Abstract Body: The discovery of iPSCs was revolutionary for regenerative medicine, especially as a source to replace a cell population with limited regeneration capability, like cardiomyocytes (CMs). However, the exact mechanism(s) governing how the microenvironment influences cardiac differentiation at a cellular scale is still widely unclear. To bridge this gap, we created an in vitro fibrous model from a decellularized porcine cardiac extracellular matrix. The aim is to assess the impact of the microenvironment, specifically the effects of protein composition and micro-architecture, on the cardiac differentiation of iPSCs. To study the role of protein composition, atrial and ventricular regions of the porcine heart are manually identified and separated as their protein composition is observed to be different. To test this, the material was subjected to proteomics analysis with LS-Mass Spectrometry where about 1000 proteins were identified in the ventricular region and only 300 proteins in atria. The decellularized tissue was tested for complete removal of nuclei by staining and further validated with DNA quantification. The dECM was blended with synthetic polymer (polycaprolactone) for electrospinning. The average collected fiber diameter was 14.8 ± 3.9 μm. The fibrous scaffolds were seeded with iPSCs and differentiated towards cardiac lineage. dECM samples show comparable signs of biocompatibility as laminin-521 coated TCPS, forming clusters throughout the differentiation process indicating that the scaffolds encourage cardiac differentiation. Further analysis is being conducted to compare the cellular expressions of differentiation markers including mesoderm formation, cardiac specification, and cardiomyocyte structural and functional markers. The effects of the ECM composition will be checked with myosin light and heavy chain expressions, as their expressions vary in the atrial and ventricular regions of the heart.