Direct and indirect endothelial regulation of neural progenitor cells : a modular approach to understand the neural stem cell niche

Abstract

The dynamic EC phenotype will produce soluble (cytokines) and insoluble (matrix) fractions that are biochemically distinct from statically produced EC phenotype. NPCs exposed to the dynamic EC-soluble fraction, comprised of cytokines and secreted proteoglycans, exhibit increased neurosphere proliferation and "stem-like" phenotypes compared to static EC cytokine profiles in neurosphere assays. Furthermore, dynamic EC cytokines promoted neuronal differentiation while static EC cytokines promoted oligodendrocyte differentiation while non-EC conditioned medium promotes astrocyte differentiation in vitro. These results indicate the utility of different EC cytokine profiles to enrich desired NPC populations. In contrast, NPC attachment to the insoluble EC fraction was poor and requires further refinement of the matrix isolation process to achieve definitive information about differential NPC response on EC-substrates.

Evaluation of NPC response to isolated EC factors facilitates an understanding of how each component of the niche individually regulates NPC fate. Improving upon this model, autocrine/paracrine signaling gradients and matrix can be generated in non-contact EC-NPC co-cultures using a novel flow device to stimulate ECs with fluid shear stress while NPCs remain statically cultured below. Significant diffuse based differences were detected in the dynamic EC cultures. Specifically an increase in PSA-NCAM+ (neuroblast or oligodendrocyte precursor cell) cells was observed in NPCs located within 10 μm of dynamically cultured ECs. This in vitro model provides increased complexity appearing to capture lineage progression that occurs within the in vivo niche. This altered EC phenotype can be manipulated by local fluid shear stress to manipulate neurogenic factors that can enrich proliferative and lineage-specific NPC populations that can be used for ex vivo expansion of these cell populations for therapeutic purposes or for in vitro model development to better understand the in vivo NPC niche.

Each year over 1.5 million Americans incur injuries to their central nervous system (CNS) through traumatic brain or spinal cord injuries. Current treatment options to repair the damaged tissue do not achieve full functional recovery. Adult neural progenitor cells (NPCs) are multi-potent precursor cells capable of repopulating the injury, and thus are being explored for their regenerative capacity. Controlled ex vivo expansion of NPC populations is required to generate sufficient cells for therapeutic transplantation. Understanding the in vivo NPC niche will facilitate the design of better ex vivo expansion methods and may lead to improved viability and repopulation following transplantation. Endothelial cells (ECs) regulate NPC self-renewal and differentiation in vitro through soluble factors, leading to improved ex vivo expansion of neural and oligodendrocyte precursor cells. Previous in vitro EC-NPC models, however, have utilized statically cultured ECs, which are phenotypically different from ECs in vivo, which are exposed to fluid shear stress within the vasculature. Therefore, the goal of this thesis was to develop an in vitro model that utilizes a more physiologically relevant EC phenotype through dynamic stimulation (10 dynes/cm2) from which soluble and insoluble factors can be isolated and used to stimulate NPC proliferation and enrichment of desired NPC phenotypes.