Investigating Heterogeneous Cell Populations with Dielectrophoresis

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

Investigating Heterogeneous Cell Populations with Dielectrophoresis

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

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

Summary: 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. : Flanagan and colleagues tested glycosylation contributions to a unique, fate-specific electrophysiological property of neural stem cells. They found the N-glycan branching pathway generating highly branched N-glycans associated with astrocyte fate. Enhanced branching shifted the electrophysiological property and fate potential of neural stem cells toward astrocytes, revealing the importance of N-glycan branching to neural stem cell differentiation. Keywords: neuron progenitor, astrocyte progenitor, glycosylation, biophysical, dielectrophoresis, membrane capacitance, mouse, branch, MGAT, L-PH

Antiinflammatory therapy with canakinumab for atherosclerotic disease

BACKGROUND: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. METHODS: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P=0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P=0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P=0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P=0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P=0.31). CONCLUSIONS: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. Copyright © 2017 Massachusetts Medical Society