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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.

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.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Project summary/abstract In barrier tissues, developmental transcription factors are often reactivated in cases of disease or inflammation to drive expression of a suite of embryonic genes. While this reuse of pathways can prompt the increased proliferation or migration characteristic of both states, other times the reason for repeated activity is less clear. As an example, HIF1α activity during epidermal development within the hypoxic womb of most mammals does not contribute to generation of proper skin structure, but reactivation after wounding is critical for appropriate healing. One possible explanation for this bimodal activity is that the first wave of activation, during development, might regulate the second wave. Prior work has demonstrated that epithelial stem cells are capable of learning from their experiences, encoding epigenetic memories to respond to a stimulus more quickly the second time encountered. These epigenetic memories are evident at the chromatin level as maintained accessibility of stress-induced loci. If a similar phenomenon is active during development, it could serve to prime epidermal stem cells to respond to inflammatory cues later in life. This hypothesis will be tested by modulating HIF1α activity during embryonic epidermal development and carefully assessing resultant molecular changes. In utero lentiviral delivery of Cre to Hif1a fl/fl embryos will selectively prevent epidermal Hif1a induction in the post-gastrulation embryo. Preliminary work supports that HIF1α is dispensable for skin morphogenesis but drives substantial transcriptional changes in epidermal stem cells. Notably, expression of hypoxia-induced target genes and chromatin-modifying enzymes is altered in the absence of HIF1α. After further characterizing molecular changes, focusing on the chromatin landscape, next experiments will leverage inducible systems to selectively perturb the first wave of HIF1α activity, leaving postnatal activity intact. Inflammatory challenge of these animals will reveal whether embryonic HIF1α activity has lifelong effects on stem cell function, as hypothesized. Moreover, bioinformatic analysis will uncover loci throughout the adult genome which are poised in naïve homeostatic skin but are only expressed and essential in HIF1α-activated situations such as wounding. When compared with epigenetic changes occurring in Hif1a-null embryonic epidermis, I will learn if this poising occurs during embryogenesis and is reliant upon hypoxia. Hif1a has not yet been implicated in epigenetic regulation of the skin, and exploring this function could have important implications for wound healing and disease states of the skin where the protein is again stabilized. The proposed project will be conducted in the lab of Dr. Elaine Fuchs, PhD at the Rockefeller University. The Fuchs lab provides a supportive environment with ample resources to explore the connections between development and inflammation of the skin. Performing the above experiments will establish a robust foundation in cellular and molecular biology techniques with skills in data analysis, critical thinking, leadership, mentorship, and communication, providing strong support for a future independent scientific career in academia.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary/Abstract Objectives: Blood-Labyrinthine Barrier (BLB) dysfunction is implicated in a range of inner ear disorders (BLB- IEDs), including Meniere's disease, autoimmune inner ear disease, and sensorineural hearing loss. These conditions disrupt inner ear fluid homeostasis, leading to vertigo, hearing loss, tinnitus, and imbalance. The BLB comprises microvascular endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes (PVM/Ms), which are essential for auditory and vestibular function. This project aims to investigate the role of the BLB in inner ear homeostasis and disease pathophysiology while developing hiPSC-based models to identify therapeutic targets. Research Design: Using human induced pluripotent stem cell (hiPSC) technology, we will model BLB-specific microvascular interactions and explore novel therapeutic interventions. Our approach focuses on generating hiPSC-derived BLB pericyte spheroids by directing neural crest stem cells toward a pericyte fate using vestibular neuronal spheroid-conditioned medium (VNS-CM). Additionally, we will develop hiPSC-derived PVM/M spheroids by differentiating yolk sac macrophage-like cells into BLB-specific PVM/Ms. These models will be integrated into advanced microfluidic devices to create physiologically relevant 3D BLB spheroids, laying the groundwork for BLB assembloid development in future R01 studies. Methodology: We will characterize BLB-specific structural, molecular, and functional properties of hiPSC- derived pericytes and PVM/Ms using advanced imaging, molecular biology, and functional assays. Structural characterization will involve transmission electron microscopy (TEM) to examine ultrastructural features. Molecular profiling will be conducted through immunocytochemistry and RT-PCR to confirm BLB-specific gene and protein expression. Functional validation will include transepithelial electrical resistance (TEER) and dextran permeability assays to assess barrier integrity, as well as cytokine response assays to evaluate BLB-selective properties under inflammatory conditions. By integrating stem cell engineering and microfluidic technologies, we will construct 3D spheroids that replicate BLB molecular and functional characteristics, providing a robust platform for disease modeling, mechanistic studies, and therapeutic screening for BLB-IEDs. Clinical Relevance: By addressing a critical gap in BLB research, this project will advance our understanding of BLB dysfunction across multiple inner ear disorders. Our hiPSC-derived models will facilitate drug screening for patient-specific responses to treatments such as diuretics, histamine modulators, and corticosteroids, reducing the current trial-and-error approach. Additionally, these assembloids will enable disease modeling of BLB-IEDs, offering new insights into disease mechanisms and therapeutic development. This research aligns directly with the NIDCD's mission to support biomedical and behavioral research in hearing and balance disorders, ultimately improving public health and quality of life.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Epilepsy is a common neurological disorder that affects approximately 50 million people worldwide. Approximately 30% of patients with epilepsy have treatment-resistant (refractory) seizures, presenting a major clinical challenge and burden. The acquired and genetic forms of epilepsy represent the two major classes of epilepsy, and these arise mainly from neurological insults and genetic mutations, respectively. Temporal lobe epilepsy (TLE) is the most common form of acquired epilepsy, and mesial temporal lobe epilepsy (MTLE) is the most common subtype of TLE. Dravet syndrome (DS), the most common form of genetic epilepsy, is a catastrophic pediatric disorder which is most frequently caused by mutations in the SCN1A voltage-gated sodium channel. The mechanisms that contribute to the eventual development of seizures and associated comorbidities in MTLE and DS are still incompletely understood, and further research on the cellular and molecular changes that underlie these disorders is necessary in order to facilitate the development of improved treatments. Extracellular vesicles (EVs) are small, membranous particles that are naturally released by cells. EVs play an important role in intercellular communication and have been shown to possess anti-inflammatory and neuroprotective properties. Accordingly, the administration of EVs isolated from healthy, non-pathogenic cellular sources such as mesenchymal stem cells (MSEs) and neural stem cells have been demonstrated to reduce pathology in models of MTLE, stroke, TBI, and neurodegenerative disorders. Our preliminary data also suggests that endogenously-released EVs in the brain (i.e. brain derived EVs or BDEVs) from naïve wild-type mice have anti-inflammatory and cell protective properties. However, in certain disease states, BDEVs can become dysregulated and contribute to neuroinflammation and disease pathology. Little is known about the role of BDEVs (i.e., protective versus pathogenic) during the development of epilepsy. To date, only two studies have examined BDEVs in rodent MTLE models. While both studies identified changes in the expression of BDEV miRNAs following status epilepticus, neither study examined whether the functional properties of the BDEVs were altered. Furthermore, whether BDEVs are altered in genetic epilepsies and contribute to disease development is completely unknown. Hence, the objective of this exploratory R21 proposal is to establish whether BDEV properties are altered in mouse models of MTLE and DS. Importantly, the analysis of two models with distinct epileptogenic mechanisms will establish conserved and epilepsy subtype-specific BDEV contributions. The data generated in this study will provide new information on the role of BDEVs in the development of acquired and genetic forms of epilepsy, and may potentially identify novel targets for therapeutic intervention.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Summary Obesity, now at pandemic levels, significantly increases the risk of numerous comorbidities, including sepsis- a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. A major contributor to the obesity epidemic is the widespread consumption of ultra-processed foods (UPFs), which promote excessive nutrient intake and are linked to chronic metabolic and inflammatory disturbances. These conditions activate nutrient-sensing pathways that connect nutrient excess with systemic inflammation, contributing to obesity-associated immune dysregulation. We hypothesize that obesity-associated metabolic stress enhances the production of pro-inflammatory small extracellular vesicles (sEVs), which in turn drive systemic inflammation and contribute to organ injury during sepsis. Small EVs are released by various cell types into the extracellular space and circulate in body fluids, where they are taken up by local or distant recipient cells. Our data from obese pediatric patients and healthy controls suggest that obesity imparts inflammatory traits to circulating sEVs. These vesicles, when internalized by immune cells such as macrophages, modulate inflammatory gene expression. Preliminary findings further indicate that the RNA cargo within sEVs plays a central role in regulating these immune responses. In this study, we will: 1. Define the upstream regulatory pathways that confer pro-inflammatory properties to liver-derived sEVs and evaluate their impact on sepsis outcomes in pre-clinical models. 2. Identify key molecular mediators in macrophages that drive sEV-induced inflammatory responses. Although sEV biology is rapidly evolving, the clinical implications of these vesicles remain largely unexplored. Leveraging our unique mouse models, human sEV samples, and human induced pluripotent stem cell (iPSC)- derived hepatocytes and macrophages, we aim to uncover how liver-derived sEVs shape systemic immune responses and drive organ dysfunction in sepsis. This work will clarify how obesity amplifies inflammation during critical illness and may identify novel biomarkers and therapeutic targets.

NHLBI - National Heart Lung and Blood Institute

Project Summary / Abstract Congenital diaphragmatic hernia (CDH) is a birth defect where the fetal diaphragm does not form properly and organs from the abdomen move into the chest, affecting lung development. As a result, one-third of U.S. infants with CDH dies, mainly due to the abnormal development of lung blood vessels (vascular remodeling) and high blood pressure after birth. Although surgery after birth can fix the defect in the diaphragm, it cannot reverse the damage already done to the lungs before birth. Experts agree that a therapy that promotes lung development and reverses vascular remodeling before birth is urgently needed, but currently no such treatment exists. Our team has established a new therapy for fetal lung regeneration using stem cell-derived nanoparticles, called extracellular vesicles (EVs). These particles contain bioactive molecules (proteins, lipids, and miRNAs) that are key for normal lung development. In experimental studies using models of CDH, we showed that prenatal EV treatment improves all aspects of lung development, including formation of lung vasculature. However, the mechanisms behind EV beneficial effects on fetal lung vessels in CDH remain undefined. The objectives of this proposal are to determine how EV therapy mediates lung vascular regeneration and which CDH babies are most likely to benefit from it. Our central hypothesis is that EVs reverse fetal lung vascular remodeling by delivering miRNAs that are critical for normal vessel development, benefiting those with the most severe disease. We will test our hypothesis with three specific aims: Aim1 will study how EVs promote the regeneration of CDH lung endothelial cells, the most critical component of blood vessels; Aim2 will employ cutting-edge technology (spatial transcriptomics and proteomics) to determine the molecular pathways that are modulated by EV treatment throughout the CDH lung; Aim3 will test the effects of prenatal EV treatment on varying degrees of CDH severity by assessing lung morphology with histology and advanced imaging (micro-CT), and lung function after birth with ventilation and echocardiography studies. This project combines the expertise of specialists in EV biology and regenerative medicine, lung development and CDH modeling, spatial transcriptomics and proteomics, fetal surgery and clinical outcomes. This team will advance the knowledge on CDH lung disease and lay the foundation for the clinical translation of a novel EV- based strategy that will improve survival and quality of life for CDH babies and their families.

ExxonMobil Foundation

Funds education initiatives and global health programs including malaria prevention, women's economic opportunity, and STEM education.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The University of Puerto Rico at Aguadilla (UPR-Aguadilla) requests funding to acquire a Nikon AX Galvo Confocal Laser Scanning Microscope (LSCM) with a TI2-E inverted platform and a four- laser configuration (405/488/561/640 nm) to establish transformative imaging capabilities at our resource-limited institution serving 96% Pell Grant recipients. This state-of-the-art instrument addresses a critical infrastructure gap, enabling high-resolution fluorescence imaging, live-cell microscopy, and quantitative analysis essential for competitive biomedical research and undergraduate education. The LSCM will directly support four active research projects spanning parasitology (monogenean host-specificity studies), plant pathology (coffee biocontrol development), environmental chemistry (metalloprotein biomarkers), and neuroscience (astrocyte dysfunction in diabetic epilepsy) while integrating into core laboratory courses including Immunology (BIOL 4009) and Undergraduate research courses (BIOL 3108 and QUIM 4999). Our multidisciplinary faculty, in partnership with the Neuroimaging and Electrophysiology Facility (NIEF) Excellence Imaging Center, offers expertise in confocal microscopy, encompassing advanced imaging and specialized sample preparation techniques. This collaboration ensures effective implementation of the technology, sustained technical support, and high-quality training programs that will enhance research productivity and broaden educational impact. The broad, long-term objective is to transform UPR-Aguadilla from a primarily teaching institution into a research-active campus capable of producing graduate-school-ready students equipped with cutting-edge technical skills. Access to advanced confocal microscopy will stimulate new research collaborations, enhance faculty productivity, and provide 30-40 students annually with hands-on experience in modern imaging technologies currently absent from our curriculum. The instrument will strengthen our partnership with the emerging Natural History Museum of Puerto Rico for specimen digitization and support comprehensive outreach programs targeting 25-50 high school students annually through "Seeing Science Up Close" workshops. Expected outcomes include 1- 2 peer-reviewed publications within three years, establishment of 1-2 new institutional collaborations, and measurable enhancement of biomedical research capacity. This investment will significantly advance STEM education and research opportunities at UPR-Aguadilla while expanding access to cutting-edge scientific instrumentation for students pursuing biomedical careers and contributing to the development of skilled researchers in the biomedical sciences.

NCI - National Cancer Institute

Project Summary Pancreas cancer is now the third leading cause of cancer related deaths, due to late diagnosis and therapy resistance. While pancreatic ductal adenocarcinoma (PDAC) has been the main focus of the field, cells of different subtypes of disease can be mixed in PDAC tumors. Single-cell data demonstrate there are subclones within PDAC with a basal gene signature, which align with adenosquamous. The basal-squamous signature is associated with worse prognosis and resistance to gemcitabine. This resistance demonstrates the need to understand the establishment and maintenance of basal-squamous pancreas cancer so effective therapies may be developed. We completed RNA-sequencing analysis to find genes upregulated in basal-squamous patient samples compared to classical patient samples to identify candidates that may be involved in basal- squamous establishment and maintenance in a physiologically relevant model. We identified novel candidate genes, whose expression correlate with canonical basal-squamous genes, that we hypothesize may play a role in basal-squamous pancreas cancer growth and identity. The Reya lab previously defined Musashi2 (Msi2) as a functional marker for cancer stem cells in pancreas cancer and recently published a novel mouse model to study pancreas cancer development from a common precancerous pool of cells. This model induces the expression of stabilized MycT58A in Msi2+ cells (Msi2-Myc mice). Msi2-Myc mice can form multiple subtypes of pancreas cancer and reliably form basal-squamous tumors (68% of mice). We will use Msi2-Myc derived precancers, human PDAC cell lines, and PDAC patient samples to determine if candidate genes are sufficient for basal-squamous establishment. We will overexpress candidate genes in these models to determine if there is an emergence and/or acceleration of the basal-squamous state through in vitro and in vivo models. We will also determine if they are necessary to maintain the basal-squamous state and growth. We will knockdown candidate genes by shRNA in human squamous cell lines, Msi2-Myc derived tumor cells, and basal-squamous patient samples, and determine the impact on the basal-squamous state and growth in in vitro and in vivo models. We will conduct CLIP-sequencing to find direct targets and integrate with RNA-seq to elucidate the mechanism by which the basal-squamous state is maintained. Preliminary data show that knockdown of candidate genes in Msi2-Myc derived mouse cells and human cell lines significantly reduces growth in vitro and in vivo, suggesting their role in maintaining growth. By RNA-seq and qPCR analysis of human cell lines and histology and immunofluorescence of resultant Msi2-Myc tumors, we have preliminary evidence that suggest genes of interest may be necessary to maintain basal-squamous identity. Based on these data, the aims of this proposal are to test the hypotheses that candidate genes are (1) necessary for the maintenance of basal-squamous pancreas cancer growth and identity and (2) sufficient to drive the establishment of basal- squamous pancreas cancer.

FIRST Robotics

Funds robotics teams and STEM mentorship programs for K-12 students.