controls, suggesting that microthread-based composite layers exhibit similar active functional properties. Cell orientation in composite layers suggests an increase in nuclear alignment within 100 μm of fibrin microthreads and suggests that microthreads influence the alignment in adjacent areas. In this study, we developed composite layers with tunable, mechanical patch properties that improve cell alignment and support cell functionality.

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. We previously developed cell-seeded biological sutures to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. Herein, we implanted both cell-seeded and non-seeded fibrin based biological sutures into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on our previous work and established cardioprotective effects. Non-seeded or hMSC-seeded sutures were implanted into healthy athymic rat hearts. Prior to cell seeding, hMSCs were passively loaded with quantum dot (QD) nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by QD tracking, and the fibrotic tissue area was evaluated. Non-seeded biological sutures decreased SAC near the suture from 0.20±0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC-seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non-seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm2). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm2, p<0.001), with QD-labeled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non-seeded sutures, leading to improved regional mechanics of the implantation region.

Stem cell therapy has the potential to regenerate cardiac function after myocardial infarction. In this study, we sought to examine if fibrin microthread technology could be leveraged to develop a contractile fiber from human pluripotent stem cell derived cardiomyocytes (hPS-CM). hPS-CM seeded onto fibrin microthreads were able to adhere to the microthread and began to contract seven days after initial seeding. A digital speckle tracking algorithm was applied to high speed video data (>60 fps) to determine contraction behaviour including beat frequency, average and maximum contractile strain, and the principal angle of contraction of hPS-CM contracting on the microthreads over 21 days. At day 7, cells seeded on tissue culture plastic beat at 0.83 ± 0.25 beats/sec with an average contractile strain of 4.23±0.23%, which was significantly different from a beat frequency of 1.11 ± 0.45 beats/sec and an average contractile strain of 3.08±0.19% at day 21 (n = 18, p < 0.05). hPS-CM seeded on microthreads beat at 0.84 ± 0.15 beats/sec with an average contractile strain of 3.56±0.22%, which significantly increased to 1.03 ± 0.19 beats/sec and 4.47±0.29%, respectively, at 21 days (n = 18, p < 0.05). At day 7, 27% of the cells had a principle angle of contraction within 20 degrees of the microthread, whereas at day 21, 65% of hPS-CM were contracting within 20 degrees of the microthread (n = 17). Utilizing high speed calcium transient data (>300 fps) of Fluo-4AM loaded hPS-CM seeded microthreads, conduction velocities significantly increased from 3.69 ± 1.76 cm/s at day 7 to 24.26 ± 8.42 cm/s at day 21 (n = 5–6, p < 0.05). hPS-CM seeded microthreads exhibited positive expression for connexin 43, a gap junction protein, between cells. These data suggest that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction. In addition, extended culture allows cells to contract in the direction of the thread, suggesting alignment of the cells in the microthread direction.