(H-287) Cardiac-Specific Extracellular Matrix Scaffold for Cardiac Patch Engineering

Tissue-engineered stem cell patch holds great potential to repair cardiac function and ensure cell retention and survival upon transplantation by functioning as cell delivery vehicles. It also has the potential to enhance cell communication and maturation by creating a microenvironment for cells. Here, we have optimized a fabrication method of a biomimetic anisotropic cardiac-specific extracellular matrix (ECM) scaffold. Cardiac-specific ECM sheet generated by human induced pluripotent stem cells-derived cardiac fibroblasts (hiPSC-CFs) is decellularized to mimic the physiological structure and components in the cardiac environment. We hypothesize that the optimized hiPSC-CFs derived ECM scaffold (hiPSC-CF-ECM) can provide sufficient thickness and mechanical strength for in vivo retention, and create a microenvironment enhance the structural and functional maturation of human induced pluripotent stem cells derived cardiomyocyte (hiPSCs-CMs) by restoring cell-ECM crosstalk. The optimized hiPSC-CF-ECM holds great potential for hiPSC-CMs maturation and cardiac patch engineering.

hiPSC-CFs were cultured up to 10 weeks on micro-patterned polydimethylsiloxane (PDMS) substrates and were decellularized to create anisotropic ECM scaffolds. DNA quantification was performed to estimate cell proliferation throughout time points. Structural components of ECM were evaluated by immunofluorescence staining to observe anisotropy and to measure thickness of the scaffold. Major ECM components were quantified by enzyme-linked immunosorbent assay (ELISA), and a comprehensive compositional evaluation was analyzed through liquid chromatography–mass spectrometry (LC-MS). Mechanical properties of the hiPSC-CF-ECM scaffold were measured by tensile stretching using Intron Testing System. The structural and functional markers of hiPSC-CMs cultured on the ECM scaffolds were determined by immunofluorescence staining and imaged using confocal microscopy. 

hiPSC-CF-ECM scaffold showed an anisotropic organization of nanofibrous ECM and a significantly increased thickness at 6 weeks (Figure 1A - 1C). Similarly, DNA assay showed that cell proliferation significantly increased at 6-week, indicating positive correlation between ECM accumulation and cell proliferation (Figure 1D). The amount of major ECM components showed no significant differences after 6 weeks of culture (Figure 2). A comprehensive compositional evaluation by LC-MS analysis revealed the expressions of the peptides presented in cardiac-specific fibrillar collagens, non-fibrillar collagens, and matricellular proteins (Figure 3). Mechanical characterization by uniaxial tensile stretching of 6-week hiPSC-CF-ECM revealed that the ECM alignment enhanced the mechanical strength of the scaffolds compared with randomly organized scaffolds (Figure 4). Lastly, hiPSCs-CMs cultured on optimized 6-week hiPSC-CF-ECM were aligned following the guidance of ECM nanofibers and displayed mature organization of key structural proteins (Figure 5). The process for anisotropic hiPSC-CF-ECM fabrication was successfully optimized to achieve a thick and strong scaffold that contains structural proteins for resembling cardiac microenvironment. This completely biological, anisotropic, and cardiac-specific ECM holds great potential for cardiomyocyte maturation and cardiac patch engineering.