Neural stem cells (NSCs) have great potential to treat a variety of neurological diseases and injuries due to their ability to secrete beneficial factors and to form the major cell types of the central nervous system. However, we lack a complete understanding of what these cells are and how they behave to various environments especially in a volatile degenerating niche. Our lab is focused on studying cellular characteristics and mechanisms that govern NSC fate and methods to increase the transplantation efficiency so that we may more consistently and reliably utilize these cells as cellular therapeutics. Various projects that our lab is interested in are detailed below.
Membrane electrophysiological properties identify fate potential bias in neural stem cell populations
Dielectrophoresis (DEP) provides a novel, label-free method of characterizing and enriching progenitors from a heterogeneous mixture of neural stem and progenitor cells (NSPCs). We have identified that neurogenic and astrogenic populations of cells differ in their whole cell membrane capacitance – the ability for the cell’s membrane to store charge. We are currently studying the relationship between membrane capacitance and cell fate as well as unique methods to study neurological repair utilizing DEP-based cell sorting techniques detailed below.
Characterizing electrophysiological and molecular properties of the cell surface
DEP-measured membrane capacitance signatures reflect fate bias of NSPCs – astrogenic NSPCs tend to have higher capacitance signatures compared to neurogenic NSPCs. The physical or molecular components of the cell membrane that underlies membrane capacitance is not clearly understood. We have identified that glycosylation of proteins can have a profound effect on membrane capacitance and are continuing to explore the relationship between glycosylation patterns, membrane capacitance, and cell fate.
Increasing the efficiency of DEP-based cell sorting
We’ve been able to exploit differences in membrane capacitance of astrogenic or neurogenic NSPCs to create devices that enrich specific progenitors. While this method has been used in other stem cell lineages, the complex fabrication process of these devices or instrument schemes as well as low output of cells makes adoption of this technique difficult. In collaboration with colleagues in the Biomedical Engineering department, we are working to develop more efficient microfluidic tools that enable the high throughput characterization and separation of neural stem cells and are fine-tuning the sorting parameters for NSPCs.
Enriching specific neural progenitors for studying neural repair in damaged tissue
Most stem cell transplantation studies engraft a heterogeneous population of NSPCs or cells that have been differentiated and enriched for specific cell types prior to transplantation. Usually, this results in inconsistent use and outcome of NSCs. Enriching progenitor populations by DEP provides a unique, label-free method of separating cells that does not affect cellular viability, proliferation, or differentiation potential. This method will allow us to study the contribution of various compositions of astrogenic or neurogenic NSPCs to neural repair. We currently have projects underway characterizing mixed populations of cells and assessing whether DEP-enriched cells have beneficial aspects for functional recovery in spinal cord injury.
Analyzing how glycosylation patterns affect neural stem cell interactions
Glycosylation plays a significant role in NSC development and differentiation. Specifically, N-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 defects, tremors, and seizures. Additionally, almost all proteins on the cell surface are N-glycosylated, and their retention at the cell surface and function is highly dependent on its glycosylation status affecting how a cell interacts with another cell or its environment. We have identified that increased N-glycosylation branching correlates with astrogenic development in the central nervous system in vivo and increases astrogenesis in vitro. There is a wide variety of complex N-glycan structures, but previous studies have suggested enzyme Mgat3-mediated bisecting N-glycans and enzyme Mgat5-mediated highly-branched N-glycans as potential glycan-type influencers in NSC fate potential. In collaboration with Dr. Michael Demetriou, we are currently exploring how altered ability to produce these specific N-glycans affects choice to differentiate into the main neural lineages (neurons, astrocytes, and oligodendrocytes) in vitro, and also utilizing an in vivo stroke model to assess how glycosylation-modified NSCs behave in terms of fate potential, damage attenuation, and transplantation efficiency since changes in glycosylation of cell surface receptors is likely to affect how cells respond to the transplantation niche.
Bio-scaffolds for tissue engineering
Stroke and spinal cord injury cause significant damage to the CNS typified by formation of a localized area of necrosis and inflammation surrounded by surviving uninjured tissue. Most cell transplantation studies to treat stroke or spinal cord injury have utilized injection of cells into the surrounding uninjured tissue to avoid the massive cell death associated with placement of cells directly into the injury epicenter. However, injection into the area surrounding the injured site runs the risk of damaging healthy tissue and does not address the significant cell loss in the primary area of injury. Inclusion of a scaffolding material with transplanted cells improves cell survival in the injury epicenter, likely by decreasing inflammatory cascades and providing adhesive and other support for the transplanted cells. We developed injectable scaffold materials based on fibrin that support NSPCs and neurons in vitro and improve recovery after spinal cord injury in vivo, even without the inclusion of transplanted cells. Fibrin is part of the body’s normal wound healing cascade, so is non-toxic during polymerization and degradation. We are now investigating fibrin and additional injectable scaffold materials in a series of in vitro and in vivo experiments to define optimal scaffolds for CNS repair. Projects in this area include testing effects of scaffolds on NSPC survival, proliferation, and differentiation in vitro and in vivo, combination of scaffolds with enriched NPs and APs for analysis in vitro and in vivo, and quantitative analysis of scaffold material properties.
Mechanical forces influence neural stem cell differentiation
Neural stem and progenitor cell (NSPC) fate is strongly influenced by mechanotransduction as modulation of substrate stiffness affects lineage choice. Other types of mechanical stimuli, such as stretch (tensile strain), occur during CNS development and trauma, but their consequences for NSPC differentiation have not been well understood. Our lab has identified static stretch as an extracellular force that impacts NSPC differentiation into oligodendrocytes – a phenomenon likely mediated through integrin-extracellular matrix linkages. In addition, collaborations with Dr. Medha Pathak have identified that stretch-activated ion channels have a significant role in NSPC fate potential along the neuron-astrocyte switch. Further studies to characterize how different mechanical stimuli or different substrates can affect NSPC differentiation will be necessary for designing better-suited biomaterials and understanding how properties of the transplantation niche will affect NSPC engraftment.