Neural stem and progenitor cells (NSPCs) and endothelial cells (ECs) are in close contact during development and throughout adulthood. Their communication is vital for proper brain development and the maintenance of adult neural stem cell niches. However, there is a lack of knowledge regarding the cross-talk between human NSPCs (hNSPCs) and human ECs (hECs). In this work, we utilize 2-dimensional and 3-dimensional co-cultures to understand how hECs regulate hNSPCs and how hNSPCs impact hECs and vessel formation. Using immunostaining along with single cell RNA sequencing (scRNAseq), we found hEC contact promotes an increase in type B hNSPCs, which are stem cells of the adult brain, via Notch signaling. In addition, hEC contact decreases hNSPC astrocytic differentiation and proliferation. Focusing on the opposite direction of signaling, we found hNSPCs stimulate vessel formation via secreted material including extracellular vesicles (EVs). We identified additional potential vessel-promoting factors expressed by human NSPCs and not mouse NSPCs as well as factors up-regulated by hNSPCs after hEC contact. Lastly, we studied the role of N-glycosylation in brain development using Magt5 null mice, which lack a key enzyme involved in N-glycan branching, and found Mgat5 null mice display increased neuronal differentiation and depletion of the NSPC pool. This work provides a better understanding of NSPC function, including how their proliferation and differentiation are controlled and how they interact with other cells in the niche, such as ECs.

Neural stem/progenitor cells (NSPCs) generate all the differentiated cell types in the central nervous system. However, mechanisms controlling differentiation into specific cell types, such as neurons and astrocytes, and how differentiation is regulated by extracellular cues must be identified. Gaining a better understanding of how NSPC differentiation is affected by their environment will be essential for developing these cells as therapeutics for brain repair. Our lab identified a novel biophysical signature, membrane capacitance, that indicates neuron- or astrocyte-bias of NSPCs. I found that N-glycosylation, specifically the N-glycan branching pathway, differs between astrocyte- and neuron-biased NSPCs. My studies showed that N-acetylglucosamine (GlcNAc) treatment to enhance N-glycan branching shifted membrane capacitance and altered fate of NSPCs. I identified the MGAT5 branching enzyme as a critical regulator of NSPC differentiation in vitro and in vivo. My studies showed that loss of MGAT5 significantly impacted neural development, causing enhanced neuron differentiation but depletion of the NSPC pool that led to a reduction in neurons in the MGAT5 cortex in vivo. I found that MGAT5 null NSPCs in vitro formed more neurons and fewer astrocytes than their WT counterparts. To identify a potential mechanism for how branching impacts fate, I used a proteomic screen to identify cell surface proteins regulated by branching and found a reduction in cell adhesion proteins in GlcNAc-treated NSPCs. My functional assays probing cell-cell and cell-ECM adhesion showed that cellular adhesion is diminished when NSPC N-glycan branching is enhanced. Taken together, my studies identified N-glycan branching as a critical component of fate-specific membrane capacitance, a novel regulator of NSPC differentiation, and a means by which NSPC differentiation can be affected by the extracellular environment.

Stroke is a leading cause of long-term disability and there is a high unmet clinical need for therapies that allow patients to recover lost function. Neural stem cells are good candidates for treating stroke since they can self-renew, secrete beneficial trophic factors, and differentiate into mature central nervous system (CNS) cells; however, most cells die after transplantation. In this work, injectable biomaterials were optimized as transplantation scaffolds for human neural stem/progenitor cells (hNSPCs) with the goal of improving transplanted cell survival. Biomaterials including fibrin, hyaluronic acid, laminin, collagen, and reflectin were combined and tailored to promote hNSPC function. Parameters for scaffold optimization included sensitivity to mechanical stimuli since we discovered that NSPC differentiation can be regulated by static stretch when cells adhere to specific substrate materials and mechanosensing in these cells is regulated by the stretch-activated ion channel Piezo1. Further characteristics of the scaffold included material properties, polymerization and degradation kinetics, ability to support vascularization and hNSPC function, and injectability into naïve and damaged CNS tissue. A novel salmon fibrin-hyaluronic acid-laminin combination scaffold for hNSPC tissue engineering was developed in vitro, and this work marked the first report for the use of both human neural and vascular cells within a biomaterial to promote vascularization. This developed transplant construct was used in vivo in a preclinical transient middle cerebral artery occlusion (tMCAO) stroke model in rats as a potential therapeutic.

Cell heterogeneity is essential in organism development and is a key feature in many diseases. Identifying the defining features of distinct cell types within a heterogeneous population can improve our understanding of development and disease progression and lead to the discovery of better therapeutics. Conventional label-based cell separation methods are often used to isolate distinct cell types from a heterogeneous population. However, this approach has a number of disadvantages, including a lack of unique biomarkers to identify cells of interest. This hampers mechanistic studies aimed at deciphering the unique properties of specific cell types in heterogeneous cell populations. Label-free cell separation techniques generate enriched cell populations necessary for investigating distinct cell functions without the need for unique cell type-specific labels. This body of work focuses on the utilization of dielectrophoresis (DEP), a label-free electrokinetic method, to study heterogeneous cell populations. It details the development of the Hydrophoretic Oblique Angle Parallel Electrode Sorting (HOAPES) device, a high-throughput DEP-based cell sorter that can separate cells based on intrinsic electrical properties. This novel cell sorter combined multiple microfluidic modules to enable continuous separation of distinct cell types and was used to analyze heterogeneous populations of neural stem and progenitor cells (NSPCs) and glioblastoma (GBM) cells. The HOAPES device utilizes continuous fluid flow to enable high throughput separation, providing sufficient cells for downstream assays to assess both sorting performance and identify distinct cell properties associated with sorted cell phenotype. Furthermore, this method helped uncover correlations between cell fate, cell surface N-glycosylation and electrophysiological properties of NSPCs. Similarly, links were identified between chemotherapeutic resistance, glycosylation, and membrane electrophysiological properties of GBM cells.

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