Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system’s technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.National Institute of Allergy and Infectious Diseases (U.S.) (Grant U24 AI118668

Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential

p38 signaling and receptor recycling events in a microfluidic endothelial cell adhesion assay.

Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs) is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV) and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome

Direct Drug Cocktail Analyses Using Microscale Vortex-Assisted Electroporation

Combination therapy has become one of the leading approaches for treating complex diseases because it coadministers clinically proven drugs to concurrently target multiple signaling pathways of diseased cells. Identification of synergic drug combinations at their respective effective doses without unwanted accumulative side effects is the key to success for such therapy. In this work, we demonstrate the feasibility of the vortex-assisted microfluidic electroporation system for direct drug cocktail analyses where drug substances were individually delivered into cytosols in a sequential and dosage-controlled manner. Through quantitative analyses, the synergic combinational dosage ratios of the chemotherapeutic drug and the anticancer flavonoid were identified. When integrated with high-throughput label-free rare cell purification techniques, the presented system has the potential for development of personalized medicines as the system would be capable of comprehensively assessing drug combinations directly on patients’ cellular samples

Receptor number analysis after the inhibition of receptor recycling followed by incubation in soluble REDV and comparison with and without shear exposure via flow through REDV-coated microchannels.

<p>Error bars denote standard errors for each point based on 3 repetitions. Upon pre-incubation in100 µg/mL of REDV without recycling inhibition, the static and dynamic cases are not statistically different (<i>p</i> = 0.161), indicating that recycling plays a role in the rapid presentation of receptors observed in the uninhibited condition. Statistical comparisons 50, 75, and 100 µg/mL points within each condition are provided as Supplementary Information.</p

Receptor number analysis of HUVECs where p38 was inhibited before (static) and after flow through microchannels at a shear stress of 1.1 dyn/cm² (dynamic).

<p>Upon pre-incubation in 100 µg/mL of REDV without p38 inhibition, the number of receptors in the dynamic case is significantly higher than in the static case (<i>p<0.001</i>). With p38 inhibition, the static and dynamic cases are not significantly different (<i>p</i> = 0.907). Statistical comparisons of the 50, 75, and 100 µg/mL points within each condition showed no significant difference (<i>p>0.001</i>).</p

Receptor number analysis of activated β₁ integrins on HUVECs incubated prior with various concentrations of REDV (static) and after flow through microchannels at a shear stress of 1.1 dyn/cm² (dynamic).

<p>Error bars denote standard errors for each point based on 3 repetitions. There is no statistical significance between the static and dynamic cases even at the 0 µg/mL REDV point where there appears to be a difference (<i>p</i> = 0.23).</p