(G-276) Engineering Vascular Cells to Vascularize Biomimetic Organ and Tissue

Introduction:: The vasculature plays a critical role in many essential biological processes of organs and tissues to deliver and transport nutrients and gases to maintain tissue survival. It is also an essential component in the field of tissue engineering. Without a proper vasculature within an engineered tissue, tissue engraftment and survival remain poor. Hence, the research community has invested a significant effort in generating well-connected vasculature network within implanted tissues or in organ-on-a-chip models using advanced technologies such as 3D lithography patterning, and 3D printing. Nevertheless, the vasculature networks generated by such techniques are large-caliper vessel structures and poorly phenocopy realistic in vivo vessel networks of small capillaries. This is largely due to the limited capacity of capillary vessel formation in adult endothelial cells (ECs). Adult ECs need to be co-cultured with stromal cells to form capillary vessels. In contrast, the ECs during early embryo development can form robust capillary networks without stromal cells. We hypothesize that during early development, ECs express unique transcriptional factors that allow them to form robust capillary vessels. Through sequencing, we identify a unique transcription factor ETV2 that renders adult ECs their robust attribute of capillary vessel formation. Consequently, we engineer adult ECs by re-expressing this transcriptional factor to empower them with a robust capacity to form interconnected capillary vessels and use these engineered vascular cells to construct long-lasting and durable capillary vessel networks both in vivo and in vitro to enhance physiological functions of different organs such as colon organoids, and islets.

Materials and Methods:: Upon screening for different transcription factors, we identified ETV2 as a potent vasculogenic transcriptional factor that was present during early development ECs but not in adult ECs. Utilizing a lentiviral transduction, we re-expressed ETV2 in adult ECs isolated from various organs such as dermal, pulmonary, adipose, and cardiac tissues to assess their ability to form stable capillary vessel networks in vitro. Using different organoid 3D culture models such as normal, and tumor colon organoids, we studied the interactions of the engineered adult ECs with the organoids and monitored the organoid growth and maintenance in co-culture with the engineered vasculature. We also utilized the engineered adult ECs to vascularize human cadaveric islets in an organ on chip model and monitored the functions of the vascularized human islets to secrete insulin. Finally, we implanted the engineered adults ECs and assessed the capacity of the engineered adult ECs to form durable and stable vessel networks in vivo.

Results, Conclusions, and Discussions:: Results and Discussion: By re-expressing ETV2 into adult ECs, the engineered adult ECs upregulated several genes involved in vasculogenesis. These genes remained persistent in adult ECs isolated from various organs such as dermal, pulmonary, adipose, and cardiac, rendering them robust capacity to form long-lasting vessel networks in vitro without any supporting stromal cells. When cocultured with patient-derived organoids such as colon organoids, the engineered vascular cells increased the growth rate of both normal and malignant colon organoids while control adult ECs failed to support normal and colon organoid growth. After embedding the human islets with the engineered adult ECs in a microfluidic device, a functional and lumenized vessel network was rapidly formed and surrounded the human islets. Porting the devices enabled us to detect insulin secreted by human islets in response to a glucose challenge. Once these engineered adult ECs were implanted subcutaneously into immune-compromised mice, these engineered vascular cells formed stable vessels for 5 months without any sign of regression or angioma formation. The vessel networks formed by the engineered adult ECs were also decorated with murine mural cells suggesting that these ECs are not only capable of forming vessels by themselves, but they can also recruit murine mural cells to establish stable and hierarchical vessel structures similar to in vivo vessels.

Conclusions: Utilizing a vasculogenic transcriptional factor that is only present in ECs during early vascular development, we engineered versatile vasculogenic adult ECs that form robust capillary vessels. These vessels also support co-cultures of organoids derived from multiple tissues such as colons and pancreatic islets in both 3D culture and in a fluidic biomimetic platform. These vasculogenic adult ECs are also capable of forming long-lasting vessels upon transplantation in mice. Together our findings suggest that these engineered adult ECs are a powerful tool to enable us to form robust capillary vessels in both in vitro and in vivo. Using these engineered vascular cells, we can unravel the molecular cross-talks between organoids, tissues and endothelial cells to provide additional insights to further improve strategies to engineer vascularized tissues for transplants and regenerative medicine.


Acknowledgements (Optional): :

References (Optional): : References:

[1] Nguyen, D.H.T., et al. (2013). “Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro.” Proceedings of the National Academy of Sciences. 110(17): 6712-6717.

[2] Palikuqi, B., Nguyen, D.H.T., et al. (2020) “Adaptable and hemodynamic human vasculogenic endothelial cells for organogenesis and tumorigenesis”. Nature. 585: 426-432.