The Role of Microvascular Signaling in the Neurogenic Niche

Rita Matta, Yale University Graduate School of Arts and Sciences


Stroke is among the leading causes of death and disability worldwide, partly due to the lack of effective therapies to facilitate the recovery of damaged brain tissue. Stem cell therapies used to treat neurological diseases are promising, owing to their innate ability to enhance endogenous repair mechanisms and promote functional recovery. However, the maintenance of stem cells in a quiescent state throughout delivery remains a significant challenge. This challenge only exacerbates the difficulty of therapeutic strategies attributed to the low survival rate of engrafted cells within the inflamed, cytotoxic brain. Tissue engineering provides the opportunity to develop cell delivery strategies that maintain cell quiescence and reduce inflammation during and post-delivery, thereby promoting cell survival, migration, and success following engraftment. The subventricular zone (SVZ), located lateral to the lateral ventricle, is the largest region in the adult brain where proliferating neural stem cells (NSC) reside. For NSC to differentiate in response to injury into the functionally specific cell types that comprise healthy brain tissue, they must first migrate rostrally into the olfactory bulb (OB). This process is dependent upon signaling from microvascular endothelial cells (EC) and pericytes (PC) within the SVZ, ultimately directing NSC along the rostral migratory stream (RMS) to the OB. Diffusible secreted signals from EC can increase survival, proliferation, and differentiation of SVZ NSC in vitro as well as in vivo. Here, we investigate the role of vascular cells in NSC functionality, particularly, NSC migration and survival. Our results demonstrate that, with the microvascular structure, EC, and not PC, promote NSC migration and cluster formation, both by cell-cell contact and soluble factor secretion. Using a 3D scaffold that mimics the biomechanics, biochemistry, and biostructure of specific regions of the brain, we can visualize the migration of NSC clusters throughout the pores of this functionalized scaffold towards EC. Due to N-cadherin’s established role in NSC polarization and cytoskeletal rearrangement, we demonstrate that EC secreted MMP2 leads to NSC clustering, increased N-cadherin expression, and enhanced NSC migration. When the NSC cluster leader cell was ablated using a microfluidic system, the cluster no longer can migrate, even when in the presence of EC soluble factors, confirming that NSC clustering is a prerequisite for migration. The novelty of the compositional, architectural, and mechanical mimicking scaffold has allowed us to probe biofunction and inform us about important signals to incorporate into a delivery structure. Due to the positive impact EC have on NSC, we use polymeric microbeads for their co-encapsulation to be delivered into the brain. We demonstrate that NSC encapsulated with EC have increased NSC survival and maintained quiescence, prior to and post injection to a non-injury model, as compared to NSC encapsulated alone. Once injected into the brain, NSC encapsulated with EC present reduced immune cell activation and enhanced cell survival as compared to freely injected cells. Furthermore, NSC encapsulated with EC delivered to two rat stroke models demonstrate enhanced cell infiltration and migration into the stroke damaged tissue with the use of extracellular matrix (ECM) as a suspension vehicle. Our work provides convincing evidence that engineered mimics of the neurovascular niche may serve as a neuroprotective delivery vehicle, reducing inflammation upon transplantation, ultimately improving the state of current delivery systems. As we aim to enhance the construction of our bioengineered niche, we observe the impact of vascular cells on NSC survival during injury-like conditions, specifically when deprived of glucose. We demonstrate that EC, but not PC, promote NSC cell proliferation and reduce cytotoxicity during glucose deprivation by direct cell-cell contact and soluble factor secretion. This effect is diminished when NSC VEGFR3, abundantly expressed by NSC in the SVZ, is blocked. In addition, we demonstrate that NSC and EC co-cultures have elevated levels of VEGF-C, not seen for NSC alone. To further assess NSC survival in vivo, we delivered microbeads to a mouse stroke-injured brain, where NSC encapsulated with EC have high VEGF-C expression around the injection site compared to microbeads with NSC encapsulated alone. Our results demonstrate a novel role for VEGF-C/VEGFR3 in promoting NSC survival during injury which can significantly enhance current therapies. In summary, our work can aid the creation of a novel cell delivery therapeutic for stroke, promoting NSC migration and survival upon transplantation. Together our findings highlight the potential for neural-vascular coupling to promote functional and long-term recovery in the stroke injured brain.