(89d) Enhancing Vascularized Biomimetic Organ and Tissue By Engineering Vascular Cells with Early Development Transcription Factor

Duc–Huy T. Nguyen¹, Brisa Palikuqi¹, Sina Rabbany², Shahin Rafii¹, Robert Schwartz¹

¹Department of Medicine, Weill Cornell Medical College, New York, Hempstead New York 10065, USA.

²Department of Bioengineering, Hofstra University, New York 11549, USA.

Background: Several efforts have been invested in constructing a vascular capillary network by using adult endothelial cells (ECs) in the field of tissue engineering and translational medicine. However, adult ECs possess limited capability to form a robust capillary network. At best, some stromal cells can slightly enhance the ability of adult ECs to form a limited interconnected capillary network. As 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, several advanced technologies such as 3D lithography patterning, and 3D printing have been used to generate a vascular tree. However, these vascular trees only contain large-caliper vessel structures sparsely decorated the interstitial space of the tissue and far from a realistic in vivo tissue where densely populated vessel networks of small capillaries penetrate every tissue compartment such that no cells in the living tissue is more than 200µm away from a capillary vessel.

In contrast, the endothelium during early development possesses a remarkably robust capability to form well interconnected capillary networks without the presence of supporting stromal cells. We hypothesize that unique transcriptional factors might be present in the early development endothelium that render the ECs their robust attribute of forming robust capillary vessels. Therefore, we sought to engineer the adult ECs by expressing transcriptional factors present during early vascular development to empower the adult ECs with a robust capacity to form interconnected capillary vessels and use these engineered vascular cells to construct long-lasting and durable capillary vessel network both in vivo and in vitro to enhance physiological functions of different organs such as colon organoids, islets, and hepatocytes in biomimetic organotypic models.

Materials and Methods: Using lentiviral transduction and screening experiment, we identified ER71, a transcriptional factor present early in the vascular development, as a potent vasculogenic transcriptional factor. Once re-expressed in adult endothelium from various organs such as dermal, pulmonary, adipose, cardiac, the adult ECs quickly re-acquire their attributes of early vascular cells, enabling them to form a robust interconnected capillary vessel network of small caliper. To verify whether these engineered vascular cells can form sustained capillary network, we implanted the engineered vascular cells in mice and monitored the durability of the capillary network over 5 months. To confirm whether these vascular cells form capillary vessel networks in vitro, we embedded them inside large macrofluidic devices (scalable up to 2cm in dimensions). In addition, we also co-culture the engineered vascular cells with normal, tumor colon organoids, human cadaveric islets, and human primary hepatocytes in these macrofluidic platforms.

Results and Discussion: By introducing transcriptional factor ER71 into mature ECs, the engineered ECs upregulated several genes involved in angiogenesis and vasculogenesis. These upregulated genes remain persistent in ECs across various organs such as dermal, pulmonary, adipose, and cardiac, rendering them robust capacity to form long-lasting small caliper capillary networks. Once implanted into the subcutaneous of immune-compromised mice, these engineered vascular cells formed a long-lasting vessel network up to but not limited 5 months without regression or formation of angiomas.

When cocultured with patient-derived organoids such as colon organoids, the engineered vascular cells interacted and increased the growth rate of both normal and malignant colon organoids while control adult ECs fail to support the organoids. To further extend the utility of the engineered vascular cells, we examine the role of the engineered vascular cells to vascularize human islets. After embedding the human islets with the engineered ECs in macrofluidic devices, a functional and lumenized capillary network is formed surrounding the human islets inside the macrofluidic devices. Porting the macrofluidic devices enabled us to detect insulin secretion of human islets in response to a glucose challenge. Similarly, we also successfully vascularized human hepatocyte aggregates and sustained human hepatocyte functions in macrofluidic devices as shown by stable protein secretion and Cytochrome P450 activities.

Conclusions: The early development transcription factor ER71 was identified to enable adult ECs to form interconnected capillary vessel networks. These small caliper capillary vessels were found to be durable and long-lasting, more than 5 months in vivo and in vitro. These networks were shown to have enhanced physiological functions for different organs such as colon organoids, islets, and hepatocytes. With the engineered ER71 vascular cells, this methodology enhances the extent of capillarization within biomimetic organotypic models over traditional large caliper vessel network generated by 3D printing and lithography techniques. Utilizing the engineered vascular cells, the molecular cross-talks between organoids, tissues with proper capillary vessel network can be instigated to provide additional insights to further improve strategies to engineer vascularized tissues for transplants and regenerative medicine.

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.