Engineering vascularized tissues by synthetic control of paracrine signaling

Introduction:: The vascularization of engineered tissues remains a significant challenge for the biomedical community. While numerous biomaterial platforms have demonstrated that spatiotemporal control over growth factor presentation can direct vascularization, clinical translation of these approaches has been limited due to the high cost and short shelf-life of growth factors. In contrast, clinical trials have explored the use of gene or cell therapies to induce vascularization, but the inability to control gene expression or cell behavior results in vasculature that is short-lived and non-functional. Inspired by the process of vascular morphogenesis, in which tight regulation of paracrine signals controls the formation of functional vasculature, we hypothesize that the ability to precisely regulate gene expression and paracrine signaling in engineered cells will lead to improved control over vessel formation and maturation for applications in ischemic disease and regenerative medicine.



Recent advances in mammalian synthetic biology have produced inducible genetic circuits that enable user control over cell behavior and function. While these circuits have been applied to engineer immune cells for cancer therapy, their utilization in tissue engineering remains unexplored. Thus, in this study, we seek to engineer vascular cells with inducible genetic circuits in order to direct the vascularization of engineered tissues by controlling paracrine signaling.




Materials and Methods:: Standard molecular cloning techniques were used to construct genetic circuits in which synthetic zinc finger transcriptional regulators (SynZiFTRs)¹ control the expression of various paracrine factors implicated in vasculogenesis (e.g., VEGF, FGF2) (Fig 1A). SynZiFTR circuits, which can be induced by small molecules, were transduced into human dermal fibroblasts. Orthogonality, dose-dependent expression, and bioactivity were evaluated for each engineered fibroblast cell line by using ELISA and Western blot. Engineered fibroblasts were co-cultured with human umbilical vein endothelial cells within fibrin, methacrylamide-functionalized gelatin, and methacrylated dextran hydrogels that were either free-floating or encapsulated within microfluidic devices (Fig 1A). Vasculogenesis was initiated by inducing paracrine factor expression from the engineered fibroblasts by adding different combinations and concentrations of small molecules to the cell culture media. Immunofluorescent staining and confocal microscopy were used to visualize the resulting vascular networks that self-assembled within the hydrogels, and a custom MATLAB code was used to extract metrics of network complexity (e.g., vessel density, %branching) and perfusion.


Results, Conclusions, and Discussions:: Fibroblasts engineered with SynZiFTR circuits exhibited dose-dependent expression of paracrine factors upon small molecular induction, as well as orthogonality between the different engineered cell lines. Induction of paracrine signaling resulted in vascularization of both natural and synthetic hydrogels. Tuning VEGF production resulted in dose-dependent increases in vessel density in microfluidic devices (Fig 1B). Interestingly, while combinatorial VEGF and SDF1α production increased vessel density compared to VEGF alone, VEGF in combination with FGF2 did not yield additional increases in vessel density. However, induction of FGF2 lead to a significant increase in vessel perfusion (Fig 1C). Taken together, these results demonstrate that engineered cells can be used to tailor vessel density and functionality within biomaterials by enabling control of paracrine signal production.



By combining synthetic biology, cell engineering, and tissue engineering tools, we have established an engineered tissue platform in which we can induce vascularization by controlling the exchange of paracrine signals between endothelial cells and fibroblasts. The ability to control paracrine factor concentration over several orders of magnitude enables the vascularization of disparate biomaterials with varied vasculogenic potential, as well as the ability to tune vascular density. Furthermore, the use of multiplexed and orthogonal genetic circuits provides additional handles to tune vascular architecture and functionality. Ongoing work will explore how the timing of vasculogenic and maturation signals influences vessel formation and functionality within the engineered tissue platform.


Acknowledgements (Optional): :

References (Optional): : 1) Li, Israni et al., Science 378, 1227-34 (2022).