Neural stem cell fate

We study characteristics of the cell plasma membrane, which is the first interface between a cell and its environment, to better understand how differentiation and fate potential are influenced by extracellular cues. We found that cell surface glycosylation plays a significant role in neural stem cell (NSC) development and differentiation.

Glycosylation is the process by which sugars (glycans) are added to proteins and lipids destined for the cell surface. Glycosylation is instrumental in neural development, as several genetic defects in glycan processing enzymes are associated with severe neurological abnormalities, including neural tube closure defects, locomotor deficiencies, tremors, and seizures. Almost all proteins on the cell surface are N-glycosylated, and their function and retention at the plasma membrane are highly dependent on glycosylation. This directly affects how a cell interacts with another cell or its environment.

We found that the N-glycan branching glycosylation pathway regulates the formation of neurons and astrocytes from NSCs (Yale et al., Stem Cell Reports 2018). Highly branched N-glycans on NSCs direct cells toward an astrocytic fate, whereas cells with decreased branching form more neurons (Yale et al., Stem Cell Reports 2018, Yale et al., Stem Cell Reports 2023). When branching is disrupted during brain development, accelerated neuronal differentiation leads to progenitor depletion that affects formation of the cerebral cortex (Yale et al.. Stem Cell Reports 2023).

These data identify a new role for glycosylation in stem cell differentiation and future studies will    focus on the glycosylation enzymes, extracellular proteins and intracellular pathways involved.

 

Human neural stem cells and their interactions in the niche

Understanding the function of human NSCs can open windows into human brain development and identify novel therapeutic options for neurological conditions.

Human NSCs in the brain reside in specialized niches and recreating key elements of these niches in vitro will enable study of these critical cells in a more physiological setting. We developed a 3D in vitro human cell and scaffold model that mimics the mechanical properties of brain tissue and contains key brain extracellular matrix (ECM) molecules. We included human NSCs and human endothelial cells to generate a neurovascular model since these are primary cell types in the niche. Understanding the interaction of human NSCs and human endothelial cells is important since vessel disruption is a hallmark of many neurological insults. For example, stroke is a leading health concern and causes significant damage to the CNS typified by vessel injury and formation of a localized area of necrosis and inflammation surrounded by surviving brain tissue.

Studying the interaction of human NSCs and human endothelial cells led to the finding that NSCs stimulate vessel formation (Arulmoli et al., Acta Biomaterialia 2016). Analysis of human NSCs by RNA sequencing showed that these cells make many factors known to impact vessel formation and function. These studies identify crucial interactions between human NSCs and human endothelial cells that can impact vessel formation and tissue vascularization.

We are currently focused on determining the human NSC signaling molecules affecting human vessel formation and investigating the reciprocal effects of human endothelial cells on human NSCs.

 

Label free technologies to identify cell phenotype and enrich distinct cell populations

Detecting the phenotype of live cells is challenging and usually relies on cell type-specific markers. However, markers for cells of interest are often lacking or not sufficiently specific. We utilize a label free approach to study cell phenotype using dielectrophoresis (DEP)(Adams et al., Methods 2018; Lee et al., Current Stem Cell Reports 2018). DEP refers to the motion of particles (or cells) in a non-homogeneous electric field. Cells that vary in their inherent cellular properties can have different responses to the electric field, resulting in distinct patterns of movement that can be used to measure cell characteristics or enrich cell populations.

Characterizing electrophysiological and molecular properties of the cell surface

Varying the frequency of the applied electric field in alternating current DEP enables the generation of DEP spectra for each cell population. These spectra can in turn be used to calculate electrophysiological properties of the cell membrane and cytoplasm. Using DEP, we found that NSCs that differ in fate bias also differ in membrane capacitance, which is the ability of the membrane to store charge (Flanagan et al., Stem Cells 2008; Labeed et al., PLoS One 2011; Lu et al., Integrative Biology 2012; Nourse et al., Stem Cells 2014). Astrogenic NSCs tend to have higher capacitance signatures compared to neurogenic NSCs. The physical or molecular components of the cell membrane that underly membrane capacitance have not been well studied, but we found cell surface glycosylation has a profound effect on membrane capacitance (Yale et al., Stem Cell Reports 2018).

We are continuing to explore the relationship between glycosylation patterns, membrane capacitance, and cell fate. 

(Video from Dr. Alan Jiang)

Enriching specific neural progenitors

Since DEP can be used to induce cell movement based on cell membrane electrophysiological properties, we tested whether we could enrich progenitors tied to either neuronal or astrocytic fates using DEP-based microfluidic devices. Enriching these progenitors can lead to a better understanding of their molecular profiles and functional properties, which can provide insight into neural development and improve the use of these cells to treat neurological conditions.

Using DEP-based cell sorting devices we have successfully enriched different progenitor populations (neurogenic and astrogenic) from mouse and human NSCs (Nourse et al., Stem Cells 2014; Simon et al., Biomicrofluidics 2014; Jiang et al., Biomicrofluidics 2019; Adams et al., Biosensors and Bioelectronics 2020). Enriching progenitor populations by DEP provides a unique, label-free method of separating cells that does not affect cellular viability, proliferation, or differentiation potential (Lu et al., Integrative Biology 2012). This approach allows us to study the specific cellular properties of NSCs that differ in fate to better understand brain formation and the role these cells can play in neural repair.

We are currently using DEP-enriched NSCs to determine the molecular profiles of distinct progenitor populations.

 

Increasing the efficiency of DEP-based cell sorting

DEP-based sorting of NSCs has enabled characterization of distinct progenitor cell types. However, a limitation of DEP microfluidic devices can be low throughput of sorted cells, leading to a lack of sufficient cell numbers for some biological or medical applications. We have addressed this in two ways.

Firstly, we found that expansion of NSCs after DEP sorting retained progenitor enrichment and generated billions of cells, giving sufficient numbers for cell transplantation or other critical studies (Simon et al, Biomicrofluidics 2014). Secondly, we developed next generation DEP sorting devices that continuously sort, generating much greater numbers of sorted cells (Jiang et al., Biomicrofluidics 2019; Adams et al., Biosensors and Bioelectronics 2020). These high throughput continuous sorting devices have been used to successfully enrich progenitors from mouse and human NSCs and provide a significant increase in sorted cell number compared to traditional devices.

We are in the process of continuing innovation in DEP-based continuous sorting devices to increase the fractionation of cells in each sort to learn more about distinct cell phenotypes.

 

 

Glioblastoma stem cells

Glioblastoma is a particularly tragic neurological condition. The cancer is quite aggressive, and despite tumor resection and treatment with radiation and chemotherapeutics median survival is 2 years or less. A major problem is resistance to chemotherapeutics, contributing to recurrence and a lack of subsequent treatment options. Several lines of evidence indicate that the resistant cells are glioblastoma stem cells.

We are applying our DEP-based platforms to the study of resistance in glioblastoma, with the hope of identifying means to further investigate the cellular and molecular characteristics of these cells. These studies could lead to novel treatment options targeting chemotherapeutic resistant cells.