Bioengineering a long-lasting human neurovascular niche model with perfused vascular networks

Introduction: The neurovascular niche (NVN) is a specialized region of the brain that contains brain microvascular networks (BMVNs) and neural stem cells (NSCs) capable of differentiating into new neurons. This makes the NVN an attractive therapeutic target for neurodegenerative disorders. ECs within the BMVNs are known to orchestrate NSC self-renewal and differentiation; however, this dynamic interaction is not well understood in the human brain. Bioengineered models of the NVN are promising to bridge this gap in knowledge but are currently limited in the stability of BMVN and NSC co-culture, which lasts for only two weeks in culture1. This makes it difficult to model neurodegenerative diseases and drug response, which are usually chronic. Our research aims to improve the stability of BMVNs formed in a microfluidic model of the NVN through genetic manipulation of ECs and media optimization to prevent early vascular lumen regression. Our hypothesis is that long-lasting BMVNs would support better NSC survival and differentiation, thus improving the current NVN models.

ETS variant transcription factor 2 (ETV2) is expressed during development to induce vascular cell fate and initiate lumen formation2. Research has found that adult ECs expressing ETV2 form perfused vascular networks that are more adaptable and retain stable, hollow lumens for extended periods of time. E4ORF1 is an adenoviral-derived gene that has been proven to reduce EC dependence on serum and increase robustness3. Through genetic manipulation of ETV2 and E4ORF1 in ECs, we seek to prolong the stability of BMVNs to create long-lasting microfluidic models.

Materials and

Methods: ECs are transduced with a tdTomato lentivirus so that they fluorescence red to enable easy tracking of BMVN formation. The microfluidic model is created by resuspending a single cell suspension of ECs, PCs, and ACs in the cell ratio 6:0.5:1 million cells/mL in a fibrin hydrogel and seeding the resulting solution into a microfluidic device. Interstitial flow across the bulk hydrogel material is continuously applied to aid in BMVN formation and perfusion. Microfluidic devices are fed daily to re-establish interstitial flow, and within 7-10 days cells self-organize into BMVNs with hollow, perfused lumen.

ETV2-ECs are made via transduction with a doxycycline-inducible lentivirus that will upregulate ETV2 expression only in the presence of doxycycline. After antibiotic selection, ETV2-ECs are treated with doxycycline for minimum 6 days in 2D culture before seeding into a microfluidic device to evaluate BMVN formation as previously described. E4ORF1-ECs are made via lentiviral transduction and subsequent antibiotic selection. E4ORF1-ECs are seeded into microfluidic devices to evaluate their 3D performance as previously mentioned. ETV2-ECs and E4ORF1-ECs both also are transduced with tdTomato so that they continuously fluoresce red.

Results, Conclusions, and Discussions: Preliminary results found that ETV2-ECs treated with doxycycline after seeding in a microfluidic device do not form well-connected BMVNs and instead are spiked in morphology. Pre-treating ETV2-ECs with doxycycline for at least 6 days resulted in approximately 30-1000 fold increase in ETV2 expression depending on dose. When seeded in 3D, ETV2-ECs maintain hollow lumen for longer than untransduced controls; however, this ability is highly dependent on ETV2 expression prior to 3D seeding. If ETV2 expression is too high, we observed a slower 2D growth rate, strange 2D morphology, and varying BMVN formation. This preliminary data suggests that while timed upregulation of ETV2 can improve the stability of formed BMVNs within a microfluidic device, ETV2 expression must be fine-tuned as over expression will hinder BMVN formation and stability.

Preliminary experiments with E4ORF1-ECs confirm that these cells demonstrate superior ability to survive in serum-free environments. When grown to a monolayer then serum-starved for 36 hours, E4ORF1-ECs maintain traditional EC cobblestone morphology while 25% of control cells detached and remaining cells demonstrated stressed, spiked morphology. When seeded with supporting cells in 3D, E4ORF1-ECs form vascular networks that appear to have a lumen but are not well organized. These networks, though not well connected with a clear luminal flow path, did survive for longer than controls within the microfluidic device. Static (no interstitial flow) experiments have demonstrated that increasingly organized E4ORF1 vascular networks are possible but requires media optimization. These results suggest that E4ORF1-ECs are increasingly durable and demonstrate potential to retain stable BMVNs for extended periods of time in microfluidic culture but require further media optimization to improve vascular network organization and connectivity.

Both ETV2-ECs and E4ORF1-ECs demonstrate promise in creating long-lasting, perfused vascular networks. While this research is in its early stages, its implications stretch beyond modelling of the human NVN and can be applied to create long-lasting tissue engineered models of any organ with durable vascular networks.

Acknowledgements (Optional):

References:
1. Winkelman, M. A. & Dai, G. Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation. Sci. Adv. 9, eaaz9499 (2023).
2. Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis | Nature. https://www.nature.com/articles/s41586-020-2712-z.
3. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene | PNAS. https://www.pnas.org/doi/10.1073/pnas.0805980105

This work is funded by NASA