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U.S. National Science Foundation

The Established Program to Stimulate Competitive Research (EPSCoR) is designed to fulfill the mandate of the National Science Foundation (NSF) to promote scientific progress nationwide. EPSCoR eligibility status is yearly updated and reported in the EPSCoR website (see EPSCoR eligibility). Through this program, NSF establishes partnerships with government, higher education, and industry that are designed to affect sustainable improvements in a jurisdiction's research infrastructure, Research and Development (R&D) capacity, and hence, its R&D competitiveness. The FEC program (formerly known as EPSCoR Track-2 program ) builds interjurisdictional collaborative teams of EPSCoR investigators in Science, Technology, Engineering, and Mathematics (STEM) focus areas consistent with the currentNational Science Foundation Strategic Plan. Projects are investigator-driven and must include researchers from at least two EPSCoR eligible jurisdictions with complementary expertise and resources necessary to address challenges, which neither party could address as well or as rapidly independently. FEC projects have a comprehensive and integrated vision to drive discovery and build sustainable STEM capacity that exemplifies institutional, geographic, and disciplinary diversity. The projects STEM research and education activities seek to broaden participation through the strategic inclusion and integration of all individuals, institutions, and sectors. Additionally, EPSCoR recognizes that the development of early-career faculty is critical to sustaining and advancing research capacity.

U.S. National Science Foundation

The Experimental Program to Stimulate Competitive Research (EPSCoR) is a program designed to fulfill the National Science Foundation's (NSF) mandate to promote scientific progress nationwide. The EPSCoR program is directed at jurisdictions that have historically received lesser amounts of NSF Research and Development (R&D) funding. Thirty-one jurisdictions including twenty-eight states, the Commonwealth of Puerto Rico, the U. S. Virgin Islands, and Guam currently are eligible to participate. Through this program, NSF establishes partnerships with government, higher education, and industry that are designed to effect lasting improvements in a state's or region's research infrastructure, R&D capacity and hence, its national R&D competitiveness.Research Infrastructure Improvement Program: Track-2 (RII Track-2) awards provide funds in the range of $1.5 to 2.0 million per year for up to 3 years to consortia of EPSCoR jurisdictions. The awards promote opportunities for collaborations among EPSCoR jurisdictions in all areas of science, engineering, and education supported by the National Science Foundation (NSF). RII Track-2 proposals must describe a clear, comprehensive, and integrated vision to drive discovery, and train a skilled workforce capable of solving science and engineering challenges of regional, thematic, and national relevance. Proposals should also include a strong rationale for the establishment of the consortium and clearly demonstrate that the consortium is well-positioned to produce results that cannot be obtained by any single partner working independently. The Science, Technology, Engineering, and Mathematics (STEM) research and education activities should broaden participation by different types of institutions, individuals, and sectors in the project.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract : Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a chronic bacterial infection. It remains a significant global health burden1, killing more than a million individuals annually. In 2025, there was a 15% rise in TB cases in the USA, with a major outbreak in Kansas and active TB cases in Maine. The current TB treatment options are limited, require months-long treatment regimens, and are largely ineffective against drug-resistant strains. Even after successful treatment, 40 to 60% of TB survivors suffer from TB-associated lung damage, causing chronic respiratory symptoms, long-term morbidity and mortality. However, currently there are no approved TB therapies that mitigate, prevent or reverse TB-associated lung damage, highlighting a major unmet clinical need. Alveolar epithelial destruction is a central feature of TB-associated lung damage, but the mechanisms driving this destruction remain poorly understood. Autopsy studies, animal models, and in vitro systems suggest that Mtb compromises the function of alveolar epithelium, leading to the loss of alveolar epithelial integrity. Our preliminary studies show that JHU083, a glutamine metabolism inhibitor, reduces lung bacillary burden in Mtb-infected mice and increases the expression of stemness markers by alveolar epithelial progenitor cells. Our central hypothesis is that Mtb infection compromises function of alveolar epithelial cells, resulting in the loss of both AT1 and AT2 cells, leading to epithelial destruction and lung damage. This project seeks to answer three fundamental questions: (1) Which alveolar epithelial cell functions are crucial for epithelial integrity in vivo? (2) What are the metabolic drivers of epithelial destruction in Mtb-infected lungs? (3) Can alveolar epithelial cells be metabolically reprogrammed to restore epithelial integrity in Mtb-infected lungs? Using a multidisciplinary approach—including single-cell transcriptomics, metabolomics, immunophenotyping, ex vivo co-culture systems and in vivo models of TB—will transform our understanding of TB pathogenesis by providing the first comprehensive in vivo dissection of the molecular and cellular drivers of alveolar epithelial destruction— a fundamental yet overlooked contributor to TB-associated lung damage and a major unmet clinical challenge. The successful completion of this work will transform our understanding of TB pathogenesis and catalyze host- directed strategies to mitigate, prevent or reverse lung damage in TB survivors.

NEI - National Eye Institute

PROJECT SUMMARY/ABSTRACT Damage and dysfunction of photoreceptors in the retina is one of the leading causes of vision loss and there are currently no treatment options for such retinal degenerative diseases. With the recent breakthroughs in human stem cell technology, it has become possible to recreate a 3D mini retina in a dish called retinal organoids (ROs) from human pluripotent stem cells (hPSC) that share several similarities with human retina including generating electrical signals to light stimuli. However, there are several limitations that remain unidentified or poorly understood which precludes ROs as a robust in vitro model of human retina for studying diseases, testing therapeutics and as a renewable source of human photoreceptors for stem cell replacement therapies. Our goal in this proposal is to determine the limitations in ROs with respect to cone photoreceptor signaling both at the level of signal transduction as well as signal transmission across the photoreceptor synapse to the remaining neural circuitry. In addition, we will also identify the limitations of ROs as a viable alternative for modeling retinal diseases in a dish with the goal of testing gene therapy in a dose-dependent manner. In Aim 1 we will determine the relative contributions of cone photoreceptor-extrinsic, cone photoreceptor-intrinsic as well as developmental factors that limit a uniform population of functional cones with robust phototransduction profiles. In Aim 2 we will delineate the extent to which there is reliable transmission of electrical signaling between cone photoreceptors and the second-order neurons in ROs using a combinatorial approach of anatomical, electrophysiological, and genetic tools. In Aim 3, we will leverage patient-derived ROs and gene-edited ROs to model two distinct retinal diseases that affect cone signaling and use a viral-mediated delivery approach to quantify a dose-response potency assay for gene therapy. Together these aims will help establish ROs as a powerful in vitro model of human retina and as a robust platform for modeling retinal diseases and testing gene therapy.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT Heterozygous de novo missense variants in KCNH1 are associated with the rare neurodevelopmental disorders Temple‐Baraitser Syndrome (TBS) and Zimmermann‐Laband Syndrome (ZLS), which share features including developmental delay, facial and digital abnormalities, and frequent epileptic seizures. KCNH1 encodes KV10.1, which is a voltage-gated potassium channel (Kv) widely expressed in central neurons. To date, more than 40 pathogenic KCNH1 variants have been reported. Preliminary evidence supports a gain-of-function (GOF) effect for most variants. The mechanistic insight we have on these mutations is limited to patch-clamp recording experiments with heterologous cells. The cellular mechanisms responsible for KCNH1-associated neuronal defects, particularly how intrinsic ion channel dysfunction causes abnormal human neuron excitability, remain completely unexplored. Additionally, there are no effective disease-modifying therapies for KCNH1-related disorders. Selective small molecule treatment is challenging given the high degree of conservation among related KV channels especially hERG, which in human heart is the major cause of life-threatening proarrhythmic off-target drug effects. Alternative approaches for targeting KCNH1, such as antisense oligonucleotides (ASOs) may be more selective and avoid off-target effects that compromise safety. However, proof-of-concept evidence that an ASO can reverse KCNH1 GOF defects in neuronal excitability does not exist, nor do any human-based model systems of KCNH1-associated disease. Here, we will use our expertise in ion channel biology, CRISPR/Cas9 gene editing and induced pluripotent stem cell (iPSC) technologies to perform a systematic characterization of KCNH1 mutations in human iPSC-derived excitatory and inhibitory neurons. In Aim 1 we will study excitatory and inhibitory neurons differentiated from a cohort of iPSC lines heterozygous for 3 distinct and functionally diverse KCNH1 mutations to determine their neurophysiological properties. We will evaluate expression of KV10.1 channels and measure single cell and population-based firing properties using whole-cell patch clamp and multi-electrode array (MEA) based recordings, respectively in cultured neurons. In Aim 2 we will evaluate the ability of a KCNH1 targeting ASO to normalize activity of iPSC-neurons heterozygous for pathogenic KCNH1 variants. We will assess potency, efficacy and time-dependence of gene suppression by monitoring the reduction in KCNH1 mRNA and protein expression following ASO treatment. We will use single cell (patch clamp recording) and population (MEA) electrophysiology to test the hypothesis that ASO treatment will normalize the resting potential and firing rate. We will optimize methods to isolate KV10.1 current in ASO- treated iPSC-neurons and assess functional knockdown of the current by the ASO. The goals of this exploratory R21 proposal are to: a) establish the first human iPSC-based models of KCNH1-associated diseases, b) provide a systematic evaluation of the impact of disease-associated mutations on neuronal physiology, and c) take the first step of developing a rational therapeutic for these devastating diseases.

U.S. National Science Foundation

Ethical and Responsible Research (ER2) research projects use fundamental research to produce knowledge about what constitutes or promotes responsible or irresponsible conduct of research and why, as well as how to best instill responsible conduct of research into researchers, practitioners, and educators at all career stages. In some cases, projects will include the development of interventions or applications to ensure ethical and responsible research conduct. The program funds research projects that identify: (1) factors that are effective in the formation of ethical science, technology, engineering, and mathematics (STEM) researchers; (2) approaches to developing those factors in all STEM fields that NSF supports; and (3) why and how those factors and approaches increase responsibly conducted research. Proposals from or involving substantial collaboration with minority-serving institutions, women's colleges, or organizations primarily serving persons with disabilities are strongly encouraged. Proposals that include international collaborations are encouraged if the unique resources, expertise, facilities, or locations of international partners enhance the merit of the proposed work. International partners are required to find non-NSF funding. Please see NSF s PAPPG for further guidance on international collaborations.

NIAID - National Institute of Allergy and Infectious Diseases

Enterovirus D68 (EV-D68) was described in 1962 as a respiratory illness but has recently had increased circulation and disease severity. EV-D68 is associated with acute flaccid myelitis (AFM), paralysis in children characterized by spinal cord lesions. There are no specific treatments for AFM and most children have long-lasting neurological deficits. Neuropathogenesis of EV-D68 is likely a combination of virus- and immune- mediated cytotoxicity, though relevant models to investigate this are limited. We recently showed that neonatal mice paralyzed by EV-D68 have abundant T cell recruitment to the spinal cord. When T cells were depleted, mice were protected from paralysis, suggesting a role for T cells in AFM. The role of T cells in human AFM remains unknown. The absence of human models to study T cell interactions in the central nervous system (CNS) hinders progress in identifying viral targets, mechanisms of neural injury, and immune contribution to pathogenesis. Major histocompatibility complexes (MHC) present antigens to T cells to induce the cytotoxic response and effector and memory T cells. Cell surface MHC Class I (MHC-I) is upregulated on neurons and glial cells after CNS injury. However, MHC-I modulation during viral infection of the CNS has not been studied in a multicellular human model. While viral infections are canonically expected to increase surface MHC-I, many viruses downregulate MHC-I as an immune evasion strategy. Our data suggests that EV-D68 infection downregulates, but does not eliminate, MHC- I in infected hSCO. We developed a human spinal cord organoid (hSCO) model for EV-D68 infection from induced pluripotent stem cells (iPSC). hSCO differentiate into multiple cell types of the spinal cord, including neurons and glial cells, and can be infected by EV-D68. Importantly, hSCO are in suspension without Matrigel, a substance that has hindered incorporation of T cells into CNS organoids due to effects on T cell activation and migration. We will utilize our human organoids to understand MHC-I modulation during EV-D68 infection and to investigate interactions between T cells and EV-D68 infected cells of the human CNS. Our overarching hypothesis is that EV-D68 neuropathogenesis is mediated by effects of CD8+ T cells, which we will test by 1) defining the mechanism of EV-D68 downregulation of MHC-I and 2) examining interactions between T cells and EV-D68 infected hSCO. Results will define mechanisms of EV- D68 neurovirulence in a novel human model by elucidating interactions between infected hSCO and the adaptive immune system. Findings will increase understanding of EV-D68 pathogenesis and potentially identify new virus-specific or immune-specific therapeutic targets for AFM.

NINDS - National Institute of Neurological Disorders and Stroke

Project Abstract: Mitchell Syndrome is a progressive childhood-onset neurodegenerative disorder characterized by sensory ataxia, hearing loss, skin changes, and eventual paralysis and encephalopathy, typically leading to death within the second decade of life. The first patient with Mitchell Syndrome was treated at Washington University in St. Louis (WashU), which makes WashU uniquely situated for researching this rare disease. WashU sees around 30% of known patients, has characterized the disease's natural history, and has extensive resources such as post-mortem tissues and a biofluid biobank. Our long-term goal is to develop effective treatments for Mitchell Syndrome. The disease is caused by an autosomal dominant variant in ACOX1, leading to a gain-of-function in the acyl-CoA oxidase 1 (ACOX1) protein. Our preliminary studies indicate that the variant impacts both sensory neurons and oligodendrocytes. Patient-derived induced pluripotent stem cells (iPSCs) offer a scalable, homogeneous platform for modeling rare diseases and evaluating precision-medicine therapeutics. We aim to develop iPSC-derived models of oligodendrocytes and sensory neurons to recapitulate Mitchell Syndrome as a tool to evaluate potential treatments. Preliminary data from patient-derived iPSC lines show transcriptional and metabolic abnormalities linked to the disease variant. We propose two specific aims: Aim 1: Evaluate ACOX1 gain-of-function in iPSC-derived oligodendrocytes. Aim 2: Investigate ACOX1 gain-of-function in iPSC-derived sensory neurons. Mitchell Syndrome intertwines lipid metabolism, oxidative stress, and neuronal/glial degeneration. This project aims to provide essential models for therapeutic evaluation, leveraging WashU's unique expertise and resources. At the culmination of this project we will have two scalable, disease-relevant, human models of Mitchell Syndrome that can be used for mechanistic studies, therapeutic development, and biomarker exploration – critical steps on the way to treat this lethal and tragic disease.

NCI - National Cancer Institute

PROJECT ABSTRACT The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, which include polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). MPNs carry an inherent risk of progression to advanced MPN, consisting of accelerated- phase disease (AP; 10-19% blasts in the peripheral blood or bone marrow), as well as blast phase disease (BP; ≥ 20% blasts in the peripheral blood or bone marrow). The prognosis of patients with advanced MPN remains quite poor, with median survival of 2.6 months. Importantly, chemotherapy regimens used to treat Acute Myeloid Leukemia (AML) such as standard induction chemotherapy (which are often used in advanced MPN) appear to have limited efficacy in this setting. Thus, the treatment of advanced MPN is a major unmet clinical need. We recently carried out a phase I/II study to test the safety and efficacy of combination therapy with the JAK1/2 inhibitor Ruxolitinib and the hypomethylating agent Decitabine in patients with advanced MPN (MPD-RC 109 study; NCT02076191). This combination (RUX-DAC) was based on data demonstrating synergy between these drugs in in vitro preclinical studies. 46 patients were accrued to the phase I and II studies. 37 patients were response evaluable. Complete response (CR) occurred in 10%, Complete Response with incomplete count recovery (CRi) in 24%, Partial Response (PR) in 24%. 42% of patients had no response to therapy. Using samples available from the MPD-RC 109 study, as well as samples from a contemporaneous clinical trial of 28 patients with advanced MPN treated with the RUX-DAC regimen carried out at the MD Anderson Cancer Center (NCT02257138), and samples collected from advanced MPN patients treated with the RUX-DAC regimen as a standard of care at Memorial Sloan Kettering Cancer Center, we seek to assess and validate genetic and epigenetic determinants of response to RUX-DAC in this cohort of homogenously treated advanced MPN patients. Specifically, we seek to assess whether the mutational profile of advanced MPN patients explains and predicts response to therapy. We further seek to assess whether alterations in genomic architecture in advanced MPN occur in patients who respond to therapy. Finally, we seek to determine if the baseline global methylation profile correlates with response to therapy, as has been demonstrated for other myeloid malignancies. Data resulting from these studies could be used to guide therapeutic decisions and identify patients for whom combination RUX-DAC therapy has the highest likelihood of procuring a response, as well as to open new lines of biologic and therapeutic inquiry into this disease.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold promise for cardiac disease modeling, drug testing, medical device testing, and regenerative medicine. However, their limited functional maturity in vitro remains a major barrier to widespread application. We recognize the crucial role of distinct and specific mechanical loads in cardiac morphogenesis and seek to co-opt signals downstream of mechanical engagement to drive maturation of hPSC-CMs. In particular, we seek to establish a causal link between mechanical stimulation and hPSC-CM maturation by focusing on the contribution of extracellular matrix (ECM) proteins, the primary family of proteins that mediate and confer mechanical force to the cell. Our lab has developed a novel human, chambered cardiac muscle pump model (hChaMP) capable of simulating both stretch and shear forces akin to a native cardiac cycle. We have begun to incorporate epicardial-derived cells (namely cardiac fibroblasts, CF) into the hChaMP (termed epi-ChaMP via a previously funded R01), as CFs remodel the ECM in response to mechanical stimulation. We also have expertise in cutting-edge computational modeling approaches to refine mechanical stimulation parameters and in fully characterizing the composition of the ECM following mechanical stimulation. Given our unique ecosystem, we can test the hypothesis that CM maturation is augmented in the epi-hChaMP via potent signaling of an evolving ECM deposited by CF in response to dynamic volumetric pressure. We will do so by developing and validating a computational fluid-structure interaction model that accurately replicates native cardiac pressure profiles in the epi-hChaMP (Aim 1), testing the mechanistic role of ECM deposition and remodeling by FBs in driving cardiomyocyte and tissue-scale maturation under physiologic loading (Aim 2), and by applying a statistical optimization framework to define dynamic volumetric loading regimes that maximize functional maturation of epi-hChaMP tissues. The proposal directly addresses reviewer feedback through clearer model differentiation, enhanced methodological descriptions, and inclusion of a non- cardiac fibroblast control. Completion of this project will reveal unappreciated contributions of ECM to CM maturation (Basic Science Innovation) and will yield a robust in vitro human muscle pump with unprecedented physiological relevance (Applied Science Innovation).

NIA - National Institute on Aging

PROJECT SUMMARY Sarcopenia, characterized by the loss of skeletal muscle mass and function with age, affects 10-22% of the global adult population and accounts for 1.5% of total formal healthcare costs in the United States. Sarcopenia significantly increases rates of mortality, hospitalization, cognitive impairment, falls, and depression, particularly among the elderly. With no approved pharmacological treatments, current interventions are limited to increased exercise and enhanced nutrition. However, clinical trials for these interventions have consistently failed to increase muscle mass despite improving muscle strength. Increases in muscle mass generally require the incorporation of new muscle nuclei derived from muscle stem cells (MuSCs). Since the discovery of muscle stem cells in 1961, many regulators of MuSC activity have been elucidated, but there has been limited success in identifying druggable targets for improving muscle healthspan. Recent discoveries in other stem cell systems, such as blood, intestine, and skin, have highlighted the key role of the autonomic nervous system (ANS) in regulating stem cell biology. While the autonomic innervation of skeletal muscle is well studied, we know little about the direct effects of the ANS on MuSCs. We propose to investigate the functional link between ANS neurons and MuSCs using a novel model in Drosophila melanogaster. We have developed methods to leverage the Drosophila genetic toolkit to image and manipulate both the ANS and MuSCs in adult flies and have obtained preliminary evidence showing a robust direct innervation of MuSCs by the axons of autonomic neurons in adult fly muscles. We also find that this innervation degrades with age. Our proposal will refine whole-body imaging methods and genetic tools for robust and faithful labeling of the ANS and MuSCs. We will investigate changes in the counts and spatial distribution of MuSCs and innervating autonomic neurons across the lifespan and during the course of regeneration from injury. We will identify the signaling molecules mediating the regulation of MuSCs by the ANS and adapt existing tools for the suppression or enhancement of neurotransmitter release in the ANS or their receptors in MuSCs. Lastly, we will modify existing protocols for isolating MuSCs form dissected muscle preparations to allow the extraction and sequencing of transcripts from single MuSCs to survey gene expression changes downstream of ANS modulation in both aging and injury repair contexts. By genetically dissecting the relationship between ANS and MuSCs, our proposed work will identify new molecular regulators of muscle aging, paving the way for alternative therapeutic approaches aimed at treatment of MuSC decline with age and improving muscle health.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Stroke is the most common neurological disorder and leading cause of long-term disability in the US. While some recovery occurs spontaneously and can be facilitated by rehabilitation, recovery is often incomplete. Therefore, there is a need to better understand the mechanisms of post-stroke recovery in order to develop more targeted interventions. In the past, efforts to examine the mechanisms of stroke recovery have utilized animal models with focal and homogeneous cortical lesions. In contrast, human patients present with lesions in a range of locations and sizes, limiting our ability to translate findings from experimental models into human patients. The overall objective of this project is to better understand the impact of lesion location and extent on the mechanisms of post-stroke recovery. Specifically, we will seek to determine whether the role of the contralesional hemisphere after stroke is dependent upon lesion location and extent. We will seek to address this objective by examining rats randomized to receive four distinct stroke models that will independently vary lesion size relative to the corticospinal tract and lesion location in the cortex or internal capsule. We will examine these animals using a complimentary combination of methods including electrophysiology, functional imaging, and cortical inactivation to test our central hypothesis that corticospinal tract integrity will determine the specific neural circuits associated with recovery. Specifically, we will test the bimodal balance recovery model which posits that the mechanisms of recovery will depend upon corticospinal tract integrity with recovery from an incomplete infarct facilitated by restoring interhemispheric balance and recovery from a complete infarct facilitated by the unaffected hemisphere. Initially, we will examine the difference in longitudinal changes in task-related neural activity associated with lesion location and extent (Aim 1). Next, we will examine the impact of different lesion locations and sizes on changes in corticocortical connectivity (Aim 2). We will use both an assessment of direct effective connectivity as a longitudinal surrogate for anatomical connectivity (Aim 2A) as well as a measure of resting- state functional connectivity that is similar to assessments of resting state functional connectivity MRI scans in human patients (Aim 2B). Finally, we will use chemogenetic techniques to determine whether cortical inactivation reinstates deficits following recovery (Aim 3). If successful, the project will provide evidence for patient-specific mechanisms of recovery that primarily depend upon corticospinal tract integrity. The novelty and potential impact of the project stems from the use of multiple distinct lesions that seek to better model clinical stroke, and the combination of longitudinal measures that will allow us to assess the functional relevance of potential recovery mechanisms. Achieving these goals will ultimately allow us to design more personalized therapies seeking to maximize post-stroke recovery by applying patient-specific neurorehabilitation strategies.

NIMH - National Institute of Mental Health

Project Summary Schizophrenia is a severe form of mental illness, characterized by psychotic symptoms, large cognitive impairments, and often life-long functional disability. While advances in early detection enable us to identify youth at clinical high risk for psychosis (CHR-P), a critical problem remains. To date, there are no gold- standard treatments to prevent the onset of the full disorder. A major barrier to developing such interventions is the clinical heterogeneity of the CHR-P population. This stems from the underlying complexity of the illness state itself, an active system of interacting factors. While we know many factors are associated with risk, we lack a clear understanding of the mechanisms by which these factors interact and reinforce each other to create the pathways that drive the transition to psychosis. This knowledge gap prevents the development of targeted, precision interventions. This project addresses this challenge by adopting a powerful alternative to traditional research models. We will apply cutting-edge network theory to a large sample of over 2,000 CHR-P individuals, modeling the high-risk state as a system of interacting factors. This approach allows us to move beyond simply listing risk factors to identifying those that are most central and influential in driving the illness forward. Specifically, we will construct the first multi-domain network in this population, comprehensively mapping the interplay between clinical symptoms, neurocognitive deficits, social functioning, and environmental stressors to reveal how they interact to accelerate illness progression. To achieve this, our research plan is threefold. First, we will establish the comprehensive baseline network structure of the CHR-P state, providing a foundational map of its interacting components (Aim 1). Second, we will isolate the critical network differences between youth who later develop psychosis and those who do not, pinpointing the specific interactions most predictive of illness onset (Aim 2). Third, we will incorporate longitudinal data to model how these network connections change over time, mapping the precise temporal pathways that constitute the progression to disease (Aim 3). This research is expected to provide a data-driven roadmap to the most potent and direct targets for preventative intervention. By identifying the system’s key drivers, our work will transform our understanding of psychosis risk. Ultimately, this will accelerate the development of personalized, mechanism-based treatments designed to disrupt the pathways to psychosis and prevent its onset in vulnerable young people.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Up to 22% of pregnant women either receive opioid pain medications or misuse opioids, consequently exposing their fetuses to potential adverse outcomes such as congenital heart defects, stillbirths and disrupted early cardiovascular development (eCVD). While cessation of opioid use might not be possible, effective and individualized pain management during pregnancy is critical and strongly warranted. However, the precise details of how opioid timing, dose, and type affecting eCVD remain poorly understood. There is an urgent need to investigate the impact of opioids on eCVD in models that faithfully simulate human embryonic development. Without such knowledge, establishing a guide for opioid treatment during pregnancy to mitigate adverse cardiovascular outcomes in neonates, remains unlikely. We have developed a novel cell platform using human pluripotent stem cell (hPSC)-derived vascularized organoids (vCOs) to elucidate the effects of drugs on eCVD. The combination of a genetically modified embryonic stem cell (ESC) reporter line expressing cardiomyocyte (CM), endothelial cell (EC), and smooth muscle cell specific fluorescence proteins in combination with our newly established differentiation protocol, allows us to evaluate the impact of opioids on CM and EC development and their role in eCVD. Genetic profiling confirms that our platform mimics normal eCVD during the first six weeks of human embryogenesis. The platform's high throughput nature and applicability in human induced pluripotent stem cells positions it as a promising translational tool to predict the cell-type-specific effects of various opioids on structure, function, vascular network formation during patient specific eCVD. We now seek to acquire robust experimental evidence demonstrating our platform's efficacy in modelling the impact of maternal opioid use on offspring eCVD. Our central hypothesis posits that antenatal opioid exposure disrupts both structural and functional eCVD, and that hPSC-derived vCOs provide a personalize and robust platform for predicting these detrimental effects. Our proposal seeks to accomplish the following key objectives: (1) to validate our newly developed cell platform and its predictive capabilities, (2) to assess the impact of opioids on eCVD and survival in vivo using a mouse model, and (3) to develop a personalized risk profile for opioid-induced eCVD defects using hPSC-derived vCOs. This hypothesis is supported by preliminary data indicating an increased number of miscarriages in opioid-treated pregnant mice, along with cardiac malformations in their offspring. In addition, we observed opioid-dependent transcriptomic alterations and disturbed CM and EC interactions in vCOs. The proposed research aims to provide a comprehensive understanding of the mechanisms underlying opioid- associated congenital cardiovascular defects, and to explore strategies to prevent their occurrence. This knowledge will form the groundwork for developing evidence-based, personalized therapeutic interventions for future clinical applications.

Exelon Foundation

Supports STEM education, workforce development, and community resilience programs in Exelon utility service territories in the Mid-Atlantic and Midwest.

NIH

Abstract Cardiometabolic diseases (CMDs), including obesity, have reached epidemic proportions, affecting > 70% of the US adults and > 50% of persons worldwide. Exercise training is cost-effective to mitigate the modifiable CMD risk factors for our veterans. During the previous funding cycle, we demonstrated that habitual exercise activates hemodynamic shear-responsive molecular transducers to catalyze the anti-inflammatory metabolites in vascular endothelium. However, exercise training also modulates parasympathetic and sympathetic outflow to ameliorate vascular dysfunction and arterial stiffness. Recently, interaction between autonomic nervous system and immune cells was a reported to promote the lymphoid organs to mediate atherosclerosis in the aortic adventitia. Over the past decades, we and others have primarily focused on the vascular endothelium and smooth muscle cells in vascular remodeling. Specifically, shear stress-responsive endothelial nitric oxide synthase (eNOS) is well-known to catalyze nitric oxide production and its metabolites (NO⋅  NO2- + NO3-),9 and oxidative stress induces vascular smooth muscle cells to undergo the transformation from the contractile to fibrotic phenotypes. However, in the aortic adventitia, extracellular matrix (ECM) deposition has been observed in the Angiotensin II (Ang II)-infused hypertensive mice, and immune cells; namely T cells, were identified to prime perivascular fibrosis. While Ang II activates sympathetic nervous system, the mechanism whereby exercise reduces neuro-immune cell interaction to mitigate Ang II-mediated aortic inflammation and vascular fibrosis remains unknown. In this context, we hypothesize that habitual exercise mitigates Ang II-mediated neuro-immune interaction to reduce inflammatory macrophages and activation of fibroblasts. Our hypothesis is supported by our preliminary findings: 1) Ang II-induced sympathetic nerve axons and norepinephrine release to activate the β2-adrenergic receptor (β2-AR)-positive macrophages; 2) Ang II increases monocytes in the bone marrow (BM) and monocyte-derived macrophages; and 3) four weeks of voluntary wheel running (VWR) mitigates Ang II-mediated vascular fibrosis, pulse wave velocity (a surrogate for arterial stiffness), and blood pressure. To test our hypothesis, we have three aims: In Aim 1, we plan to elucidate Ang II-mediated sympathetic nerve-macrophage interaction. In Aim 2, we plan to Investigate Ang II-mediated Ccr2+macrophages to activate fibroblasts. In Aim 3, we plan to demonstrate exercise-mitigated sympathetic and macrophage interaction to reduce vascular fibrosis. We will determine the role of the β2-AR using macrophage- specific β2-AR KO mice and investigate macrophage-fibroblast communication. We will profile BM hematopoietic stem cells and progenitors, and perform BM transplantation to elucidate exercise-mitigated β2- AR+ macrophage. Overall, elucidating exercise-mitigated neuroimmune interaction paves the way for identifying therapeutic targets to modify cardiometabolic disorders for our war fighters, veterans, and their families.

STEM Paths Innovation Network

Expanded Learning Strategy

STEM Paths Innovation Network

Expanded Learning Strategy

NCI - National Cancer Institute

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Despite aggressive therapy, survival remains poor because GBM is sustained by glioma stem-like cells (GSCs) that are highly plastic, therapy-resistant, and able to rewire signaling pathways. Epigenetic regulators are key drivers of this plasticity, but the specific molecular events that connect epigenetic changes to druggable signaling pathways are still not well understood. Our preliminary data identify the arginine methyltransferase CARM1 as a critical regulator of GSC biology. CARM1 methylates the transcription factor NFIA, which represses NGFR expression and downstream NTRK signaling. When CARM1 is lost, NGFR levels increase, GSCs shift toward a radial glial-like lineage state, and cells become more sensitive to the brain-penetrant NTRK inhibitor Entrectinib. These findings suggest a direct mechanistic link, CARM1¨NFIA(R389)¨NGFR, that connects an epigenetic modification to lineage programming and therapeutic vulnerability. First, we will test how CARM1-dependent methylation of NFIA controls NGFR/NTRK signaling and pathway responsiveness. We will use CRISPR editing, NFIA point mutants, proteomics, and chromatin assays to define the molecular mechanism. Second, we will evaluate the impact of CARM1 loss on tumor growth and drug sensitivity in vivo using orthotopic xenografts in immunodeficient mice treated with Entrectinib. Animal studies are necessary because cell culture systems cannot reproduce the intracranial microenvironment, blood.brain barrier.dependent drug exposure, tumor progression, and survival outcomes required to evaluate this therapeutic strategy. Immunodeficient mice are required to permit reliable engraftment of human patient-derived GSCs and to determine whether the molecular and pharmacologic effects observed in vitro translate to tumors growing within the brain. Together, these aims will dissect both the molecular mechanism and therapeutic implications of CARM1 loss in glioblastoma. The innovation of this proposal focuses on post-translational modification of a transcription factor as a switch that controls a clinically relevant signaling pathway. We will determine how CARM1 regulates GSC lineage states and creates a new vulnerability that can be targeted with an FDA-approved drug. Overall, this project will provide mechanistic insight into how epigenetic enzymes reprogram glioblastoma and establish a framework for exploiting these changes therapeutically. If successful, the study will open a new avenue for treating glioblastoma by linking epigenetic regulation to targeted therapy

NINDS - National Institute of Neurological Disorders and Stroke

SUMMARY Incurable neurodegenerative diseases are a growing public health crisis. The ability to generate substantial quantities of disease-pertinent neuron types, with and without predisposing mutations, holds great promise for probing disease mechanisms and developing therapies. However, current protocols yield neurons that fail to mature in vitro and stall at an embryonic identity. This reflects a fundamental gap in knowledge concerning regulatory programs that drive neuronal maturation and limits the potential of stem-cell-based interrogations of age-related neurodegenerative disease. The nervous system employs alternative splicing (AS) to massively expand transcriptomic diversity and protein function. In particular, conserved AS programs consisting hundreds of exons are coactivated at distinct stages during neurodevelopment, including postnatal neurons. In my postdoctoral work, I have found that differentiated neurons, fail to activate the postnatal AS program, and I hypothesize that this postnatal AS program is a conserved, pan-neuronal mechanism driving neuronal maturation. My preliminary data includes contracted and accelerated physiological maturation of mouse embryonic stem cell-derived motor neurons upon global activation of postnatal splicing, suggesting feasibility of my hypothesis. This proposed study aims to expand and generalize the notion that RNA biogenesis strategies such as AS, drive neuronal maturation in human reprogrammed neurons: Aims 1 and 2 ask if activation of the adult alternative splicing program will advance the maturation of human motor and cortical neurons. This will be achieved through overexpression of master splicing factors in postmitotic neurons, evaluation of transcriptomic changes using bulk and single cell approaches, and assessment of physiological maturation. Thereafter, I utilize my approach to build a novel model to study age-related 4R tauopathies: Aim 3 takes advantage of my unique strategy to yield mature tau isoforms and elevated 4R tau in cortical neurons carrying MAPT variants, and to identify mechanisms to reduce tau pathology. Using this unprecedented stem cell-based model, I will assay tau burden, understand gene expression driving disease onset, and target cis-regulators in the MAPT that will decrease tau pathology. Existing reprogramming strategies are incomplete and do not overcome the barrier of the intrinsic aging clock in differentiated human neurons. Thus, it remains vital to continue investigating additional pathways to understand and modify maturation timescales. My undertaking has critical importance in this context: I will explore a novel function for alternative splicing during neurodevelopment, improve understanding of mechanisms that control maturation of human neurons, and demonstrate that my approach is a major advancement for studying age-related neurodegeneration. The insights and technology generated here will have important applications for the exploration of neurodegeneration and will be broadly useful to the scientific community for modeling neurons in health and disease.

NEI - National Eye Institute

Abstract. In glaucoma and other optic neuropathies, retinal ganglion cell (RGC) axons become damaged, leading to cell death and permanent blindness. While some species, including fish, salamanders, and birds, exhibit a remarkable potential for regenerating lost neurons, mammals seem to have lost this capability. In species that can regenerate after injury, changes in early expressed transcription factors (TFs) convert a portion of pre- existing Müller glia (MG) into proliferative stem-like progenitors. Subsequently, cell-fate specifying TFs trigger cell cycle exit and retinal specification. Since precise knowledge of the TFs controlling development and regeneration is incomplete, there is a critical need for further investigation into how TFs enable Müller cells to respond to injury and how waves of TFs lead to proliferation and ultimately to RGC specification. Thus, our main objective is to use human pluripotent stem cell (PSC) -derived 3D retinal organoids (rORGs) as a model to explore endogenous glia-to-neuron repair in the retina. In AIM1a, we will isolate and study lineage-traced Müller glia from rORGs under quiescent conditions and after stimulation with TFs promoting proliferation (β- catenin, LIN28) and/or neurogenesis (ASCL1, NEUROG2). This will be done primarily by AAV-delivered TF overexpression. In AIM1b, additional targets will focus on recently described regeneration roadblocks (NFIA, -B, -X, and ATF7IP-JUNB-ZNF207[AJZ]) that converge around STAT signaling. CRISPR interference will suppress these targets, which we expect to enhance multipotent progenitor cell formation. In nature, injury appears to participate in regeneration, so in AIM2, we will use a cell type-specific drug-inducible Caspase9 (iCasp9) RGC cell ablation model to study the effects of cell injury on MG activation. This will make it much easier to observe the disappearance and re-appearance of ablated and regenerated lineage trace reporter RGCs. In addition, cell damage/death may induce the signaling pathways necessary for regeneration, and our approach will allow us to study that at different stages. In AIM3, we will pivot from enhancing MG-derived retinal progenitors to making actual RGCs. As with early TF-focused experiments, developmentally relevant TFs will be delivered by AAV to steer progenitors toward an RGC fate. Overall, we aim to identify pro- regenerative factors, with the primary goal of restoring the histological architecture of an intact functional retina, which will hopefully lead to new approaches for restoring vision for the millions of individuals who have optic neuropathy-related vision loss.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, with over 890,000 new cases annually. However, there have been few advancements in treatments for HNSCC over the past decade, leaving cisplatin to remain the standard chemotherapeutic agent. Despite the efficacy and affordability of cisplatin, resistance is a major setback in the successful treatment of HNSCC. Many HNSCC patients experience relapse and develop a resistance to cisplatin, diminishing its effectiveness over time. Cisplatin resistance (CR) can occur through various mechanisms, including the enrichment of cancer stem cells (CSCs). This process could be facilitated through liquid-liquid phase separation (LLPS), resulting in CR. In cells, LLPS regulates various biochemical processes by forming membrane-less condensates. This process is controlled by intrinsically disordered regions (IDRs), in which their flexibility allows them to interact with multiple binding partners to control various cellular functions. Recent studies have linked LLPS to the formation of super- enhancers (SEs), in which this interplay is crucial for cell identity and tumorigenesis. FOSL1 (Fos-like Antigen- 1) is a protein that plays a significant role in regulating gene expression, cell proliferation, and differentiation. Structurally, FOSL1 has two IDRs that could contribute to LLPS with other regulators and transcription factors. In our previous studies, we have shown that FOSL1 is upregulated in HNSCC CSCs and revealed that FOSL1 establishes SEs to maintain the high expression of cancer stemness genes (e.g., SNAI2, CD44, and FOSL1 itself) to promote tumorigenesis and metastasis. Additionally, we found that FOSL1 promotes CR by enhancing the CSC population in HNSCC mouse models. Furthermore, we demonstrated that FOSL1 can undergo phase separation with other key regulators to establish SEs. In our preliminary studies, we observed an increase in FOSL1 condensate formation in CSCs compared to non-CSCs in HNSCC. We also demonstrated that IDRs in FOSL1 are required for its phase separation capabilities. Based on these findings, we hypothesize that FOSL1 undergoes LLPS at SEs associated with cancer stemness genes to promote CSC maintenance and facilitate CR in HNSCC. To test this hypothesis, the following aims will be investigated: (1) Aim 1 will identify key regulators involved in FOSL-dependent LLPS in HNSCC. This will be achieved using FOSL1 recombinant proteins and nuclear extract of HNSCC CSCs to obtain FOSL1 nuclear condensates and identify the key regulators that partition with FOSL1 LLPS through proteomic analysis. ChIP-seq and RNA-seq will be used to identify key cancer stemness genes in HNSCC CSCs. Moreover, immunofluorescence and RNAscope will be used to determine if the regulators and oncogenes co-localize within the FOSL condensate; (2) Aim 2 will examine the functionality of the FOSL1 mutant without LLPS ability and its capabilities of promoting tumorigenesis, metastasis, and CR in vitro and in vivo. These findings will elucidate how FOSL1-dependent LLPS maintains the functional properties of CSCs to promote CR in HNSCC and identify potential drug targets to improve the efficacy of chemotherapies.

NCI - National Cancer Institute

Project Summary Title: Explore niche-leukemic stem cell interactions and evaluate niche-directed leukemia treatments. Retention of minimal residual leukemic stem cells (LSCs) within the bone marrow (BM) microenvironment, known as the niche, plays a pivotal role in therapeutic resistance and leukemia relapse. Our long-term goal is to unravel the intricacies of the niche and regulatory mechanisms governing human LSCs, identifying potential therapeutic targets within the tumor microenvironment to enhance leukemia treatment efficacy. We observed that dipeptidyl peptidase 4 (DPP4) deletion significantly alters LSC distribution in the AML BM and identified N-cadherin-expressing BM mesenchymal stem cells (N-cad+ MSCs) as critical in shaping LSC localization, essential for AML cell migration, stemness, and survival. We also discovered significant interactions between DPP4 on AML cells and glypican-3 (GPC3) on N-cad+ MSCs, regulating Cxcl12 activity and gradient. We hypothesize that molecular interactions between N-cad+ MSCs and LSCs are crucial for orchestrating LSC properties and are essential for effective human AML treatment. The objectives of this proposal are to elucidate the intricate crosstalk between N-cad+ MSCs and LSCs and evaluate niche-directed treatment strategies in both human and mouse AML models. Aim 1: Elucidate the molecular interactions between LSCs and niche cells. We will use inducible Gpc3 knockout in N-cad+ MSC mouse models to determine GPC3's role in AML development and LSC properties and study its impact on the crosstalk between N-cad+ MSCs and LSCs. Histological imaging and functional assays using AML patient BM biopsies will explore GPC3's role in the human LSC niche. Aim 2: Investigate the impact of N-cad+ MSC-derived Cxcl12 signaling on human LSC activity. We will perform scRNA-seq and histological imaging analysis of patient BM biopsies to identify whether N-cad+ MSCs are major CXCL12 sources in the BM niche for human LSCs. We will use AMD3100 treatment to block CXCL12 signaling in human LSCs, enabling us to evaluate the distinct properties of DPP4high and DPP4low LSCs in response to CXCL12. Aim 3: Evaluate niche-directed treatment strategies. We will compare chemotherapy efficacy between N-cad+ Cxcl12−/− and control AML mice and evaluate the stemness, survival, and localization of residual LSCs post- chemotherapy. Preclinical trials will assess the effects of niche-directed therapies on LSC activity, disease progression, and overall survival in AML patient-derived xenograft models using chemotherapy- resistant/relapsed AML cells.

NIH

Significance to VA: Based on our recent reported search criteria for mild cognitive impairment (MCI) and Alzheimer's disease (AD) from electronic health records (EHR) within Veterans Affairs Healthcare System (VAHS), we identified 339,007 Veterans with MCI and 572,063 Veterans with AD, but there is no effective, safe therapy for MCI and AD. The newly FDA approved anti-amyloid antibody therapies (AAT) have limited effects on halting cognitive decline and exhibit serious side effects of cerebral hemorrhages. Innovation and Impact: We propose to test a potent Rho-associated coiled-coil kinase (ROCK) 1 and 2 inhibitor as a novel AD therapeutic agent, based on our findings that prescriptions of ROCK inhibitor were associated with a ~50% lower risk of developing MCI and AD, compared to non-users, adjusted with age, sex and comorbidities from 25 million Veterans' medical records. We will use AD patient-derived induced pluripotent stem (iPS) cell-differentiated human neurons and three AD mouse models to perform proof-of- concept preclinical studies. We will also perform proteomics and snRNAseq analyses of mouse brains treated with ROCK inhibitors to identify molecular changes. Specific Aims: Aim 1. To evaluate the efficacy of ROCK inhibition in AD mouse models and human neurons. Pharmacokinetic-pharmacodynamics (PK-PD) relationship will be established in mice after chronic dosing via oral gavage or highly palatable foods mixed with ROCK inhibitor to quantify reduction in neurodegeneration, astrogliosis, microgliosis, and memory impairment in 3 AD mouse models. We will also use isogenic iPS cells carrying either familial AD (FAD) mutant or wild-type PSEN1 and then differentiate them into neuro-spheroids. Outcomes from PSEN1 mutant iPS cells will be compared to those from Psen1 Knock-In mice. Aim 2. To identify molecular changes following ROCK inhibition in AD mouse models and human neurons. We will identify changes of proteins related to Tau phosphorylation (e.g. GSK3β), Aβ clearance (e.g. clusterin), pro- and anti-inflammatory cytokine and chemokines, gliosis, apoptosis and neuronal loss (e.g. AKT1). We will profile plasma and brain proteomics of AD mice after chronic dosing and will identify transcriptomic changes using snRNAseq. We will determine whether changes in proteomics and transcriptomics correlate with clinic-pathological outcomes in these mutant mice following ROCK inhibition. Methodology: We will use isogenic iPS cells carrying either an FAD mutant or wild-type PSEN1 and differentiate them into neuro-spheroids. We will also use wild-type mice and three AD mouse models for PK-PD analyses of the ROCK inhibitor, based on their relevant pathological phenotypes, PS19 (overexpressing mutant Tau), Psen conditional double knockout mice (increased pTau), and Psen1 L435F knock-in (KI) mice (increased Aβ42/40 ratio and amyloid pathology). Conventional biochemical analysis and cutting edge mass spectrometry-based proteomic profiling and snRNAseq will be used to obtain and integrate outcomes from mouse brains, such as levels of pathological proteins, astrogliosis, microgliosis, neurodegeneration, and memory impairment in AD mouse models. Path to Translation/Implementation: Our repurposed FDA-approved drug in this study derives from the analysis of large quantities of VA clinical records and shows little harmful side effects in clinical uses. We will execute our in vivo proof-of-concept studies under chronic dosing paradigms. We hope to translate our findings to future drug development by performing toxicity studies in rats and further seeking FDA approval for future clinical trials to test its safety and efficacy as a repurposed AD drug.