Biology subjects

McDevitt, T. C.

Publications and source records attributed to McDevitt, T. C..

16 recordsLinked to original sources

bioRxiv2024

Distinct differentiation trajectories leave lasting impacts on gene regulation and function of V2a interneurons

During development, early regionalization segregates lineages and directs diverse cell fates. Sometimes, however, distinct progenitors produce analogous cell types. For example, V2a neurons, are excitatory interneurons that emerge from different anteroposterior progenitors. V2a neurons demonstrate remarkable plasticity after spinal cord injury and improve motor function, showing potential for cell therapy. To examine how lineage origins shape their properties, we differentiated V2a neurons from hPSC-derived progenitors with distinct anteroposterior identities. Single-nucleus multiomic analysis revealed lineage-specific transcription factor motifs and numerous differentially expressed genes related to axon growth and calcium handling. Bypassing lineage patterning via transcription factor-induced differentiation yielded neurons distinct from both developmentally relevant populations and human tissue, emphasizing the need to follow developmental steps to generate authentic cell identities. Using in silico and in vitro loss-of-function analyses, we identified CREB5 and TCF7L2 as regulators specific to posterior identities, underscoring the critical role of lieage origins in determining cell states and functions.

developmental biology↗

bioRxiv2024

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development

Proper regulation of gene dosage is critical for the development of the early embryo and the extraembryonic tissues that support it. Specifically, loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. In this study, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. We generate an allelic series for CDX2 in human induced pluripotent stem cells (hiPSCs) consisting of WT, heterozygous, and homozygous null CDX2 genotypes, differentiate these cells in a 2D gastruloid model, and subject these cells to multiomic single nucleus RNA and ATAC sequencing. We identify several genes that CDX2 dose-dependently regulate cytoskeletal integrity and adhesiveness in the extraembryonic mesoderm population, including regulators of the VEGF, canonical WNT, and non-canonical WNT signaling pathways. Despite these dose-dependent gene expression patterns, snATAC-seq reveals that heterozygous CDX2 expression is capable of inducing a WT-like chromatin accessibility profile, suggesting accessibility is not sufficient to drive gene expression when the CDX2 dosage is reduced. Finally, because the loss of CDX2 or TBXT phenocopy one another in vivo, we compare differentially expressed genes in our CDX2 knock-out model to those from TBXT knock-out hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability, including ANK3 and ANGPT1. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and suggest these genes may underlie the defects in vascular development and allantoic elongation seen in the absence or reduction of CDX2 in vivo. Summary StatementUsing 2D human gastruloids, CDX2 is shown to dose-dependently influence genes related to tissue permeability, cell-cell adhesions, and cytoskeletal architecture during extraembryonic mesoderm development.

developmental biology↗

bioRxiv2023

TBXT dose sensitivity and the decoupling of nascent mesoderm specification from EMT progression in 2D human gastruloids

In the nascent mesoderm, levels of Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how this varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dose reduction during early human gastrulation using human induced pluripotent stem cell (hiPSC)-based models of gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing of 2D gastruloids comprised of WT, TBXT heterozygous (TBXT-Het), or TBXT null (TBXT-KO) hiPSCs reveal that varying TBXT dosage does not compromise a cells ability to differentiate into nascent mesoderm, but that the loss of TBXT significantly delays the temporal progression of the epithelial to mesenchymal transition (EMT). This delay is dependent on TBXT dose, as cells heterozygous for TBXT proceed with EMT at an intermediate pace relative to WT or TBXT-KO. By differentiating iPSCs of the allelic series into nascent mesoderm in a monolayer format, we further illustrate that TBXT dose directly impacts the persistence of junctional proteins and cell-cell adhesions. These results demonstrate that EMT progression can be decoupled from the acquisition of mesodermal identity in the early gastrula and shed light on the mechanisms underlying human embryogenesis.

developmental biology↗

bioRxiv2021

Hepatitis C Virus Infects and Perturbs Liver Stem Cells

Hepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk beyond viral clearance remains unclear. We identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA-sequencing uncovered extensive transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data adds a pathogenesis factor - infection of liver stem cells - to the biology of HCV infection that explains persistent liver damage and enhanced cancer risk through an altered stem cell state.

microbiology↗

bioRxiv2021

Liver Organoid and T Cell Coculture Models Cytotoxic T Cell Responses against Hepatitis C Virus

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV nonstructural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

immunology↗

bioRxiv2021

Allele-specific gene editing rescues pathology in a human model of Charcot-Marie-Tooth disease type 2E

Many neuromuscular disorders are caused by dominant missense mutations that lead to dominant-negative or gain-of-function pathology. This category of disease is challenging to address via drug treatment or gene augmentation therapy because these strategies may not eliminate the effects of the mutant protein or RNA. Thus, effective treatments are severely lacking for these dominant diseases, which often cause severe disability or death. The targeted inactivation of dominant disease alleles by gene editing is a promising approach with the potential to completely remove the cause of pathology with a single treatment. Here, we demonstrate that allele-specific CRISPR gene editing in a human model of axonal Charcot-Marie-Tooth (CMT) disease rescues pathology caused by a dominant missense mutation in the neurofilament light chain gene (NEFL, CMT type 2E). We utilized a rapid and efficient method for generating spinal motor neurons from human induced pluripotent stem cells (iPSCs) derived from a patient with CMT2E. Diseased motor neurons recapitulated known pathologic phenotypes at early time points of differentiation, including aberrant accumulation of neurofilament light chain protein in neuronal cell bodies. We selectively inactivated the disease NEFL allele in patient iPSCs using Cas9 enzymes to introduce a frameshift at the pathogenic N98S mutation. Motor neurons carrying this allele-specific frameshift demonstrated an amelioration of the disease phenotype comparable to that seen in an isogenic control with precise correction of the mutation. Our results validate allele-specific gene editing as a therapeutic approach for CMT2E and as a promising strategy to silence dominant mutations in any gene for which heterozygous loss-of-function is well tolerated. This highlights the potential for gene editing as a therapy for currently untreatable dominant neurologic diseases.

genetics↗

bioRxiv2021

Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.

neuroscience↗

bioRxiv2020

Silencing of E-cadherin in induced human pluripotent stem cells promotes extraembryonic fates accompanying multilineage differentiation

Summary/AbstractIn embryonic development, symmetry breaking events and the mechanical milieus in which they occur coordinate the specification of separate cell lineages. Here, we use 3D aggregates of human pluripotent stem cells (hPSCs) encapsulated in alginate microbeads to model the early blastocyst prior to zona pellucida hatching. We demonstrate that 3D confinement combined with modulation of cell-cell adhesions is sufficient to drive differentiation and collective migration reminiscent of the pre-implantation embryo. Knockdown of the cell adhesion protein CDH1 in encapsulated hPSC aggregates resulted in protrusion morphologies and emergence of extra-embryonic lineages, whereas unencapsulated CDH1(-) aggregates displayed organized radial delamination and mesendoderm specification bias. Transcriptomic similarities between single-cell RNA-sequencing data of early human embryos and encapsulated CDH1(-) aggregates establishes this in vitro system as a competent surrogate for studying early embryonic fate decisions and highlights the relationship between cell-cell adhesions and the mechanical microenvironment in directing cell fate and behavior. HighlightsO_LIGeneration of embryonic scale 3D morphogenesis using hydrogel encapsulation C_LIO_LIManipulating adhesion triggers emergence of specific morphologies and cell fates C_LIO_LIAcquisition of germ layer cell fates mimics early human embryonic diversity C_LI

developmental biology↗

bioRxiv2020

Deep neural net tracking of human pluripotent stem cells reveals intrinsic behaviors directing morphogenesis

Lineage tracing is a powerful tool traditionally used in developmental biology to interrogate the evolutionary time course of tissue formation, but the dense, three-dimensional nature of tissue limits the ability to assemble individual traces into complete reconstructions of development. Human induced pluripotent stem cells (hiPSCs) enable recapitulation of various aspects of developmental processes, thereby providing an in vitro platform to assess the dynamic collective behaviors directing tissue morphogenesis. Here, we trained an ensemble of independent convolutional neural networks to identify individual hiPSCs imaged via time lapse microscopy in order to generate longitudinal measures of individual cell and dense cellular neighborhood properties simultaneously on timescales ranging from minutes to days. Our analysis reveals that while individual cell parameters are not strongly affected by extracellular microenvironmental conditions such as pluripotency maintenance regime or soluble morphogenic cues, regionally specific cell behaviors change in a manner predictive of organization dynamics. By generating complete multicellular reconstructions of hiPSC behavior, our cell tracking pipeline enables fine-grained understanding of developmental organization by elucidating the role of regional behavior stratification in early tissue formation.

bioengineering↗

bioRxiv2020

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.

cell biology↗

bioRxiv2020

Cardiac Cell-Derived Matrices Impart Age-Specific Functional Properties to Human Cardiomyocytes

Cell-derived matrices (CDMs) isolated from cultured cells provide complex and tissue-specific biochemical and physical cues derived from the extracellular matrix (ECM) that are lacking in typical tissue culture environments. However, current methods enhance ECM adhesion and thickness via introduction and promotion of singular matrix proteins, skewing the matrix composition, and confounding comparisons between CDMs. Here we developed a protocol that enhances CDM stability and deposition, respectively, by combining an L-polydopamine surface coating with Ficoll macromolecular crowing prior to hypotonic decellularization. This methodology was applied to the study of age-dependent phenotypic and functional changes observed in cardiac ECM by comparing the morphologic, electrophysiological and metabolic response of cardiomyocytes in response to CDMs produced by fetal and adult cardiac fibroblasts. Furthermore, mass spectrometry proteomics identified the enrichment of collagen VI in fetal CDMs, which we determined via siRNA-mediated silencing during CDM production to be necessary for maximal oxidative respiration in cardiomyocytes.

cell biology↗

bioRxiv2020

Bi-directional Impacts of Heterotypic Interactions in Engineered 3D Human Cardiac Microtissues Revealed by Single-Cell RNA-Sequencing and Functional Analysis

Technological advancements have enabled the design of increasingly complex engineered tissue constructs, which better mimic native tissue cellularity. Therefore, dissecting the bi-directional interactions between distinct cell types in 3D is necessary to understand how heterotypic interactions at the single-cell level impact tissue-level properties. We systematically interrogated the interactions between cardiomyocytes (CMs) and cardiac non-myocytes in 3D self-assembled tissue constructs in an effort to determine the phenotypic and functional contributions of cardiac fibroblasts (CFs) and endothelial cells (ECs) to cardiac tissue properties. One week after tissue formation, cardiac microtissues containing CFs exhibited improved calcium handling function compared to microtissues comprised of CMs alone or CMs mixed with ECs, and CMs cultured with CFs exhibited distinct transcriptional profiles, with increased expression of cytoskeletal and ECM-associated genes. However, one month after tissue formation, functional and phenotypic differences between heterotypic tissues were mitigated, indicating diminishing impacts of non-myocytes on CM phenotype and function over time. The combination of single-cell RNA-sequencing and calcium imaging enabled the determination of reciprocal transcriptomic changes accompanying tissue-level functional properties in engineered heterotypic cardiac microtissues.

bioengineering↗

bioRxiv2020

Developmental co-emergence of cardiac and gut tissues modeled by human iPSC-derived organoids

During embryogenesis, paracrine signaling between tissues in close proximity contributes to the determination of their respective cell fate(s) and development into functional organs. Organoids are in vitro models that mimic organ formation and cellular heterogeneity, but lack the paracrine input of surrounding tissues. Here, we describe a human multilineage iPSC-derived organoid that recapitulates cooperative cardiac and gut development and displays extensive cellular and structural complexity of both tissues. We demonstrate that the presence of endoderm tissue (gut/intestine) in multilineage organoids contributed to the development of the cardiac tissue, specifically cardiomyocyte expansion, compartmentalization, enrichment of atrial/nodal cells, myocardial compaction and functional fetal-like maturation. Overall, this study demonstrates the ability to generate specific cooperative tissues originating from different germ lineages within a single organoid model, an advance that will further the examination of multi-tissue interactions during development and disease.

developmental biology↗

bioRxiv2020

Axial Elongation of Caudalized Human Pluripotent Stem Cell Organoids Mimics Neural Tube Development

Axial elongation of the neural tube is critical during mammalian embryogenesis to establish the anterior-posterior body axis1, but this process is difficult to interrogate directly because it occurs post-implantation2,3. Here we report an organoid model of neural tube extension by caudalized human pluripotent stem cell (hPSC) aggregates that recapitulates the morphologic and temporal gene expression patterns of neural tube development. Axially extending organoids consisting largely of longitudinally elongated neuroepithelial compartments also contained TBXT(+)SOX2(+) neuromesodermal progenitors, PAX6(+)nestin(+) neural progenitor populations, and MEOX1(+) paraxial mesoderm populations. Wnt agonism stimulated singular axial extensions in a dose-dependent manner, and elongated organoids displayed regionalized rostral-caudal HOX gene expression, with spatially distinct hindbrain (HOXB1) expression from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR-interference-mediated silencing of the TBXT, a downstream Wnt target, increased neuroepithelial compartmentalization and resulted in multiple extensions per aggregate. Further, knock-down of BMP inhibitors, Noggin and Chordin, induced elongation phenotypes that mimicked murine knockout models. These results indicate the potent morphogenic capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early nervous system development in humans.

bioengineering↗

bioRxiv2020

Single cell determination of cardiac microtissue structure and function using light sheet microscopy

Native cardiac tissue is comprised of heterogeneous cell populations that work cooperatively for proper tissue function; thus, engineered tissue models have moved toward incorporating multiple cardiac cell types in an effort to recapitulate native multicellular composition and organization. Cardiac tissue models comprised of stem cell-derived cardiomyocytes require inclusion of non-myocytes to promote stable tissue formation, yet the specific contributions of the supporting non-myocyte population on the parenchymal cardiomyocytes and cardiac microtissues have yet to be fully dissected. This gap can be partly attributed to limitations in technologies able to accurately study the individual cellular structure and function that comprise intact 3D tissues. The ability to interrogate the cell-cell interactions in 3D tissue constructs has been restricted by conventional optical imaging techniques that fail to adequately penetrate multicellular microtissues with sufficient spatial resolution. Light sheet fluorescence microscopy overcomes these constraints to enable single cell-resolution structural and functional imaging of intact cardiac microtissues. Multicellular spatial distribution analysis of heterotypic cardiac cell populations revealed that cardiomyocytes and cardiac fibroblasts were randomly distributed throughout 3D microtissues. Furthermore, calcium imaging of live cardiac microtissues enabled single-cell detection of cardiomyocyte calcium activity, which showed that functional heterogeneity correlated with spatial location within the tissues. This study demonstrates that light sheet fluorescence microscopy can be utilized to determine single-cell spatial and functional interactions of multiple cell types within intact 3D engineered microtissues, thereby facilitating the determination of structure-function relationships at both tissue-level and single-cell resolution. Impact StatementThe ability to achieve single-cell resolution by advanced 3D light imaging techniques enables exquisite new investigation of multicellular analyses in native and engineered tissues. In this study, light sheet fluorescence microscopy was used to define structure-function relationships of distinct cell types in engineered cardiac microtissues by determining heterotypic cell distributions and interactions throughout the tissues as well as by assessing regional differences in calcium handing functional properties at the individual cardiomyocyte level.

bioengineering↗

bioRxiv2018

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.\n\nSIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

bioengineering↗