Biology subjects

Loh, K. M.

Publications and source records attributed to Loh, K. M..

16 recordsLinked to original sources

bioRxiv2026

Comprehensive Lineage Tracing Maps the Landscape of Cell Fate Decisions in Mouse Embryogenesis

A comprehensive cell fate map of mammalian embryogenesis has remained out of reach due to the scale, cellular diversity, and non-deterministic nature of development in utero. Here, we use PEtracer to continuously install heritable genetic marks as cells divide, reconstructing lineage trees that resolve [~]75% of cell divisions across >1.5 million cells from 16 mouse embryos collected at half-day intervals from E7.5-E10.0. We pair these trees with deep transcriptional profiling to chart the landscape of cell fate decisions during gastrulation and early organogenesis. Using these data, we quantify cell fate biases, restriction timing, progenitor pool sizes, and lineage relationships across the embryo, revealing strikingly reproducible lineage architecture across replicate embryos despite the regulative flexibility of mammalian development. We further show how lineage, spatial position, and signaling jointly determine fate outcomes and timing, with their relative influence varying by tissue. This dataset provides a quantitative framework for understanding cell fate specification and a lineage-resolved reference for generating and contextualizing developmental hypotheses at organismal scale.

developmental biology↗

bioRxiv2026

Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

A preeminent goal of virology is to discover cellular genes that mediate virus entry. Genome-wide loss-of-function screens can illuminate single genes necessary for virus entry, but are stymied by genetic redundancy. Here we report a genome-wide CRISPR activation screening strategy to discover single genes that are sufficient for viral entry into normally-uninfectable cells. Sequential rounds of viral infection vastly enhanced screening sensitivity. This sequential screening strategy was generalizable to two unrelated viruses--Ebola and rabies viruses--and could broadly accelerate the discovery of viral entry factors.

microbiology↗

bioRxiv2025

VEGF/ERK activation and PI3K inhibition together drive a vein-to-artery transition in an in vitro model of human angiogenesis

Artery endothelial cells (ECs) arise through different pathways, including differentiation from mesodermal cells (vasculogenesis) or from already established vein or capillary plexus ECs (angiogenesis), the latter being most common during embryonic development and regeneration. Understanding the vein-to-artery (v2a) transition could improve revascularization therapies, but progress is limited by a lack of human models. Here, we develop a human pluripotent stem cell (hPSC) differentiation protocol that models the v2a EC conversion. Comparing v2a and mesoderm-to-artery (m2a) transcriptomes with publicly available single cell RNA sequencing (scRNA-seq) data from human embryos showed they reflected angiogenesis- and vasculogenesis-derived artery ECs, respectively. This reductionist system revealed that VEGF activation alongside PI3K inhibition was sufficient for vein ECs to acquire arterial identity within 48 hours. We model a critical step in vascular development and define the minimal signals required for artery differentiation from veins, providing a framework to promote this conversion in revascularization or therapeutic contexts.

developmental biology↗

bioRxiv2025

A human arteriovenous differentiation roadmap reveals vein developmental mechanisms and vascular effects of viruses

Extracellular signals and cell-fate trajectories during vein development remain elusive, despite trailblazing insights into artery development. Here we exploit human pluripotent stem cell differentiation and mouse embryology to present a model that answers longstanding questions: vein endothelial cell (EC) differentiation unfolds in two steps driven by opposing extracellular signals. First, VEGF differentiates mesoderm into "primed" ECs, newly-defined progenitors that co-express certain arterial (SOX17) and venous (APLNR) markers. Second, primed ECs execute vein differentiation upon VEGF/ERK inhibition; however, upon VEGF activation they can instead form artery ECs. The arteriovenous plasticity of primed ECs was supported by intersectional lineage tracing. Future venous genes including NR2F2 harbor poised chromatin in primed ECs, but are only transcribed upon VEGF/ERK inhibition. SOXF transcription factors, including SOX17, confer primed ECs with vein differentiation competence. Collectively, this two-step vein differentiation model--entailing primed EC intermediates and VEGF/ERK inhibition to trigger vein differentiation--has implications for VEGF-modulating therapies.

developmental biology↗

bioRxiv2025

Two parallel lineage-committed progenitors contribute to the developing brain

The hindbrain is a life-sustaining brain region. In one model, a common neural progenitor generates all brain regions. Here our studies of mouse embryos and human pluripotent stem cells (hPSCs) support a different model: two parallel brain progenitors emerge simultaneously during gastrulation, anterior neural ectoderm (forebrain/midbrain progenitor) and posterior neural ectoderm (hindbrain progenitor). Not only are they lineage-committed to respectively form forebrain/midbrain vs. hindbrain in vitro, but they also have diverging chromatin landscapes foreshadowing future forebrain/midbrain vs. hindbrain identities. Leveraging these differences, we differentiated hPSCs into hindbrain rhombomere 5/6-specific motor neurons, hitherto difficult to generate in vitro. We postulate the brain is a composite organ emanating from two lineage-restricted progenitors; these dual progenitors may be evolutionarily conserved across 550 million years from hemichordates to mammals.

developmental biology↗

bioRxiv2025

Metabolically purified human stem cell-derived hepatocytes reveal distinct effects of Ebola and Lassa viruses

Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo. In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.

cell biology↗

bioRxiv2024

DNMT3AR882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy. STATEMENT OF SIGNIFICANCEUnderstanding which driver mutations are required for cancer initiation, maintenance, or both phases remains poorly understood. Here, we uncover that highly prevalent pre-leukemic DNMT3A mutations are only required during disease initiation, but become dispensable after leukemic transformation, uncovering the context-specific role of this driver mutation with important therapeutic implications.

cancer biology↗

bioRxiv2024

TMX1, a disulfide oxidoreductase, is necessary for T cell function through regulation of CD3zeta

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NF{kappa}B, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3{zeta}, a subunit of TCR complex; however, overexpression of CD3{zeta} rescues the phenotype, suggesting that TMX1 is not required for CD3{zeta} function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3{delta}, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3{delta} leads to impaired TCR assembly and subsequent CD3{zeta} destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

immunology↗

bioRxiv2024

Mesoscale chromatin confinement facilitates target search of pioneer transcription factors in live cells

Pioneer transcription factors (PTFs) possess the unique capability to access closed chromatin regions and initiate cell fate changes, yet the underlying mechanisms remain elusive. Here, we characterized the single-molecule dynamics of PTFs targeting chromatin in living cells, revealing a notable "confined target search" mechanism. PTFs like FOXA1, FOXA2, SOX2, OCT4 and KLF4 sampled chromatin more frequently than non-pioneer factor MYC, alternating between fast free diffusion in the nucleus and slower confined diffusion within mesoscale zones. Super-resolved microscopy showed closed chromatin organized as mesoscale nucleosome-dense domains, confining FOXA2 diffusion locally and enriching its binding. We pinpointed specific histone-interacting disordered regions, distinct from DNA-binding domain, crucial for confined target search kinetics and pioneer activity within closed chromatin. Fusion to other factors enhanced pioneer activity. Kinetic simulations suggested transient confinement could increase target association rate by shortening search time and binding repeatedly. Our findings illuminate how PTFs recognize and exploit closed chromatin organization to access targets, revealing a pivotal aspect of gene regulation.

molecular biology↗

bioRxiv2023

Liver size is predetermined in the neonate by adding lobules at the periphery

Organs vary in size between and within species to match organismal needs1,2. Decades-old theoretical work has proposed that scaling of organs and body parts relative to the body relies on the features of energy-transport systems, the vascular system in mammals3. Yet, experimental studies on whether or how vascularization helps determine organ size have lagged behind. The mammalian liver is a remarkable example, as liver size scales proportionally with high precision between individuals4. Here, we use quantitative clonal mapping, volumetric imaging, and genetic perturbations combined with novel molecular and genetic tools to identify the temporal and spatial constraints that establish mouse liver size. We find that adult liver size is predetermined during a neonatal period when new functional units, termed lobules, are added to the organ. New lobules are vascularized by prominent sprouting angiogenesis of the hepatic vein, restricted to the periphery of the organ. When Wnt signals are ablated in the single cell-layered mesothelium at the periphery, lobule growth fails, and the organ adopts a compromised size set point. Remarkably, within a week after birth and well before hepatocyte division stops, vein sprouting rapidly declines and lobule addition concludes, setting a limit on the final liver size. These findings posit that vascularization in the neonate constrains and helps determine adult liver size. Together, these results propose a novel, vasculature-centric experimental framework for studying organ size control and scaling in mammals.

developmental biology↗

bioRxiv2021

Increased ACTL6A Occupancy Within mSWI/SNF Chromatin Remodelers Drives Human Squamous Cell Carcinoma

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit, ACTL6A, is amplified at an early stage in the development of squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives its interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was sub-stoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near-saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition over the genome activating SCC genes and also enhanced the recruitment of transcription factor TEAD with its co-activator YAP, promoting their chromatin binding and enhancer accessibility. Both of these mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs. HighlightsO_LIACTL6A occupancy within BAF complexes is sub-stoichiometric in normal epithelial cells. C_LIO_LISCC cells upregulate ACTL6A thus increasing ACTL6A assembly with BAF complexes. C_LIO_LIGenome-wide chromatin profiling identifies ACTL6A-dependent regulatory regions. C_LIO_LIIncreasing ACTL6A incorporation enhances TEAD-YAP binding to BAF complexes. C_LIO_LIACTL6A overexpression counteracts polycomb-mediated repression at SCC signature genes. C_LI

molecular biology↗

bioRxiv2020

An atlas of lamina-associated chromatin across thirteen human cell types reveals cell-type-specific and multiple subtypes of peripheral heterochromatin

Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamin-associated domains (LADs) have yet to be mapped genome-wide in a diverse panel of human cell types, which has limited our understanding of the role peripheral chromatin plays in development and disease. To address this gap, we mapped LAMIN B1 (LB1) across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas of peripheral chromatin with publicly available genomic data, as well as gene expression and repressive histone maps generated for this study, revealed that in all twelve cellular contexts lamin-associated chromatin is organized into at least two subtypes defined by differences in LB1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Most genes gain or lose LB1 occupancy consistent with their cell type along developmental trajectories; however, we also identified examples where the enhancer, but not the gene body and promoter, change LAD state. Imaging of fluorescently labeled DNA in single cells validated these transitions and showed intermediate radial positioning of LADs that are gene dense, relatively accessible, and dynamic across development. This atlas represents the largest resource to date for peripheral chromatin organization studies.

developmental biology↗

bioRxiv2020

Copy Number Variation at 16p11.2 Imparts Transcriptional Alterations in Neural Development in an hiPSC-derived Model of Corticogenesis

Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations of these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). Cortical neural progenitor cells derived from these hiPSCs were profiled using RNA-Seq, which identified alterations in radial glial gene expression that precede morphological abnormalities reported at later neurodevelopmental stages. Moreover, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of "footprint"-free hiPSC clones that are available by request from the Simons Foundation Autism Research Initiative. This publicly available resource of 65 human hiPSC clones can serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.

neuroscience↗

bioRxiv2019

Cross-species blastocyst chimerism between nonhuman primates using iPSCs

Through the production of chimeric animals, induced pluripotent stem cells (iPSCs) can generate personalized organs with diverse applications for both basic research and translational medicine. This concept was first validated in rodents by forming a rat pancreas in mice and vice versa. However, the potential use of human iPSCs to generate xenogenic organs in other species is technically and ethically difficult. Recognizing these concerns, we explored the generation of chimeric nonhuman primates (NHP) embryos, by injecting either chimpanzee or pig-tailed macaque iPSCs into rhesus macaque embryos. We first derived iPSCs from chimpanzees and pig-tailed macaques. We found that the chimpanzee iPSCs mixed well with human iPSCs during in vitro co-culture and differentiation. The differentiation of mixed human and chimpanzee iPSCs formed functioning cardiomyocyte layers in vitro, whereas human or chimpanzee iPSC mixed with pig-tailed macaque or mouse cells do not; these results indicate that chimpanzee and human cells are closely related in function. Considering the ethical aspects of injecting human iPSCs into nonhuman primate blastocysts, we tested whether chimpanzee iPSCs injected into 99 macaque 5-day-old embryos formed cross-species chimeras two days after injection. Strikingly, the chimpanzee iPSCs survived, proliferated and integrated near the inner cell mass (ICM) of rhesus macaque embryos. These findings highlight the broad potential of primate iPSCs in forming cross-species chimeras beyond rodents and provides a foundational basis for organ generation using human iPSCs.

developmental biology↗

bioRxiv2019

Enabling MHC-mismatched hematopoietic stem cell transplants and organ graft tolerance without chemotherapy or radiation

Hematopoietic stem cell (HSC) transplantation can replace diseased blood systems with a healthy one, thereby treating or curing genetic blood and immune disorders including autoimmune diseases and immunodeficiencies. However, toxic chemotherapy or radiation is necessary to ablate an animals existing blood system prior to HSC transplantation, leading to significant morbidity. To accomplish safer blood-system replacement we developed a combination of six monoclonal antibodies to safely and specifically deplete the HSCs, T cells and NK cells of immune-competent mice. Remarkably, immunologically-foreign (allogeneic) HSCs mismatched at half or all the MHC genes could engraft these antibody-treated mice, generating donor blood systems that stably co-existed with host blood cells. These chimeric immune systems were immunologically tolerant to heart tissue from the HSC donor, providing a safe platform for HSC transplantation as a means to solid organ transplantation. The ability to transplant MHC-mismatched HSCs without chemotherapy or radiation has significant ramifications for regenerative medicine.

immunology↗

bioRxiv2017

Accelerated human liver progenitor generation from pluripotent stem cells by inhibiting formation of unwanted lineages

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0{+/-}3.1% AFP+ human liver bud progenitors and 87.3{+/-}9.4% ALBUMIN+ hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5{+/-}3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.

cell biology↗