Poster T13 - Development of 3D Cardiovascular Tissue with Embedded Vascular Network Through Co-Differentiation of hPSCs

Introduction: The leading cause of death worldwide is cardiovascular disease, as it takes the lives of 17.9 million people per year. This is due to damaged myocardium being unable to efficiently regenerate. There is critical demand for the construction of an accurate human cardiac tissue model, which is needed for cardiovascular disease modeling, drug testing, cell therapy, and would also eliminate the need for animal testing. There are two missing components in current cardiac models: 1) the lack of functional vasculature; and 2) the lack of accurate representation of the cardiac extracellular matrix (ECM), which prohibits differentiated CMs from maturing and endothelial cells (ECs) from becoming cardiac-specific. Both shortcomings in the current in vitro systems lead to immature cardiac tissue and prevent accurate cardiac maturation. Accurate and functional vasculature is needed because it allows for the circulatory system to function properly and is responsible for the delivery of blood cells, nutrients, and oxygen to tissues, as well as the removal of byproducts, carbon dioxide, and toxic chemicals from the tissues. The purpose of my project is to develop a three-dimensional model that allows simultaneously differentiation of hPSCs into mature CMs and cardiac-specific ECs, as we believe that bi-directional signaling between CMs and ECs will enhance CM maturation and cardiac-specific endothelial phenotype during cardiac tissue development.

Materials and

Methods: Enhancement of the co-differentiation of hPSCs into mature CMs and cardiac specific ECs will be achieved through targeting the microenvironment of the cells. We are in the process of targeting the cell environment by optimizing the cell media. Spheroids were made from hPSCs and then seeded into hydrogel dots consisting of collagen and Matrigel to mimic the native cardiac ECM. We tested the effects of incorporating components of vascular differentiation protocol in our cardiac protocol. We observed how replacing the basal media of the cardiac differentiation protocol (RPMI) with the major component of the vascular differentiation protocol basal media (Advanced DMEM) would affect the expression of cardiac and vascular markers post differentiation. We also looked at how adding ascorbic acid at different concentrations into the cardiac differentiation process would alter the development of ECs and CMs. The effect of having different sized spheroids was also looked at by making spheroids with different number of cells ranging from 2.5K hPSCs to 50K hPSCs. Cardiac differentiation was then performed on the different sized spheroids. The microenvironment was also altered by incorporating flow using commercial (AIM Bioteck idenTx) microfluidic devices (MFDs) and in-house fabricated MFDs. Spheroids were made and then embedded into the hydrogel in the MFD. Interstitial flow was generated through the differences of volumes of media.

Results, Conclusions, and Discussions: In Figure 1B, we show that RPMI media is more optimal than the Advanced DMEM F12 for both vascular marker, VE-cadherin (VECAD), and cardiac markers NK Homeobox 5 (NKX2.5) and Myosin Light Chain 7 (MYL7) expression after performing the cardiac differentiation on the spheroids embedded in hydrogel. At the same time, the presence of ascorbic acid had an impact on the differentiation and showed to increase expression of all three markers. Moving forward, we will use ascorbic acid in our media. Beating was present in all sample groups. Figure 1C shows how the microenvironment, in terms of the spheroid size (number of cells initially seeded to form the spheroid), influences cardiac differentiation. The left most graph shows that beating is seen most often with the larger sized spheroids. The qRT-PCR data demonstrates that the larger spheroids have a higher expression of cardiac markers. Figure 1D demonstrates how the cardiac differentiation protocol was able to result in both ECs and CMs and demonstrates the overlap of ECs (VECAD, green) and CMs (TNNT2, magenta). The border of ECs is well seen as VECAD is a gap junction marker. CMs are seen from the fibrous strands of TNNT2. Taken together, we have developed a model to co-differentiate CMs and ECs from hPSCs. Such a model would be significant for exploring CMs-ECs interaction during early cardiovascular development. Moving forward, we will further improve the model by optimizing media/ECM, fluid flow and introduction of cardiac EC specific transcription factors.

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