Biology subjects

Koo, D.

Publications and source records attributed to Koo, D..

7 recordsLinked to original sources

bioRxiv2026

Retrosplenial cortex vulnerability links severe hypoglycemia to cognitive impairment through neuron-microglia crosstalk

Severe hypoglycemia remains a serious adverse effect of insulin therapy in individuals with diabetes and is linked to cognitive decline, yet the mechanisms by which transient metabolic stress leads to persistent neuronal dysfunction remain poorly defined. Using mouse models of acute severe hypoglycemia and integrated screening, we identified the retrosplenial cortex as a previously unrecognized brain region that is particularly vulnerable to hypoglycemia-induced neuronal damage. This injury is driven by a feedforward interaction between neuron-specific Drp1-dependent mitochondrial fission and microglial IL-1 signaling, as pharmacological or genetic targeting of either pathway suppressed the other, rescued neuronal damage, and reversed cognitive impairment. These findings identify a region-specific neuron-microglia injury circuit that links severe hypoglycemia to cognitive dysfunction and suggest a therapeutic strategy to protect brain function without compromising diabetes management.

neuroscience↗

bioRxiv2023

Optimizing Cell Therapy by Sorting Cells with High Extracellular Vesicle Secretion

Critical challenges remain in clinical translation of extracellular vesicle (EV)-based therapeutics due to the absence of methods to enrich cells with high EV secretion. Current cell sorting methods are limited to surface markers that are uncorrelated to EV secretion or therapeutic potential. We developed a nanovial technology for enrichment of millions of single cells based on EV secretion. This approach was applied to select mesenchymal stem cells (MSCs) with high EV secretion as therapeutic cells for improving treatment. The selected MSCs exhibited distinct transcriptional profiles associated with EV biogenesis and vascular regeneration and maintained high levels of EV secretion after sorting and regrowth. In a mouse model of myocardial infarction, treatment with high-secreting MSCs improved heart functions compared to treatment with low-secreting MSCs. These findings highlight the therapeutic importance of EV secretion in regenerative cell therapies and suggest that selecting cells based on EV secretion could enhance therapeutic efficacy.

bioengineering↗

bioRxiv2023

STORM imaging buffer with refractive index matched to standard immersion oil

Stochastic optical reconstruction microscopy (STORM) provides exceptional super-resolution imaging by sparsely blinking individual dye molecules in thiol-containing media. STORM is now well-established for imaging thin biological specimens, and recent technological advancements have expanded its use to thick tissues. While the use of mounting media with an oil refractive index has been shown to reduce light scattering within tissues and thus greatly improve imaging depth and resolution in optical microscopy, the refractive index of STORM imaging buffers is typically water-like and oil-index (OI) buffers have never been considered for this purpose. In this study, we report a 3-pyridinemethanol-based STORM buffer that matches the refractive index of standard immersion oil. Our OI buffer exhibits similar superior performance in terms of photoswitching of Alexa Flour 647 dye and STORM image quality in fixed cells as conventional STORM buffers, despite having a completely different refractive index. Interestingly, it shows remarkable stability for at least 25 days, and potentially longer, which will enable STORM imaging of a large number of cells on a single prepared slide, as well as larger field-of-view imaging through multiple field stitching. By achieving perfect index matching with oil immersion objectives, OI buffers can produce accurate nanoscale morphology of thin biological specimens, without the need for complex microscope calibrations across sample depth. More importantly, our STORM buffer is expected to play a crucial role in lightsheet STORM applications for thick tissues by reducing light scattering, thereby leading to improved imaging depth and localization performance.

bioengineering↗

bioRxiv2023

Defining T cell receptor repertoires using nanovial-based affinity and functional screening

The ability to selectively bind to antigenic peptides and secrete cytokines can define populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with millions of peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs and secrete cytokines on nanovials, allowing sorting based on both affinity and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired {beta}-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes we could link TCR sequence to targets with 100% accuracy. We identified with high specificity an expanded repertoire of functional TCRs targeting viral antigens compared to standard techniques. One-sentence SummaryAffinity and secretion-based screening of antigen-specific T cells using nanovials defines a functional TCR repertoire

bioengineering↗

bioRxiv2023

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

Cells secrete numerous bioactive molecules essential for the function of healthy organisms. However, there are no scalable methods to link individual cell secretions to their transcriptional state. By developing and using secretion encoded single-cell sequencing (SEC-seq), which exploits hydrogel nanovials to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells (MSCs). We found that VEGF-A secretion is heterogeneous across the cell population and lowly correlated with the VEGFA transcript level. While there is a modest population-wide increase in VEGF-A secretion by hypoxic induction, highest VEGF-A secretion across normoxic and hypoxic culture conditions occurs in a subpopulation of MSCs characterized by a unique gene expression signature. Taken together, SEC-seq enables the identification of specific genes involved in the control of secretory states, which may be exploited for developing means to modulate cellular secretion for disease treatment.

bioengineering↗

bioRxiv2022

Sorting Single T cells based on Secreted Cytokines and Surface Markers using Hydrogel Nanovials

Immune cell function is intrinsically linked to secreted factors which enable cells to communicate with neighboring or distant cells to coordinate a response. The ability to secrete cytokines also can help define the population of cells with therapeutic potential in emerging cell therapies, such as chimeric antigen receptor (CAR)-T cell therapies. Polyfunctional cells that can secrete more than one cytokine have been found to play an outsized role in therapeutic efficacy. While there are a variety of techniques to analyze cellular secretions from individual polyfunctional cells, there are no widely-available approaches to sort viable cells based on this phenotype. Here, we apply lab on a particle technology to the analysis and sorting of T cells based on a combination of secreted factors, interferon gamma (IFN-{gamma}), tumor necrosis factor alpha (TNF-) and interleukin 2 (IL-2) and surface markers (CD8+ and CD4+). Cells are selectively loaded into the antibody-functionalized cavity of micro-hydrogel particles, called nanovials, where secreted cytokines are captured and fluorescently stained. By leveraging standard fluorescence activated cell sorters and using fluorescence pulse area/height information we can distinguish between fluorescence signals on the nanovial cavities and on cells, and are able to process greater than 1 million nanovials in one hour of sorting. The frequency of multi-cytokine secreting cells was correlated with surface marker expression, and biased towards CD4+ T cells. CD8+ cells that secreted more than one cytokine, were biased towards IFN-{gamma} and TNF- with fewer CD8+ cells secreting IL-2. The majority of cells with a polyfunctional phenotype that were sorted remained viable and regrew following sorting. This nanovial cytokine secretion assay can be applied to sort antigen-specific T cells or CAR-T cells based on their functional engagement with cognate antigens or peptide-major histocompatibility complexs (MHCs), enabling discovery of functional CARs or T cell receptors and deeper investigation into the molecular underpinnings of single T cell function.

bioengineering↗

bioRxiv2021

Scalable Fabrication of 3D Structured Microparticles Using Induced Phase Separation

Microparticles with defined shapes and spatial chemical modification can enable new opportunities to interface with cells and tissues at the cellular scale. However, conventional methods to fabricate shaped microparticles have trade-offs between the throughput of manufacture and precision of particle shape and chemical functionalization. Here, we achieved scalable production of hydrogel microparticles at rates of greater than 40 million/hour with localized surface chemistry using a parallelized step emulsification device and temperature-induced phase-separation. The approach harnesses a polymerizable polyethylene glycol (PEG) and gelatin aqueous-two phase system (ATPS) which conditionally phase separates within microfluidically-generated droplets. Following droplet formation, phase separation is induced and phase separated droplets are subsequently crosslinked to form uniform crescent and hollow shell particles with gelatin functionalization on the boundary of the cavity. The gelatin localization enabled deterministic cell loading in sub nanoliter-size crescent-shaped particles, which we refer to as nanovials, with cavity dimensions tuned to the size of cells. Loading on nanovials also imparted improved cell viability during analysis and sorting using standard fluorescence activated cell sorters, presumably by protecting cells from shear stress. This localization effect was further exploited to selectively functionalize capture antibodies to nanovial cavities enabling single-cell secretion assays with reduced cross-talk in a simplified format.

bioengineering↗