Regulation of neural stem cell differentiation and brain development by MGAT5-mediated N-glycosylation.

Undifferentiated neural stem and progenitor cells (NSPCs) encounter extracellular signals that bind plasma membrane proteins and influence differentiation. Membrane proteins are regulated by N-linked glycosylation, making it possible that glycosylation plays a critical role in cell differentiation. We assessed enzymes that control N-glycosylation in NSPCs and found that loss of the enzyme responsible for generating ß1,6-branched N-glycans, N-acetylglucosaminyltransferase V (MGAT5), led to specific changes in NSPC differentiation in vitro and in vivo. Mgat5 homozygous null NSPCs in culture formed more neurons and fewer astrocytes compared with wild-type controls. In the brain cerebral cortex, loss of MGAT5 caused accelerated neuronal differentiation. Rapid neuronal differentiation led to depletion of cells in the NSPC niche, resulting in a shift in cortical neuron layers in Mgat5 null mice. Glycosylation enzyme MGAT5 plays a critical and previously unrecognized role in cell differentiation and early brain development.

Nanofibrous Carbon Multifunctional Smart Scaffolds for Simultaneous Cell Differentiation and Dopamine Detection.

Advances in stem-cell therapy rely on new, multifunctional smart scaffolds (MSS) to promote growth while simultaneously characterizing stem cells undergoing selective differentiation. Nondestructive cell characterization techniques, such as electrochemical detection of lineage-specific metabolites, play a critical role in translational stem-cell therapy by providing clinicians with real-time information to evaluate cell-readiness for transplant. However, electrochemical sensors that provide biophysical cues capable of guiding cell fate, while preserving electroactive functionality, remain unavailable. In this work, a carbon MSS is fabricated by pyrolyzing polyacrylonitrile (PAN) with optimal multiwalled carbon nanotube (MWCNT) loading to optimize electrochemical activity and with a tunable surface to promote cell growth and organization. Carbon MSS is used to (1) enhance the morphology and differentiation of mouse neural stem/progenitor cells (mNSPCs) derived from different regions of the developing brain and (2) simultaneously detect a neurotransmitter, dopamine, from a model dopaminergic cell line growing on the electrode. The study presents a carbon multifunctional smart scaffold for advancing stem-cell therapy toward clinically relevant applications.

High-throughput continuous dielectrophoretic separation of neural stem cells.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.

Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2.

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.