To study neurovascular interactions, we have developed a 3D human brain vasculature system that consists of human brain ECs, pericytes, and astrocytes. This system has interconnected open lumens and tight junctions with permeability similar to the blood brain barrier. Building on this platform, we developed a biomimetic 3D neurovascular model that can support the homeostatic balance of human neural stem cell (NSC) derived from induced pluripotent stem cells (iPSCs) for a 1 month period, including self-renewal, neuronal maturation, and NSC quiescence.

The goal of this project is to extend the lifespan of existing 3D neurovascular model from 1 to 6 months and demonstrate its utility by exposing to chronic stressors. In phase one, we will integrate the following approaches to prolong the lifespan of our current model: 1) Reprogram with key factors to rejuvenate in vivo EC functionalities and promote durable vascular lumen; 2) Optimize a serum free medium by screening small molecules critical for the stable vascular lumen; 3) Integrate technologies for prolonged culture and real-time sensing; these include fiber photometry that allows real-time recording of neuronal activity, fluorescent reporters that allow live visualization of vascular morphology, fluid flow, Ca2+ signaling, oxygen and cellular quiescence, a stable perfusion system with fluid/pressure control for prolonged culture. Combining these approaches, we will validate the stability and functionality of the neurovascular system in terms of vascular morphology, lumen interconnectivity, perfusion, permeability, and neural activity, and NSC reserves for 6 months. In phase two, we will validate the system to model chronic inflammation-mediated neurodegeneration by introducing microglia and inflammatory cytokines and validating whether this approach can faithfully replicate the immune response, neurodegeneration, and premature depletion of NSC reserves over a 6 month period.

Significance: Because a stable vasculature is critical for normal tissue function and homeostasis, the success of this project will have broad impact on building many other tissue models for prolonged culture using human cells (both male and female) – heart, kidney, muscle, cancer – to model human diseases.

1. Made lentivirus to express E4ORF1 and ETV2 in vascular endothelial cells.

2. Performed the preliminary test on the effect of E4ORF1 and ETV2 on vascular network formation.

3. Evaluated the effect of several medium supplements on the longevity of the vessels.

4. Designed and fabricated in-house microfluidic devices and implemented the microfluidic pump for constant flow.

To study neurovascular interactions, we have developed a 3D human brain vasculature system that consists of human brain ECs, pericytes, and astrocytes. This system has interconnected open lumens and tight junctions with permeability similar to the blood brain barrier. Building on this platform, we developed a biomimetic 3D neurovascular model that can support the homeostatic balance of human neural stem cell (NSC) derived from induced pluripotent stem cells (iPSCs) for a 1 month period, including self-renewal, neuronal maturation, and NSC quiescence.

The goal of this project is to extend the lifespan of existing 3D neurovascular model from 1 to 6 months and demonstrate its utility by exposing to chronic stressors. In phase one, we will integrate the following approaches to prolong the lifespan of our current model: 1) Reprogram with key factors to rejuvenate in vivo EC functionalities and promote durable vascular lumen; 2) Optimize a serum free medium by screening small molecules critical for the stable vascular lumen; 3) Integrate technologies for prolonged culture and real-time sensing; these include fiber photometry that allows real-time recording of neuronal activity, fluorescent reporters that allow live visualization of vascular morphology, fluid flow, Ca2+ signaling, oxygen and cellular quiescence, a stable perfusion system with fluid/pressure control for prolonged culture. Combining these approaches, we will validate the stability and functionality of the neurovascular system in terms of vascular morphology, lumen interconnectivity, perfusion, permeability, and neural activity, and NSC reserves for 6 months. In phase two, we will validate the system to model chronic inflammation-mediated neurodegeneration by introducing microglia and inflammatory cytokines and validating whether this approach can faithfully replicate the immune response, neurodegeneration, and premature depletion of NSC reserves over a 6 month period.

Significance: Because a stable vasculature is critical for normal tissue function and homeostasis, the success of this project will have broad impact on building many other tissue models for prolonged culture using human cells (both male and female) – heart, kidney, muscle, cancer – to model human diseases.