Biology subjects

Clark, I. C.

Publications and source records attributed to Clark, I. C..

8 recordsLinked to original sources

bioRxiv2026

Single-cell DNA cytometry with magnetic- and fluorescence-activated bead sorting

Many clinically important cell populations are defined by intracellular DNA or RNA signatures, but isolating and profiling these cells remains difficult. Probe-based in situ hybridization strategies that are compatible with cytometry have difficulty resolving low-abundance transcripts, integrated provirus, or single-copy genomic mutations. In contrast, droplet digital PCR achieves single-molecule sensitivity; however, subsequent cell isolation requires specialized microfluidic sorters that are slow and not widely available. Here, we introduce magniFIND-seq, a nucleic acid cytometry platform that combines the sensitivity of digital PCR with commercial magnetic-activated (MACS) and fluorescence-activated (FACS) sorting instruments. Single-cell genomes and transcriptomes are captured in agarose beads, target sequences are detected by digital PCR, and bead-bound amplicons are labeled with magnetic or fluorescent probes. Controlled evaporation shrinks beads from 55 to 20 m, enabling scalable magnetic separation, fast single-bead FACS sorting, and their combination for high-purity recovery of rare populations. Using magniFIND-seq, we demonstrate multiplexed single-copy detection and FACS-based isolation of simian immunodeficiency virus proviral pol and env targets. Separately, using BCR::ABL1 as a disease-defining target, we enrich chronic myeloid leukemia cells and recover single-cell transcriptomes that resolve tyrosine kinase inhibitor-resistance programs. magniFIND-seq extends the throughput and accessibility of nucleic acid cytometry by engineering compatibility with commercial instruments.

bioengineering↗

bioRxiv2026

Functional Pairing of TCR-peptide-MHC Interactomes by Single-cell Clonal Expansion

T cell antigen-specific immunity depends on pairwise interactions between T cell receptors and peptide-MHC, yet isolating the TCR-pMHC pairs that drive productive engagement remains a major obstacle for antigen-specific therapeutics and for decoding TCR specificity. We overcome this by co-encoding TCR and pMHC in a single founder cell, then clonally expanding it inside a semi-permeable capsule so that genetically identical daughter cells engage in trans. T cell activation, rather than binding affinity, is used to sort cells with functional pairs, and a single PCR on the clone's linked genomic library captures both partners. This platform, LINC-seq, recovered known cognate pairs from pooled libraries at up to 95% accuracy and performed simultaneous, library-on-library deep mutational scanning of both partners. Wild-type clonotypes ranked among the top-enriched sequences in complex mixtures, and the screens resolved co-evolutionary epistasis and cross-reactivity rules inaccessible to one-sided mutagenesis. The approach generalizes to any receptor-ligand pair whose trans-engagement drives a reporter.

immunology↗

bioRxiv2025

Rapid generation of ventral A9-like dopaminergic neurons from patterned iPSCs

In vitro modelling of highly vulnerable nigral dopaminergic (DA) neuronal subtypes in Parkinsons disease (PD), is necessary for studying disease mechanisms. Here, we optimized a new approach by expressing the pioneer neurogenic transcription factor, Achaete-scute-like 1 (Ascl1), implicated in determining dopaminergic fate. Sequential small-molecule patterning of iPSCs into early floor plate mesencephalic progenitors, followed by inducible Ascl1 expression, rapidly differentiates midbrain DA neurons. Immunocytochemistry and transcriptomic analysis of these patterned Ascl1-driven DA neurons (PA-DANs) confirmed midbrain-lineage specificity. Importantly, we found an enrichment of DA subpopulations that corresponded to the adult human ventral SOX6-positive A9 DA subtypes vulnerable in PD. Furthermore, we combined these ventral A9-like PA-DANs with human iPSC-derived midbrain astrocytes and microglia in defined ratios to generate mature 3D A9-like assembled organoids that display characteristic spontaneous neuronal activity and electrical propagation along the axon. Our method efficiently generates a mature and functional A9-like DA neuronal platform to study PD. HighlightsO_LISequential midbrain patterning and Ascl1 expression accelerates DA differentiation C_LIO_LIPA-DANs resemble human adult ventral A9-like DA subtypes vulnerable in PD C_LIO_LI3D assembled organoids show mature identity of PA-DANs, iAstrocytes and iMicroglia C_LIO_LIPA-DANs matured in 3D organoids show neuronal network activity within weeks C_LI eTOC blurbIn this study, Ullian and colleagues have developed a rapid method to differentiate dopaminergic neurons, using small molecules to generate early floor plate mesencephalic progenitors from human iPSCs and sequentially expressing a pioneer transcription factor, Ascl1, that accelerates uniform dopaminergic neurogenesis. Patterned Ascl1-driven dopaminergic neurons (PA-DANs) in 2D and 3D assembled organoids serve as a platform to study Parkinsons disease O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/685897v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d83eb3org.highwire.dtl.DTLVardef@1fc7fa5org.highwire.dtl.DTLVardef@2033e3org.highwire.dtl.DTLVardef@2e8fa3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Deterministic Cell Pairing with Simultaneous Microfluidic Merging and Sorting of Droplets

Cell-cell interactions drive immune activation, tissue repair, and stem cell fate, yet there are few methods that can create large numbers of pre-defined cell pairs to study cell crosstalk. Droplet microfluidics allows high-throughput compartmentalization of multiple cells, but random loading results in <1% of droplets containing the desired cell combinations. Here, we present a microfluidic device capable of deterministically isolating specific cell pairs using droplet merging and sorting ( merge-sorting). The system detects target combinations using fluorescence and triggers simultaneous electrocoalescence and dielectrophoretic sorting. Using fluorescent dye- loaded droplets, we achieved 98.6% purity of merged and sorted droplets. In experiments using cells stained with three distinct dyes, >90% of desired cell pairs were recovered--compared to fewer than 1% when using random Poisson loading. To demonstrate the utility of this platform for extended co-culture studies, we merged cells in an alginate solution with calcium chloride droplets, producing monodisperse alginate hydrogels in which 92% of the beads contained target cell pairs that maintained viability over 18 hours. Compared to selective merger, this approach physically isolates desired droplets, eliminating unmerged contaminants and enabling cleaner downstream workflows. The device allows off-chip pre-incubation of droplets before pairing, the merger of reagents for multi-step assays, and the isolation of desired droplet pairs -- capabilities not jointly accessible with existing approaches. In summary, merge-sort is a flexible platform to enrich specific combinations of droplets, cells, or particles for high-throughput studies of cell crosstalk.

bioengineering↗

bioRxiv2025

The Immunophenotype and Proviral Landscape of HIV-infected CD4 T Cells During Antiretroviral Therapy

In individuals on effective antiretroviral therapy, integrated HIV proviruses persist within CD4 T cells, forming a viral reservoir that rebounds if treatment is stopped. Identifying and targeting these rare, infected cells is critical for advancing therapies, but methods to study reservoir cells are limited and their unique properties remain largely unknown. We applied DAb-seq, a high-throughput method that combines single-cell DNA and surface protein sequencing, to profile over five hundred and twenty thousand CD4 T cells from the blood of six individuals on ART. Infected cells were unequally distributed in T cell subsets, and differential protein expression between infected and uninfected cells revealed significant heterogeneity across cell subsets. Attempts to identify surface markers that differentiate infected from uninfected cells found antigens that mirrored the enrichment of HIV in central memory subsets. However, while central memory T cells harbored the majority of HIV, cells with intact provirus were enriched relative to their defective counterparts in Naive and Regulatory T cell subsets, suggesting that they differentially maintain intact proviruses. In summary, we developed DAb-seq as an open-source platform for linking the proviral landscape to diverse cellular phenotypes, revealing heterogeneity in surface protein expression and provirus maintenance across infected subsets.

genomics↗

bioRxiv2025

Detection of astrocyte epigenetic memory in in vitro systems, experimental autoimmune encephalomyelitis and multiple sclerosis samples

We recently described astrocyte pro-inflammatory epigenetic memory based on multiple complementary in vivo and in vitro studies, and the analysis of multiple sclerosis samples. Based on bioinformatic analyses, ODea and Liddelow argued that the astrocyte epigenetic memory we described is the result of contamination with immune cells, particularly myeloid cells. We rebut ODea and Liddelow arguments as follows: (1) We show substantial purity of astrocytes analyzed in in vivo and in vitro systems; (2) We recapitulate astrocyte memory responses using five independent pure astrocyte in vitro systems, and show its dependency on the histone acetyl transferase p300; and (3) Using the Liddelow lab bioinformatic pipeline to implement purity and cell-quality criteria, we detect astrocyte epigenetic memory in five independent scRNA-seq experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) astrocyte datasets. These additional analyses and studies provide further support for the existence of astrocyte pro-inflammatory epigenetic memory.

neuroscience↗

bioRxiv2024

Disease-associated astrocyte epigenetic memory promotes CNS pathology

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE)1-8. However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY+p300+ memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY+ p300+ astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro; scRNA-seq and immunohistochemistry studies detected increased ACLY+ p300+ astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

immunology↗

bioRxiv2022

Microfluidics-free single-cell genomics with templated emulsification

Single-cell RNA sequencing is now a standard method used to reveal the molecular details of cellular heterogeneity, but current approaches have limitations on speed, scale, and ease of use that stem from the complex microfluidic devices or fluid handling steps required for sample processing. We, therefore, developed a method that does not require specialized microfluidic devices, expertise, or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. PIP-seq accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse-human mixing studies, is compatible with multi-omics measurements, and can accurately characterize cell types in human breast tissue when compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq revealed the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible, and scalable next-generation workflow that extends single-cell sequencing to new applications, including screening, diagnostics, and disease monitoring.

genomics↗