Three-dimensional Modeling of the Neurovascular Niche: Neural Progenitor Cell and Endothelial Cell Interactions Modulate In Vivo Transplant Behavior

The loss of neural tissue underlies the symptomatology of several neurological insults of disparate etiology, including trauma, cerebrovascular insult and neurodegenerative disease. Successful replacement of neural tissue by transplantation of neural progenitor cells (NPCs) is dependent on the ability to perfuse the newly formed tissue and control post-implantation NPC behavior. Cocultures of brain-derived endothelial cells (BECs) and NPCs have demonstrated the ability of NPCs to increase engineered vessel stability and post-injury functional vasculature formation. Since variations in parenchymal seeding protocols have affected EC behavior in other coculture systems, we sought to examine how variations in the NPC seeding protocol would affect the derived microvasculature phenotype and also elucidate associated signals in the pre-implantation environment. Two different NPC:BEC ratios---a low ratio coculture (1:10) and a high ratio coculture (2:1) with an increased initial seeding density of NPCs---were seeded onto three dimensional hydrogel matrices and implanted subcutaneously. Both cocultures showed histological hallmarks of vasculogenesis and angiogenesis in vivo, and supported an increasing density of vessels that peaked at nine weeks. Vessels within the implants anastamosed to host vasculature and implant perfusion was demonstrated in both groups. While the kinetics of vessel growth and regression were similar, increasing the initial seeding of NPCs led to a transient increase in vascular density in vivo at six weeks. Notably, the presence of additional NPCs significantly increased the integration of graft BECs into chimeric implant vessels. ELISA analysis demonstrated that in vivo changes in microvascular network behavior correlate with in vitro NPC-mediated increases in vascular endothelial growth factor (VEGF) production and decreases in brain-derived neurotrophic factor (BDNF) production. The vessel maturation factors platelet-derived growth factor (PDGF), angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) were present in comparable levels in both groups at the time of implantation and did not appear play a direct role in NPC-mediated stability. Since we were also interested in the ability of BECs to control implanted NPC behavior, we compared hydrogels seeded with either NPCs alone or a high ratio NPC:BEC coculture. Our results showed a rapid loss of green fluorescent protein positive (GFP+) NPCs within the first eight days in both groups, which could not be fully explained by the relatively low apoptosis rate. Surviving NPCs, spread out within the tissue, extended processes and downregulated GFP. Using the progenitor cell marker sex determining region Y-box 2 (sox 2), downregulation of GFP was shown to be coincident with a loss of sox 2 expression. Coculture of NPCs with BEC support cells did not alter their differentiation status in this subcutaneous implantation model but did provide protection from initial implant apoptosis.