Biology subjects

Muir, L. A.

Publications and source records attributed to Muir, L. A..

5 recordsLinked to original sources

bioRxiv2024

Data-guided direct reprogramming of human fibroblasts into the hematopoietic lineage

Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. Current protocols are limited by low efficiency and insufficient tools for evaluating reprogramming outcomes. Although functional assays are the standard for confirming cell identity, they require fully reprogrammed cells, limiting their utility during protocol development. To address this, we assembled a single-cell transcriptomic reference atlas of hematopoietic reprogramming and tested an algorithmically-predicted transcription factor recipe for HSC induction. Long-read single-cell RNA sequencing of CD34+ reprogrammed cells revealed progressive loss of fibroblast identity alongside induction of early hematopoietic and endothelial programs, with reference-atlas benchmarking placing reprogrammed cells in an intermediate transcriptomic state between fibroblasts, endothelial cells, and HSCs. Isoform-level analysis further revealed transcriptional remodeling not captured by gene-level analyses. This experimental-computational framework offers a generalizable strategy for characterizing partially reprogrammed states and guiding optimization of reprogramming protocols.

bioinformatics↗

bioRxiv2023

Automated In Vitro Wound Healing Assay

Restoring the epidermal barrier after injury requires spatial and temporal orchestration of migration, proliferation, and signaling across many cell types. The mechanisms that coordinate this complex process are incompletely understood. In vitro wound assays are common model systems for examining these mechanisms in wound healing. In the scratch assay, a cell-free gap is created by mechanical removal of cells from a monolayer, followed by monitoring cell migration into the gap over time. While simple and low-cost, manual scratch assays are limited by low reproducibility and low throughput. Here, we have designed a robotics-assisted automated wound healing (AWH) assay that increases reproducibility and throughput while integrating automated live-cell imaging and analysis. Wounds are designed as computer-aided design (CAD) models and recreated in confluent cell layers by the BioAssemblyBot (BAB) 3D-bioprinting platform. The dynamics of migration and proliferation in individual cells are evaluated over the course of wound closure using live-cell fluorescence microscopy and our high-performance image processing pipeline. The AWH assay outperforms the standard scratch assay with enhanced consistency in wound geometry. Our ability to create diverse wound shapes in any multi-well plate with the BAB not only allows for multiple experimental conditions to be analyzed in parallel but also offers versatility in the design of wound healing experiments. Our method emerges as a valuable tool for the automated completion and analysis of high-throughput, reproducible, and adaptable in vitro wound healing assays.

bioengineering↗

bioRxiv2022

Obesity disrupts innate-adaptive immune network patterning in adipose tissue

ObjectiveObesity-induced metabolic dysfunction, tissue remodelling, and chronic inflammation in visceral white adipose-tissue (WAT) are correlated with insulin resistance, type II diabetes, and metabolic disease pathogenesis [1]. In this work, we sought to establish spatio-temporal context of adipose tissue macrophage (ATM) reprogramming during obesity. MethodsWe captured single-cell RNA-sequencing, spatial transcriptomics, and histological imagining of murine WAT over the course of diet-induced obesity to study macrophage phenotype dynamics. We developed a straightforward mathematical approach to integrating multi-modal data to quantify obesityinduced changes to WAT organization. We aligned ATM phenotypes with crown-like structures (CLS) in early obesity and used spatial network analysis to uncover signalling mechanisms implicated in CLS formation. ResultsWe identified novel diversity of the lipid-associated macrophage (LAM) phenotype, whose transcriptional profile, signaling mechanisms, and spatial context serve as indicators of CLS formation in early obesity. We demonstrated that dysregulation of lipid-metabolic signalling is a critical turning point in the monocyte-LAM lineage and identified novel ligand-receptor mechanisms including Apoe, Lrp1, Lpl and App that serve as hallmarks of nascent CLS in WAT. ConclusionsMulti-modal spatio-temporal profiling demonstrates that LAMs disproportionately accumulate in CLS and are preceded by a transition-state macrophage phenotype with monocytic origins. We identified novel ligand-receptor interactions implicated in nascent CLS regions which may guide future cellular-reprogramming interventions for obesity-related sequelae. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/482134v6_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d7ac5borg.highwire.dtl.DTLVardef@74c5c7org.highwire.dtl.DTLVardef@64b722org.highwire.dtl.DTLVardef@33392e_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- We characterize a novel lipid-associated macrophage (LAM) phenotype along the monocyte-LAM lineage - Integrated imaging, single-cell sequencing and spatial transcriptomics data show that LAMs accumulate at nascent CLS - Analysis of spatial transcriptomics data reveals a novel set of ligands and receptors that implicate immature LAMs in shaping the CLS microenvironment in early obesity - We present a simple mathematical framework for studying dynamics of tissue-structure over time

cell biology↗

bioRxiv2021

Cohesin Releasing Factor WAPL Regulates Genome Structure and Function of Mature T Cells

The cohesin complex modulates gene expression and cellular functions by shaping three-dimensional (3D) organization of chromatin. WAPL, cohesins DNA release factor, regulates 3D chromatin architecture. The 3D genome structure and its relevance to mature T cell functions in vivo is not well understood. We show that in vivo lymphopenic expansion, and allo-antigen driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of Wapl in T cells reduced long-range genomic interactions, altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. Comparison of chromatin structure in normal and WAPL-deficient T cells after lymphopenic and allo-antigen driven stimulation revealed reduced loop extensions with changes in cell cycling genes. WAPL-mediated changes in 3D architecture of chromatin regulated activation, cycling and proliferation of T cells in vitro and in vivo. Finally, WAPL-deficient T cells demonstrated reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.

molecular biology↗

bioRxiv2017

Genome Architecture Leads a Bifurcation in Cell Identity

Genome architecture is important in transcriptional regulation and study of its features is a critical part of fully understanding cell identity. Altering cell identity is possible through overexpression of transcription factors (TFs); for example, fibroblasts can be reprogrammed into muscle cells by introducing MYOD1. How TFs dynamically orchestrate genome architecture and transcription as a cell adopts a new identity during reprogramming is not well understood. Here we show that MYOD1-mediated reprogramming of human fibroblasts into the myogenic lineage undergoes a critical transition, which we refer to as a bifurcation point, where cell identity definitively changes. By integrating knowledge of genome-wide dynamical architecture and transcription, we found significant chromatin reorganization prior to transcriptional changes that marked activation of the myogenic program. We also found that the local architectural and transcriptional dynamics of endogenous MYOD1 and MYOG reflected the global genomic bifurcation event. These TFs additionally participate in entrainment of biological rhythms. Understanding the system-level genome dynamics underlying a cell fate decision is a step toward devising more sophisticated reprogramming strategies that could be used in cell therapies.

cell biology↗