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NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY / ABSTRACT The Eighth International FASEB Science Research Conference on Retinoids will be held August 3–6, 2026, in Niagara Falls, NY, co-located with the FASEB conference on Nutritional Modulation of Inflammation and Immunity. Originating in 1982, this meeting continues to serve as the leading international forum for advancing fundamental and translational research on vitamin A and retinoids. The 2026 program will feature major developments in established areas such as immune regulation, development, metabolic and nutritional disorders, and visual biology, while also highlighting emerging fields including epithelial and gastrointestinal health, neurodegeneration, stem cell biology, and regenerative pathways. A strong emphasis will be placed on mechanistic and translational applications of retinoid and rexinoid therapeutics, new analytical and imaging tools, and advances in the structural biology of retinoid enzymes, binding proteins, and transporters. A special co-located joint session, Retinoid and Immunity, will unite investigators from both conferences to examine the interplay between vitamin A biology, immune function, and host–pathogen interactions. The conference will provide attendees with an integrated understanding of the genetic, environmental, and microbiome-related factors that influence retinoid uptake, metabolism, and biological responses. It will convene scientists across career stages and disciplines, including biochemistry, structural biology, nutrition, cancer research, immunology, chemical biology, stem cell and regenerative medicine, and clinical research. A central mission of the meeting is to support early-career investigators through short-talk opportunities, poster spotlights, mentoring events, and meet-the-expert sessions. Selected trainee abstracts will be highlighted through oral presentations to increase visibility and promote career development. Partial NIH support is requested to sustain the conference's long-standing mission, broaden participation—especially among early-stage investigators—and continue fostering interdisciplinary progress in retinoid science.

National Park Service

Objectives of the Internships in Marine Geography Program are to: encourage a diverse group of young adults to study and pursue careers in Science, Technology, Engineering and Mathematics (STEM) fields; provide meaningful and relevant science-based internships to individuals from underrepresented groups in STEM fields; introduce program participants to science careers at NOAA; and increase relevancy, diversity, and inclusion in the NOAA workplace.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Creating better therapies to prevent or inhibit deleterious immunological responses, such as in autoimmunity, is hindered by our incomplete understanding of their pathogenesis. For example, no therapeutic approach is currently capable of curing or providing durable prevention f rom autoimmune type 1 diabetes (T1D). This lack of progress largely stems f rom an incomplete understanding of the interactions among key human cellular players involved in human T1D, and of how these interactions and the environment contribute to disease-causing ef fects. Current tools, f rom animal models to basic culture dishes, are limited in providing this insight. We developed a novel alternative method (NAM) to address these limitations, by creating a three-dimensional (3D) extracellular matrix-based pancreatic tissue mimic, termed the Native Islet-Immune Cell Hydrogel Environment (NIICHE). This NIICHE platform delivers new insights into human immunological processes associated with T1D by collecting 3-D, real-time, noninvasive measurements of immune cell recruitment and engagement with targeted beta cells. With the NIICHE established as a robust, human-centric benchtop screening platform, this proposal seeks to expand its utility and capacity for further testing of the human T1D hypothesis and therapeutics. Specif ically, this R56 proposal seeks to establish the following key engineering and cellular goals. Aim 1) Integrate 3D printing methods for the addition/retrieval of cells and materials to impart spatial control and retrieval. Aim 2) Establish a protocol for the stable integration of sEC monolayer atop the NIICHE and validate visualization of extravasation through the endothelium in response to a chemokine gradient. Aim 3) Expand to additional T cell sources for use in the NIICHE platform and evaluation of the dif ferential immunogenicity of human beta cell subpopulations. Achievement of these goals will create a NAM that supports the distinct placement and retrieval of cells within the 3D matrix, as well as the capacity to distinctly interrogate the role of the endothelial barrier in immune cell recruitment. Finally, expanding our cellular repertoire to include additional T cell and beta cell sources will enhance the utility and predictive capacity of this benchtop system. Once established, this expanded platform can deliver unique insights into pathogenesis and serve as a screening tool for new therapeutic targets. Beyond the T1D focus proposed herein, we envision that this practical and highly translatable 3D platform has broad utility, as its capacity to quantitatively track and assess 3D cellular interactions, traf f icking, extravasation, and immune cell attack enables investigation of numerous immunocentric questions.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

SUMMARY Developmental gene expression is tightly regulated by the dynamic interplay of H3K4 methylation (H3K4me) and H3K27 methylation (H3K27me) associated with active and repressed genes, respectively. However, our understanding of the individual and combinatorial roles these histone modifications play in adult physiological contexts remains incomplete. To overcome these limitations, we have recently generated histone mutant transgenic tools to uncover a previously unappreciated role for H3K4me in adult hematopoiesis. Adult mice globally depleted for all forms of H3K4me via expression of an inducible histone H3 lysine-4-to-methionine (H3K4M) mutant allele succumbed to a severe loss of all major mature blood cell types. Unexpectedly however, H3K4M-expressing hematopoietic stem cells (HSCs) and most committed progenitors were present at normal numbers and persisted upon transplantation into recipient mice, suggesting that H3K4me is dispensable for the maintenance and early commitment of HSCs and progenitors but essential for the terminal maturation of progenitors. Mechanistically, we showed that H3K4me opposes the deposition of repressive H3K27me at differentiation-associated genes bivalently marked by H3K4me3 and H3K27me3 in HSCs or progenitors. Indeed, by concomitantly suppressing H3K27me in H3K4me-depleted mice with an H3K27M transgene, we could rescue the acute lethality, hematopoietic failure and gene dysregulation. Thus, our results reveal that H3K4me guides hematopoiesis by opposing repressive H3K27me at fate-instructive bivalent genes, providing the first evidence for the functional interaction between these crucial chromatin marks in mammalian tissue homeostasis. These preliminary data raise fundamental questions with clinical relevance that will be addressed in 3 complementary aims. In Aim 1, we will further define the consequences of H3K4me loss on the function of HSCs and progenitors using self-renewal and differentiation assays. Additionally, we will assess whether any observed defects are reversible upon restoration of H3K4me. In Aim 2, we will identify epigenetic regulators that mediate the H3K4M- dependent arrest and the H3K27M-dependent rescue by purifying proteins associated with H3K4M and H3K27M; measuring changes to all major histone modifications; and testing select candidates for their ability to phenocopy the effects of H3K4M and H3K27M. In Aim 3, we will dissect the molecular basis by which H3K4me/H3K27me safeguard hematopoiesis with a focus on fate-instructive cytokine receptors and transcription factors dysregulated in H3K4M mice but normalized in H3K4M/H3K27M mice. Moreover, we will investigate the contribution of other epigenetic marks to the H3K4M phenotype using DNA methylation inhibitors and a novel histone mutant library. Collectively, this proposal will leverage novel tools to probe the direct, physiological impact of two antagonizing chromatin marks on hematopoiesis. As arrested differentiation and disrupted H3K4me/H3K27me have been implicated in diverse hematological conditions, our results will elucidate the underlying mechanisms and may pave the way for novel therapeutic interventions.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Hirschsprung disease (HSCR) is a serious congenital enteric neuropathy characterized by a narrowed segment of distal bowel where enteric ganglia are absent and intestinal motility is lacking. This leads to a functional bowel obstruction that causes severe obstipation, proximal megacolon, life threatening enterocolitis, and need for major surgery to remove the aganglionic segment. Both the pre-operative and post-operative periods present significant challenges for patients. Before surgery, infants require rectal irrigations 1-2 times daily to evacuate stool. After surgery, at least 50% of patients experience late complications, including persistent obstructive symptoms, fecal soiling, post-operative enterocolitis, pelvic nerve injury, damage to anal sphincters, residual aganglionosis, transition zone pull-through, and unexplained dysmotility. The morbidity of HSCR, both pre- and post-operatively, highlights the limitations of our current management and emphasizes a critical unmet need to develop a novel treatment approach to this life-threatening disease. This research proposal addresses that need by introducing an innovative hypothesis on the pathophysiology of HSCR, specifically the central role of fibrosis in the aganglionic segment. The narrow aganglionic segment in HSCR is commonly described as “tonically contracted” or “spastic,” a phenomenon attributed to uninhibited innervation by extrinsic cholinergic nerves, or to altered expression of ion channels, cytoskeletal components, and gap junction proteins. We hypothesize that the aganglionic narrowing is instead due to fibrotic remodeling of the gut wall, shifting the focus away from neuronal and protein imbalance and opening new avenues to target fibrosis in HSCR. We aim to characterize the cellular and molecular features of the aganglionic segment and to test the therapeutic potential of anti-fibrotic therapy, both as a stand-alone therapy and in combination with enteric neuronal stem cell transplantation. Aim 1 leverages multiple novel methodologies to characterize matrisome composition, immune cell populations, and bowel stiffness in the aganglionic colon of HSCR mice. Aim 2 tests the effects of anti-fibrotic therapy on the success of enteric neuronal stem cell transplantation to remodel fibrosis in the gut wall, which has not been previously studied. This new pharmacologic strategy aims to improve the early, pre-operative course of the disease by relaxing the narrowed segment to facilitate the passage of stool and reduce the risk of life-threatening obstruction and fecal stasis, offering significant clinical value by reducing the morbidity associated with pre-operative complications. Understanding and treating fibrosis in the aganglionic segment could enhance the success of regenerative cell therapy by creating a more permissive microenvironment into which transplanted cells are delivered. Overall, successful completion of the proposed aims will have a significant impact on our understanding of the pathophysiology of Hirschsprung disease and on its treatment.

NIA - National Institute on Aging

Frontotemporal dementia caused by mutations in microtubule-associated protein tau (MAPT), including the N279K mutation, is a common cause of early-onset dementia. It is neuropathologically characterized by toxic aggregation of hyperphosphorylated tau, glial activation, and neurodegeneration. The factors contributing to the disease are likely numerous and poorly understood, and no disease-modifying therapies exist for FTD. Oxidative stress (OS) occurs when a cell’s innate antioxidant system is overwhelmed by reactive oxygen species, and oxidative modifications of biological molecules have important consequences on protein, DNA, and lipid function. In particular, uncontrolled lipid peroxidation can lead to ferroptosis, a specific cell death pathway which we found to be enriched in FTD postmortem brain and may contribute to neurodegeneration. We also identified an OS and neuroinflammatory phenotype in postmortem brain from FTD patients and induced pluripotent stem cell (iPSC)-derived neurons from FTD patients. Specifically, FTD iPSC-derived neurons show upregulation of the gene secreted phoshoprotein-1 (SPP1) and its protein product osteopontin (OPN), which can activate iPSC-derived microglia in vitro. Given the centrality of OS in our FTD models and the apparent association with SPP1, this proposal seeks to investigate mechanisms of OS generation and downstream sequelae in FTD. In aim 1, I will interrogate the effects of different classes of oxidative and ferroptotic stressors on FTD MAPT N279K iPSC-derived neurons. In aim 1a I will assess cell viability and lipid peroxidation. In aim 1b I will assess tau pathology and neurite outgrowth. In aim 1c I will attempt to rescue any effects seen in aims 1a and 1b by co-treating with antioxidant and ferroptosis inhibiting compounds. In aim 2 I will characterize astrocyte-neuron crosstalk in the FTD context. First, in aim 2a I will generate iPSC-derived astrocytes from FTD MAPT N279K patients or healthy control patients and treat with OPN and assess for astrocyte reactivity. In aim 2b I will generate antioxidant response gene reporter astrocytes and treat with Ctrl or FTD neuron conditioned medium to determine the role of neuron-secreted factors in astrocyte response. Finally, in aim 3 I will explore the potential of targeting OS in FTD. I will xenotransplant FTD or Ctrl neural progenitor cells into mice forebrains and treat systemically with liproxstatin, an antioxidant and ferroptosis inhibiting compound. In aim 3a I will characterize proteins involved in these pathways as well as glial reactivity and graft survival by histology. In aim 3b I will perform snRNA-seq on micro dissected grafts to map changes in gene expression profiles in response to OS targeting.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Gastrointestinal (GI) disorders are among the most common comorbidities in patients with Autism Spectrum Disorder (ASD). The Enteric Nervous System (ENS), composed of neurons (ENs) and glia, is crucial in regulating various aspects of gut physiology. Animal models show GI motility impairments linked to altered expression of ASD-associated genes. However, recent advancements in single-cell genomic technologies have revealed remarkable molecular diversity among ENs and highlighted significant differences in ENS gene expression patterns across species. These findings underscore the need for human-specific models to recapitulate the human ENS molecular heterogeneity and dissect the cell type-specific contribution to the GI endophenotype in ASD. Under the mentorship of Dr. Giorgia Quadrato and Dr. Jason Spence, leaders in the field of the human neural and intestinal organoids, respectively, Dr. Birtele will use a human induced pluripotent stem cell (iPSC)- derived model that includes both ENs and intestinal organoids (HIOs). Using a mix-and-match approach, patient- derived neurons co-cultured with healthy intestinal cells will isolate ENS-specific contributions to GI dysfunction. Conversely, healthy neurons cultured with patient-derived intestinal organoids will reveal non-neuronal contributions. Aim 1 (K99 phase) will study the role of SYNGAP1, a top ASD gene, in GI dysfunction. ENs will be derived from a SYNGAP1 haploinsufficient-patient derived and isogenic control iPSCs line under the mentorship of Dr. Martin Garcia-Castro, expert in neural crest differentiations. Under the guidance of Dr. Jason Spence, Dr. Birtele will generate mixed and matched ENs-HIOs. Dr. Birtele will analyze mixed and matched ENs-HIOs to determine cellular and transcriptional changes caused by SYNGAP1 haploinsufficiency. In Dr. Spence's lab, Dr. Birtele will transplant ENs and ENs-HIOs in vivo to assess GI motility and peristaltic function. Additionally, under the mentorship of Dr. Unmesh Jadhav, an expert in epigenomics and intestinal stem cells, Dr. Birtele will examine the effect of SYNGAP1 haploinsufficiency on intestinal stem cell chromatin accessibility profiles by performing single-cell ATAC-seq on mixed and matched ENs-HIOs. Given the high comorbidity of GI dysfunction across many genetic forms of ASD and the enrichment in expression of these genes in ENs, Aim 2 (R00 phase) I will perform an high-throughput screening for molecular and functional impairments in ENs cultures by applying gapmer antisense oligonucleotides (ASOs) under the guidance of Dr. Justin Ichida, leader in the field of ASOs, to knock-out 35 top ASD-associated genes.Top candidates identified in this initial screen will be validated using patient-derived lines differentiated into ENs and HIOs and cultured following the mix-and-match approach. By applying a similar pipeline of experimental procedures as in Aim1, I will compare the functional and molecular profiles of in vitro and transplanted organoids to dissect possible convergent molecular mechanisms through which ASD-associated genes contribute to GI dysfunction This research will uncover molecular mechanisms governing ENs function and provide critical insights into ASD-related GI dysfunction. 1

NHLBI - National Heart Lung and Blood Institute

Project summary Kinase inhibitors (KIs) represent critical advances in cancer treatment, yet their cardiac toxicity profiles are poorly understood. Recent clinical reports highlight significant cardiotoxicity associated with osimertinib, the sole approved therapy for EGFR T790M-positive non-small cell lung cancer (NSCLC). Approximately 3-5% of patients experience clinically significant cardiac adverse effects, including reduced left ventricular ejection fraction, heart failure, and arrhythmias, leading to treatment interruptions or discontinuation. Despite this significant clinical challenge and potential negative impact on patient survival, the underlying molecular mechanisms remain unexplored. Our preliminary studies in mouse models demonstrate early cardiac dysfunction linked to mitochondrial reactive oxygen species (mtROS) generation and increased NOX4 expression following osimertinib treatment. Clinical evidence also suggests that cardiac dysfunction, although often reversible, can seriously compromise the continuity of cancer treatment, emphasizing the urgent need for effective preventive strategies. We propose mitochondrial oxidative stress as a key driver of cardiotoxicity of osimertinib, warranting further mechanistic investigation. This research aims to elucidate the molecular mechanism of osimertinib- induced cardiotoxicity and to evaluate therapeutic strategies targeting mitochondrial dysfunction and oxidative stress through three specific aims. Aim 1 will determine the mitochondrial mechanisms underlying osimertinib-induced cardiotoxicity. Utilizing human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we will measure mitochondrial function (oxygen consumption rate, mtROS levels, mitochondrial dynamics) to validate our hypothesis that mitochondrial bioenergetics disruption drives cardiac injury. Aim 2 will assess the role of NOX4 in mediating cardiac dysfunction. We will use transgenic and knockout mouse models, specifically altering cardiac NOX4 expression, to define its contribution to osimertinib-induced oxidative stress and mitochondrial impairment. In aim 3 we will evaluate cardioprotective strategies with mitochondrial-targeted antioxidants and NOX4 inhibitors. We will test MitoQ (mtROS scavenger) and setanaxib (NOX4 inhibitor), individually and in combination, to assess their efficacy in preventing osimertinib- induced cardiac damage both in vitro and in vivo. This study addresses a critical clinical issue by uncovering novel molecular insights into KI-induced cardiotoxicity, specifically identifying mitochondrial oxidative stress and NOX4 as therapeutic targets. Our findings aim to mitigate cardiac side effects associated with osimertinib, enhancing the clinical safety and efficacy of targeted cancer therapies, and thereby enabling uninterrupted cancer treatment and improving patient outcomes.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cell transplantations (HSCTs) are curative therapies for a range of debilitating hematological and immune disorders. This approach relies on the ability of healthy donor hematopoietic stem cells (HSCs) to engraft within a recipient and subsequently contribute to long term multilineage hematopoiesis, effectively replacing the diseased blood system with a healthy one. The demand for HSCTs is expected to rise, as newly approved FDA gene editing therapies are now being offered for the treatment of a variety of blood- borne diseases offering patients the opportunity to be treated with their own genetically modified HSCs. Although promising, a significant challenge with this approach lies in the ex vivo culture step required for gene editing. Removal of HSCs from their niche environment and associated culturing leads to a loss of stem cell functionality, ultimately compromising their engraftment potential. To improve the success of HSCTs, it is imperative that HSC culture systems evolve to preserve stem cell functionality. To decipher the underlying signals and factors supportive and characteristic to highly engraftable HSCs, we look to the fetal liver (FL), a developmental hub for newly emerged HSCs. Notably, FL HSCs, exhibit superior engraftment capacity compared to HSCs at other developmental timepoints. This observation leads us to hypothesize that the FL is uniquely equipped to support highly engraftable HSCs, and unraveling the underlying signals, factors, and interactions characteristic to these cells and their microenvironments will guide advances in ex vivo culture conditions that are tailored to sustain HSC function. Our previous multi-modal profiling of FL hematopoietic stem and progenitor cells (HSPCs) revealed a molecular signature characteristic to the most functional FL HSCs. Within this signature we noted enrichment of ID1-3, a class of factors known to be implicated in safeguarding the balance between quiescence and replication. We plan to supplement HSPC ex vivo culture systems with biologically relevant ID gene modulators, characterize subsequent changes in engraftment potential, and investigate the mechanistic implication of this modulation. To comprehensively characterize the FL HSC niche, we will integrate Xenium in situ spatial transcriptomic analysis with immunofluorescence staining to identify highly engraftable HSCs and their surrounding cellular environment. This approach will allow for us to pinpoint candidate factors involved in preserving engraftment potential. Our FL guided approach will facilitate advances in ex vivo systems that support or even enhance the engraftment potential, thereby improving the safety and success of HSCTs. This project will be conducted at the Center for Regenerative Medicine (CReM) at the Boston University School of Medicine, under the mentorship of Drs. Kim Vanuytsel, Ruben Dries, and George Murphy, whose expertise spans hematology, spatial biology, and regenerative medicine. Through this work, I will receive interdisciplinary training that will prepare me for a successful career as an independent research scientist.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Clonal hematopoiesis (CH) arises when hematopoietic stem cells (HSCs) acquire somatic mutations that provide proliferative advantages and increases risk for cardiovascular diseases (CVDs). It remains unknown whether CH is entirely deterministic or if extrinsic and environmental factors can modify clone evolution and CH-driven atherosclerosis. By studying how sleep and exercise – evolutionarily conserved behaviors that ensure cardiovascular and immune health – modify CH and its associated atherosclerosis, we will identify new evolutionarily tested and potent therapeutic targets. Our preliminary data, in humans and mice, demonstrate that CH mutant cells are selectively and uniquely responsive to lifestyles and that sleep and exercise reprogram the immunometabolic status of CH mutant cells − but not neighboring WT cells − in the bone marrow and aorta to influence CH clonal evolution and associated atherosclerosis. We hypothesize that sleep and exercise selectively restrict the detrimental immuno-metabolic programming of CH mutant cells, thereby diminishing clone expansion and locally modifying CH mutant aortic macrophages to slow atherosclerosis. In Aim 1, we will model three mutations and CH-accelerated atherosclerosis in high fat diet fed Ldlr-/- mice and exposed them to 12 weeks of sleep fragmentation (SF), voluntary exercise, or a sedentary lifestyle. We will track the expansion of mutant (CD45.2) cells over time. The abundance, proliferation, inflammasome and metabolic status of mutant and WT HSCs in the BM and spleen will be measured. CH cell-specific genetic deletion of Il1r1 or Pfkfb3, or pharmacological blockade of IL-1β or glycolysis, will causally test how lifestyle influences these key pathways to alter CH mutant, but not neighboring WT HSCs. In Aim 2, we will test how sleep and exercise alter the progression of CH-accelerated atherosclerosis by selectively reprogramming mutant aortic macrophages through CLEC4e-inflammasome signaling and Pfkfb3-mediated glycolysis, respectively. In atherosclerotic mice with and without CH and exposed to lifestyles, we will perform confocal and PET imaging; proteomics; CD45.1/2 oligotagged scRNA-seq and CellChat analysis; extracellular flux and isotope tracer assays to uncover new mechanisms by which sleep and exercise selectively alter the crosstalk, immune, and metabolic reprogramming of mutant, but not WT, aortic macrophages. CH cell specific deletion of Clec4e or Pfkfb3 will uncover how these specific and new mechanisms causally link lifestyle to CH mutant macrophage function in atherosclerosis. Whether sufficient sleep or exercise selectively limit the detrimental effects of CH by locally reprogramming CH mutant cells, without impacting neighboring WT cells, is a novel and clinically important question. Our studies will redefine the relationship between CH and atherosclerosis and uncover evolutionarily conserved, mechanistically defined, and therapeutically actionable pathways that selectively reprogram CH mutant cells while preserving the function of healthy WT cells, enabling personalized anti-CH and anti-atherosclerotic therapies.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Splicing factors (SFs) are RNA-binding proteins that regulate alternative splicing (AS), enabling a single gene to produce a variety of mRNA transcripts and corresponding proteins. AS plays an integral role in development, cancer, and aging, and many SFs are essential for embryonic development. Therefore, SF levels must be tightly controlled to maintain proper gene expression, which can be achieved through the AS of poison exons (PEs) within their own transcripts. PEs within SF transcripts, or SF-PEs, introduce premature termination codons, triggering nonsense-mediated decay (NMD) to reduce SF protein levels, a process known as AS- NMD. Conversely, PE skipping increases SF abundance. Prior studies highlight SF-PEs as critical for cancer cell survival, but their role in non-cancerous cells remains unclear. The goal of this proposal is to determine how SF-PEs maintain SF homeostasis to modulate transcriptomes that sustain pluripotency and differentiation. Our preliminary data suggest that PEs in Srsf3 and Tra2b, two SFs linked to cancer and developmental disease, are essential for pluripotent stem cell survival and embryonic viability. However, the morphological, functional, and transcriptomic effects of PE knockout remain unclear, as does the broader role of SF-PEs in pluripotent stem cell survival. We hypothesize that SF PEs fine-tune pluripotency by buffering SF gene expression and modulating AS of target genes critical for maintaining pluripotent cell viability. Aim 1 will utilize an in vivo reverse genetics approach and long-read RNA sequencing (LR-seq) to characterize how Srsf3- and Tra2b-PEs shape mouse embryonic development. Aim 2 will investigate SF AS-NMD dynamics in vitro using a high-throughput CRISPR-based exon deletion screen to identify SF-PEs essential for iPSC viability. Conditional knockout iPSC models will be engineered to assess effects of SF-PE knockout on transcriptomes using LR-seq, SF target binding using eCLIP, and differentiation phenotypes using functional assays. Successful completion of these Aims will elucidate how SF-PEs modulate transcriptomes, safeguard cell pluripotency, and drive differentiation. This Fellowship will provide me essential training in RNA splicing, stem cell biology, genomics, and scientific communication—critical for my future career as a physician-scientist translating basic research into clinical applications.

NCI - National Cancer Institute

Project Summary PD-1 pathway targeting antibodies have improved patient outcomes in lung adenocarcinoma (LUAD). Unfortunately, most LUAD patients do not yet benefit from these therapies, and it is not clear why. Robust responses to PD-1/PD-L1 blockade require that the intratumoral CD8 T cells are in a progenitor-exhausted (TPEX) state, as TPEX cells proliferate and give rise to cytotoxic effector CD8 T cells (TEFFs). Yet, we and others have found that tumor-specific TPEX cells are primarily housed in the tumor-draining lymph node (tdLN) associated with the lung. Via migration, these cells continually replenish the tumor migration, underscoring the critical role of the tdLN as a reservoir of stem-like CD8 T cells. However, because the tdLN is the site of long- term maintenance, we hypothesize that the biology of tumor-specific TPEX and their differentiated progeny is shaped by the interactions and signals they receive in this site. Here, we propose in-depth studies on the mechanisms controlling the differentiation and maintenance of TPEX populations in the tdLN. Our proposal integrates genetically engineered LUAD models, CRISPR-based perturbations, and single-cell approaches to dissect this process. Specifically: 1. We will define how KLF2 and T-bet prevent exhaustion by repressing exhaustion-related genes (e.g., TOX) and implementing cytotoxic effector programs as T cells differentiate across the tdLN and tumor. 2. We will determine how IL-21–BATF signaling impacts on TPEX → effector CD8 T cell transitions, and the role of KLF2 in this process. We previously showed IL-21 is provided by T-follicular helper CD4 T cells in the tdLN, and we will leverage models with and without TFH responses to pinpoint how IL-21 signaling promotes CD8 T cell cytotoxicity and limits exhaustion. 3. KLF2 is transiently downregulated by TCR signals. We will determine if KLF2 downregulation is necessary for differentiation in the tdLN and the role that TCR-dependent signals play in maintaining T cell stemness in the tdLN. Our studies will investigate immune signaling pathways and transcriptional networks regulating CD8 T cells in the tdLN, elucidate mechanisms for the provision of IL-21 and its role in driving effector function, and explore the interplay between TCR and KLF2 in shaping CD8 T cell fate. These insights will shed light on immunoregulatory mechanisms that determine whether tumor-specific CD8 T cells maintain anti-tumor functions or become dysfunctional. By illuminating the biology of the tumor-specific TPEX cells in the tdLN reservoir, our goal is to identify entry points for mobilization or reprogramming through targeted interventions, to boost the efficacy of therapies against LUAD.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Essentially all transcription occurs in stochastic and episodic bursts, conferring flexibility, adaptability, and diversity to otherwise identical cells. Regulating this `bursty' transcription in a timely and context-appropriate manner is key to proper development and homeostasis. Its misregulation causes an imbalance between dynamically counteracting genes and improper gene dosage compensation, often leading to various human diseases, including cancer, cardiovascular disease, metastasis, and infertility. However, molecular mechanisms underlying transcriptional burst regulation remain elusive due to the lack of proper in vivo models and precise long-term assays. Also, the results from previous studies often conflict with each other, hampering our precise understanding and therapeutic advancements. Our overarching goal is to elucidate the molecular mechanisms underpinning spatiotemporal regulation of in vivo transcriptional bursting during development, homeostasis, and disease, and discover new factors controlling its context-specificity and adaptability. Recent studies, including our work monitoring transcriptional dynamics of endogenous Notch target genes in live adult C. elegans, contradict the previous findings: the burst duration is the major parameter regulated in vivo, whereas burst frequency is the major target of regulation in vitro. What causes these discrepancies? What modulates the burst behaviors in a context-specific manner and how? To address these questions, we will use the C. elegans gonad as an in vivo transcriptional burst study model with our innovative approach, combining long- term single-molecule live RNA imaging, machine learning-based analysis and modeling, and bioinformatics to analyze burst dynamics regulation in vivo. Focusing on the burst dynamics of powerful and well-characterized Notch pathway, we will determine the precise roles of core Notch cis- and trans-regulatory elements (CREs and TREs) like promoters, enhancers, and mediators in transcriptional burst regulation both in in vivo and in vitro contexts. We will also define the novel functions of the transcriptional co-activator LAG-3 (MAML in humans) for context-specific regulation of transcriptional dynamics, focusing on its functions for biocondensate formation and chromatin modifications. Our results will fill the critical gap in knowledge about in vivo transcriptional bursting and greatly advance our understanding of transcriptional regulation and stem cell control, with the potential to discover new therapeutic targets and strategies for Notch-related diseases and infertility.

NCI - National Cancer Institute

PROJECT SUMMARY Tumor draining LNs (tdLN), are harbingers of aggressive disease, where the presence of metastases signals risk for recurrence and poor survival in melanoma patients. The tdLN basin, however, is also antigen-rich and may promote immune reinvigoration on immunotherapy. Indeed, recent neoadjuvant trials demonstrate increased efficacy when immune checkpoint blockade (ICB) is delivered prior to surgical resection, which may depend in part on the tdLN basin. Given that large-scale clinical trials failed to demonstrate the benefit of prophylactic, complete LN dissection in high-risk, LN-positive melanoma patients, there is an opportunity to consider the therapeutic potential of tdLNs as key hubs for continued tumor immune surveillance. Future progress, however, depends upon a mechanistic understanding for how anti-tumor immune surveillance in tdLNs is maintained and the impact of standard of care clinical therapy. Recent studies, both preclinical and clinical, have identified a subset of stem-like memory (TSL) cells CD8+ T cells that are produced as a function of suboptimal antigen presentation and are enriched in tdLNs. These TSL are reinvigorated upon ICB and required for response to therapy. Despite the fact that TSL are required for response to immunotherapy in mice and associated with outcome in patients, however, we lack an understanding for what might determine their differential abundance or functionality in situ. The underlying hypothesis of the proposed work is that maintaining TSL in the draining lymphatic basin will support systemic immune surveillance in patients. We therefore leverage our deep expertise in the lymphatic system, paired with new tools to track and perturb specialized T cell populations in the context of melanoma to generate mechanistic insights that can guide future strategies for clinical management of the lymphatic basin in the context of neoadjuvant therapy. We propose that understanding the mechanisms that maintain LN TSL will lead to new strategies to boost systemic immune surveillance. Successful completion of this work will aim to 1) map the differentiation trajectory of egressing CD8+ T cells as they seed draining LNs; 2) determine the dependence of TSL on lymphatic transport; and 3) define the TSL niche in mouse and human. We expect that the basic immunological insights generated here can be used to guide the application of neoadjuvant therapy in melanoma and other solid tumors. Further, this work will nominate new candidate targets or therapeutic schedules to improve local tumor control and protect against tumor recurrence and distant metastasis.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by a progressive inability to control muscle movement. ALS patients are often comorbid with frontotemporal dementia (FTD), also known as ALS/FTD. The clinical manifestation of ALS/FTD is mediated by the selective dysfunction and degeneration of upper and lower motor neurons (MNs) that connect the CNS to the musculature, as well as cortical neurons that are responsible for speech and executive function. A broadly observed clinical feature in ALS/FTD patients is neuronal hyperexcitability. Motor neuron excitability, measured by transcranial magnetic stimulation and threshold tracking nerve conduction studies, is an early indicator of motor neuron disease that is detected pre-symptomatically, and is prognostic of disease progression and survival in both sporadic and familial cases. Despite its wide prevalence and therapeutic potential, the mechanisms that drive intrinsic neuronal hyperexcitability in ALS/FTD patients are poorly understood. Nuclear depletion and cytoplasmic aggregation of TDP-43 represents a unifying pathological feature of the overwhelming majority of ALS (~97%) and approximately 50% of all FTD patients (FTD-TDP). TDP-43 is a multifunctional RNA-binding protein that predominately resides in the nucleus and regulates RNA splicing. We have found that TDP-43 dysfunction causes spurious mis-splicing of KCNQ2 and UNC13A in ALS/FTD postmortem patient CNS tissue that strongly correlate with TDP-43 pathology, genetic predisposition, and clinical disease metrics. UNC13A encodes a protein with critical functions in synaptic vesicle release, while KCNQ2 encodes a potassium channel that regulates action potential frequency. Here, we will test the hypothesis that TDP-43 dependent splicing errors on UNC13A and KCNQ2 mRNAs trigger detrimental defects on distinct neuronal excitability properties that compromise their functionality and contribute to ALS/FTD pathophysiology. We will use induced pluripotent stem cell (iPSC)-derived cortical excitatory and inhibitory neurons, spinal motor neurons, and postmortem patient tissue to: 1) determine the extent and diversity of erroneous splicing of UNC13A and KCNQ2 in patients, 2) characterize the functional effects of these two events on neuronal physiology and, 3) develop single and bi- functional splice-modulating antisense oligonucleotides (ASOs) that can restore their normal splicing and neuronal physiology. In Aims 1 and 2 we will use single cell patch-clamp and population-based multi-electrode arrays to measure neuron intrinsic and network properties and determine the functional ramifications of TDP-43- mediated mis-splicing in human neurons. In Aim 3 we will design, screen, and identify splice-modulating ASOs conjugated by chemical linkers to simultaneously rescue both TDP-43 target mRNAs. Our studies will impact the field by highlighting a mechanistic link between TDP-43 dysfunction and neuronal excitability through aberrant mRNA metabolism, defining the functional ramifications of two of the most prevalent splicing errors of TDP-43, and by developing a new class of ASOs that could represent rational therapeutic modalities for ALS/FTD patients.

NCI - National Cancer Institute

PROJECT SUMMARY Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm that typically affects infants and toddlers. In contrast to most hematologic malignancies where RAS mutations represent late events, we and others have shown that nearly all JMML patients have initiating mutations in the RAS pathway. In the majority of patients, this is the only oncogenic mutation found in the genome. This suggests a potential opportunity to treat this disease with targeted inhibitors of RAS signaling. Indeed, we previously found that MEK inhibition (MEKi) alleviates disease in mouse models of JMML and translated this to a recently completed Phase 2 trial (NCT03190915) in children with relapsed/refractory JMML demonstrating clinical benefit of MEKi. However, only half of all patients responded, and none demonstrated molecular responses, defined by reductions in the mutant allele burden of the founding RAS mutations. We have since shown that additional RAS effector pathways (PI3K, mTOR, AKT) in addition to RAF/MEK/ERK (MAP kinase) sustain growth upon MEKi. These findings potentially explain the variable responses to MEKi observed in patients. To address these concerns, we have tested the best-in-class Pan-RAS inhibitor RMC-7977, developed by Revolution Medicines. This compound is a “molecular glue” that binds to all active RAS proteins (i.e., only in the GTP-bound state), and recruits a second protein, cyclophilin A, to sterically prevent active RAS from reaching its effectors. We have now tested RMC-7977 in primary samples from patients with JMML and in patient derived xenograft (PDX) models of JMML and acute myeloid leukemia (AML) with RAS mutations and have observed remarkable efficacy. However, the mechanisms of response and resistance to direct RAS inhibition (RASi) have not been elucidated in hematologic malignancies. We hypothesize that specific RAS genotypes including NRAS and KRAS will be more sensitive to RMC-7977 than other RAS family members including PTPN11 and NF1. We also hypothesize that the “dosage” of RAS mutations will impact sensitivity to the drug. Lastly, we hypothesize that the cell of origin in which RAS mutations arise will impact sensitivity to RASi. We will test these hypotheses using rare primary samples, PDXs and induced pluripotent stem cells. Because primary samples from infants with JMML are difficult to obtain, determinants of response to RASi cannot be adequately modeled in-vitro alone. The proposed studies will use PDX models to evaluate therapeutic efficacy, characterize mechanisms of response and resistance, and validate findings in the context of an intact hematopoietic microenvironment. These animal studies are essential for translating mechanistic discoveries into future clinical trials for children with JMML. While rare, JMML represents a genomically “simple” disease to study Pan-RAS inhibition. The results can then be applied to more common and genomically “complex” diseases like acute myeloid leukemia and myeloproliferative neoplasms that harbor RAS mutations.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Tregs play a crucial role in maintaining immunologic tolerance and preventing autoimmune diseases. Current treatments for these conditions often involve immunosuppressive medications, which can have harmful side effects and limited effectiveness. Our research aims to unlock new possibilities in stem cell science by manipulating the expression of the transcription factor FOXP3, the master regulator of Treg development, during T cell differentiation of induced pluripotent stem cells (iPSCs). This work seeks to understand how FOXP3 expression can be most effectively regulated during iPSC differentiation and the impact of specific approaches on T cell differentiation. I propose two specific aims to achieve this goal: Aim 1 explores the effects of introducing an exogenous source of FOXP3 on iPSC differentiation. We will examine how different levels, timing, and isoforms of exogenous FOXP3 expression influence Treg development and functionality. Aim 2 focuses on identifying and manipulating Notch signaling effectors to direct Treg lineage commitment. We will create a comprehensive gene regulatory network and employ machine learning through the Python library CellOracle to model transcription factor perturbations for candidate genes in silico. To achieve these aims, I have applied new strategies to an in vitro model of T cell development, the artificial thymic organoid (aka ATO), developed by our group. The ATO platform is currently the only in vitro system that robustly supports mature CD4+ T cell production through the developmental stages that mirror conventional thymopoiesis. I have effectively increased FOXP3 expression during iPSC differentiation in the ATO model using the following methods: constitutive overexpression via lentiviral transduction, CRISPR- Cas9 knock-in for stage-specific expression, and small molecule modulation. This multi-faceted approach allows for the mechanistic investigation of Treg development from iPSCs and will provide foundational knowledge for generating iPSC-derived Tregs as adoptive cell therapy for autoimmunity. Expected outcomes of this work include a detailed understanding of how FOXP3 expression levels and timing affect Treg development. We will also define the regulatory role of Notch signaling on FOXP3 expression for this process. This knowledge will facilitate the development of future Treg therapies, offering new hope for patients with autoimmune diseases. Our work will enhance the mechanistic understanding of iPSC differentiation into the Treg lineage and propel research in stem cell-based therapies for autoimmunity. By developing a robust platform for Treg generation from iPSCs, our project holds the potential to transform autoimmune disease treatment and advance the field of stem cell-based therapies.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT The finely tuned generation and function of regulatory T (Treg) cells are essential for maintaining the balance that allows for protective immunity while preventing harmful autoimmunity. Treg cells are heterogeneous, comprising specialized subsets that contribute to tissue repair and mediate context-specific immune responses. Despite their critical roles in essential biological processes, it remains unknown whether the subset-specific functions of Treg cells are driven by their developmental origins, transcriptional programs, or a combination of both. This unresolved challenge largely stems from two issues: the lack of unbiased means to identify mutually exclusive Treg cell subsets with distinct functions, and the absence of tools to trace their ontogeny. However, my recent discoveries have opened promising avenues for overcoming these obstacles. Using colorectal cancer models and human patient specimens, I identified that interleukin-10 (Il10) expression distinguishes two subsets of Treg cells with opposing functions: IL-10+ Treg cells, which exhibit anti-tumor properties, and IL-10– Treg cells, which promote tumor growth. Furthermore, I identified Dapl1 as a gene uniquely expressed by naïve CD4 T cells, thereby providing a definitive marker for extrathymically generated Treg cells. The overarching goal of this research proposal is to determine whether the developmental origins of IL-10– vs IL-10+ Treg cells contribute to their distinct functions, and to identify the transcriptional programs underlying these differences. This proposal tests the hypothesis that both of these subsets are of mixed developmental origins, with their distinct functions driven by differentially expressed transcription factors. Specifically, in Aim 1, using a novel Dapl1-based lineage tracing model, I will determine whether IL-10+ and IL-10– Treg cells arise from thymic or extrathymic (peripheral) origins and elucidate how these developmental pathways shape their functions. Additionally, in Aim 2, I will define the transcriptional programs that drive their subset-specific activities, by inducing Treg cell specific deletion of key regulators such as Zeb2 and Nfil3. By employing genetic mouse models, advanced single-cell analyses, and CRISPR-based screening, the proposed studies will reveal the nature of Treg cell functional heterogeneity, ultimately guiding the development of more precise immunotherapeutic strategies with major implications for public health. The proposed career development plan complements my training in cellular and molecular immunology with single-cell analysis and computational biology. I will take advantage of the extensive resources of the Memorial Sloan Kettering Cancer Center, part of the Tri-Institutional network with the Rockefeller University and Weill Cornell, as well as benefit from the mentorship of Dr. Alexander Rudensky and guidance from Advisory Committee members Dr. Christina Leslie, Dr. Ming Li, and Dr. Steven Josefowicz. By the end of the mentored phase, I will have acquired the necessary tools to conduct comprehensive studies at the intersection of immune cell heterogeneity and immune communication with the environment as an independent investigator.

NIAID - National Institute of Allergy and Infectious Diseases

Sepsis is a leading cause of death worldwide, with most patient mortality stemming from lingering immune dysfunction in sepsis survivors. A key feature of sepsis-associated immune dysregulation is monocyte exhaustion, a phenotype of paradoxical pro-inflammatory and immunosuppressive gene expression, impaired differentiation, and reduced antigen presentation. Monocyte exhaustion can persist for years after sepsis onset, a result of long-term immune memory. However, the mechanisms controlling such long-term memory remain to be elucidated. Whereas previous research has conceptualized innate immune memory through diametrically opposed mechanisms that either promote (train) or restrict (tolerize) monocyte responses, my preliminary data suggests that exhaustion represents a distinct memory state characterized by unique immune, transcriptional, and epigenetic features. Therefore, in contrast to the two-state model for innate memory, I hypothesize that innate memory represents a continuum of states driven by distinct epigenetic patterning, with prolonged, high- intensity immune stimulation leading to monocyte exhaustion in septic individuals. In Aim 1 of my proposed study, I will profile the unique transcriptional and epigenetic features defining monocyte exhaustion, as well as employ integrative modeling to determine how immune stressor strength, duration, and timing influence the establishment of distinct innate memory states. In Aim 2, given preliminary data showing genome-wide DNA hypermethylation in exhausted monocytes, I will test the hypothesis that inhibition of DNA demethylation enzyme TET2 is upstream of these epigenetic changes, and that treatment with TET agonists is a tractable therapeutic strategy to restore healthy epigenetic memory. Finally, in Aim 3, based on my recent identification of a novel DNMT3L isoform expressed in septic monocytes, I will test the altered chromatin affinity and regulatory activity of this isoform and establish its contribution to DNA methylation reprogramming during monocyte exhaustion. Completion of these proposed Aims will allow me to develop skills in new experimental techniques, including single-cell RNA sequencing, reduced representation bisulfite sequencing, in vivo mouse sepsis modeling, and cytometric arrays. Aims 1 and 3 will be pursued during the K99 mentored research phase at Virginia Tech in the laboratory of Dr. Liwu Li, an expert in the fields of monocyte biology and innate immune memory. Whereas my previous graduate studies focused on epigenetics and mammalian development, Dr. Li will provide valuable instruction as I expand into the topics of immunology and hematology. I will also pursue coursework at Virginia Tech in computational modeling of biological systems while engaging with professional development workshops covering such topics as scientific communication, mentorship, and R-series proposal development. The goal of this project is ultimately to pursue a career as an independent biomedical investigator in academic research; these studies will serve as a foundation for my own research program aimed at identifying the major molecular players responsible for establishing and maintaining innate immune memory.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary Streptococcus mutans is the primary bacterial agent of dental caries (cavities), a widespread disease affecting billions worldwide. The pathogen's ability to cause disease stems from its remarkable capacity to adapt to the harsh and dynamic oral environment, a process controlled by sophisticated regulatory networks. This proposal focuses on a key, yet underexplored, regulatory mechanism known as O-phosphorylation, a post- translational modification that reversibly alters protein function to control cellular processes. Our preliminary phosphoproteomic analysis of S. mutans has revealed that O-phosphorylation is widespread, affecting numerous proteins critical for stress adaptation, metabolism, and cell division. However, the functional significance of these phosphorylation events remains largely unknown. The central hypothesis of this application is that O-phosphorylation is a critical stress-responsive regulatory mechanism that modulates protein function to promote S. mutans adaptation and virulence. To test this hypothesis, we will pursue three specific aims. Aim 1 will determine the functional impact of O- phosphorylation on S. mutans physiology by systematically generating and screening the first-ever arrayed panel of phospho-mutants for any bacterial species, creating a functional map of the phosphoproteome. Aim 2 will investigate the role of the PppL phosphatase in cell division by dissecting a novel "altered-function" suppressor mutation that rescues a lethal phenotype, providing mechanistic insight into a critical cell division checkpoint. Aim 3 will examine the phosphorylation-mediated regulation of the Pgf glycosylation machinery, a key virulence pathway, to uncover a novel paradigm of regulatory crosstalk between O-phosphorylation and O-glycosylation. Successful completion of this project will fundamentally advance our understanding of bacterial signal transduction. It will deliver a novel functional map linking specific phosphorylation events to virulence, provide a detailed molecular mechanism for two key regulatory pathways controlling cell division and glycosylation, and establish a foundational resource for the field. Ultimately, this work is expected to uncover novel, druggable targets, paving the way for the development of new therapeutic strategies to combat dental caries.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Abstract Polycomb group (PcG) complexes are multi-protein, evolutionarily conserved epigenetic machineries that regulate stem cell fate decisions, cell identity and early development. The PcG machinery can be divided into two major complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). Traditionally, PcG complexes are associated with gene repression mainly via histone-modifying activities. While PRC2 catalyzes methylation on lysine 27 of histone H3 (H3K27me1/2/3) via EZH1/2, PRC1 deposits a ubiquitin group at lysine 119 of histone H2A (H2AK119ub1) via the E3-ligases RING1A/B. Interestingly, several PcG encoding genes are found to be mutated in individuals with developmental disorders. Specifically, de novo missense mutations in the genes encoding for RING1A (RING1), and RING1B (RNF2), have been found in pediatric patients with neurodevelopmental disorders. How mutations at PcG genes impair development in humans is completely unexplored. Additionally, we have discovered novel missense mutations in both genes in children with intellectual disabilities. We conducted predictive analyses using crystal structures to start understanding how these mutations affect PRC1's stability and interaction with nucleosomes. In this proposal, we will focus our efforts in one of the RNF2 mutations, which is associated with intellectual disabilities using novel knock-in ESC lines as well a new mouse model carrying a monoallelic missense mutation on RNF2. Preliminary data reveal that mutant RING1B disrupts Polycomb complex assembly, induces derepression of PRC1 and PRC2 target genes, and impaired differentiation into neurons. By ChIP-seq and mass spectrometry we will investigate chromatin occupancy and recruitment mechanisms and potential rescue strategies. Additionally, this proposal will examine how Rnf2 mutations impact hippocampal structure, and behavioral outcomes in mice. Immunohistochemistry, RNA-seq, and ATAC-seq will determine the cellular diversity and regulatory dynamics in the hippocampus, providing insights into the mutation's molecular and behavioral consequences. Overall, our proposed research aims to define the role of missense mutations in Polycomb genes in neurodevelopment in vitro and in vivo, examining epigenetic mechanisms, behavior, and neuronal architecture. This work will enhance our understanding of how missense mutations influence PRC1 function and their contribution to neurodevelopmental disorders, shedding light on the complex relationship between epigenetics and neurodevelopment. Finally, our findings could pave the way for therapeutic strategies for neurodevelopmental disorders associated with PcG mutations.

NHLBI - National Heart Lung and Blood Institute

Project Summary Heart failure (HF) is the leading cause of death; resulting in a variety of physical and biochemical changes within the heart. Better understanding how physical and biochemical cues influence CMs, can help in the development of better HF therapies. We aim to investigate how viscoelasticity and growth factor (GF) sequestering influence healthy and diseased human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) using engineered biomaterials. We hypothesize that mimicking cardiac viscoelasticity will increase maturity of iPSC-CMs and enhance GF signaling. We further hypothesize that iPSC-CMs will sense viscoelasticity through TRVP4 channel protein and ERK pathway activation. We measured human tissue viscoelasticity via stress relaxation testing, and found that slower relaxing tissues exhibited higher fibrosis, indicated by increased picrosirius red staining. We then engineered viscoelastic hydrogels to mimic cardiac stress relaxation, elastic hydrogels that exhibited no relaxation, and relaxation speeds in between. We found that iPSC-CM on viscoelastic alginate were more cylindrical in morphology, exhibiting a significant increase of 0.1 in sphericity. This morphology is similar to mature cardiomyocytes in the heart. Additionally, the cells on viscoelastic gels beat at higher speed (0.16 µm/s increase) than those on elastic hydrogels. These data indicate that iPSC-CMs exhibit a more mature morphology and function on viscoelastic hydrogels. We further investigated the effect of cell secreted GFs. We found that mesenchymal stromal cell (MSC) conditioned media, or the MSC secretome, which is known to contain high concentrations of growth factors including IL-8, FGF-6 and IGF-1, resulted in an increase of 0.8 µm/s in contraction velocity, and a trend in increasing contraction displacement. To deliver GFs, we engineered a viscoelastic hydrogel capable of sequestering growth factors via sulfate group modification. Alginate sulfation resulted in increased regeneration in a rat soleus crush injury. We hypothesize that the combined influence of our material to sequestered growth factors and exhibit viscoelasticity will lead to growth factor receptor clustering, increased growth factor signaling, and drive disease and maturity in iPSC-CMs that are healthy or have point mutations linked to hypertrophic or dilated cardiomyopathy.

NIA - National Institute on Aging

Project Summary Adult tissue homeostasis depends on the tightly regulated activity of tissue-resident stem and progenitor cells. With age, this regenerative capacity declines due to impaired progenitor function, contributing to tissue dysfunction and degeneration. One of the most striking examples of this occurs in the hair follicle, a highly regenerative mini-organ that undergoes cyclical phases of growth (anagen) and rest (telogen). Aging disrupts the cycle by prolonging telogen and diminishing the proliferative output of progenitor cells, ultimately leading to follicle miniaturization and hair loss. Despite its clinical relevance, the molecular mechanisms governing progenitor cell activation and maintenance in the hair follicle remain incompletely understood. To address this gap, I performed single-cell RNA sequencing analysis, RNA velocity analysis, and immunofluorescence staining of cycling postnatal mouse skin, identifying SOX5 as a transcription factor specifically expressed in the earliest subset of activated progenitor cells at anagen onset, localized to a key structure known as the secondary hair germ (SHG). Expression then persists throughout the anagen phase within the proliferative lower matrix before becoming undetectable until the next cycle, suggesting a temporally restricted role in activating progenitor cells and guiding their commitment to a follicular lineage. Supporting this, in vitro overexpression of SOX5 in primary human keratinocytes significantly enhances proliferation, pointing to SOX5 as a central regulator of proliferative dynamics during follicular regeneration. Based on these findings, I hypothesize that SOX5 induces anagen and protects the hair follicle against aging by regulating proliferation of the hair matrix cells and directing SHG cells towards a hair follicle lineage fate. In Aim 1, I will determine whether SOX5 is required for SHG activation and sufficient to initiate early lineage specification. I will also evaluate whether SOX5 overexpression reprograms human keratinocytes toward a follicular identity. In Aim 2, I will assess the role of SOX5 in maintaining matrix proliferation and hair follicle structure during aging using a combination of ex vivo human hair follicle organ culture and a transgenic Sox5 overexpression mouse model. By elucidating how SOX5 governs progenitor cell activation and maintenance, this work may uncover therapeutic strategies to restore hair progenitor cell function in aging and hair loss disorders. More broadly, it will contribute to our understanding of how tissue-specific progenitor programs can be leveraged to counteract age-related regenerative decline.

NIGMS - National Institute of General Medical Sciences

Quiescence is an actively maintained state, characterized by a reversible, cell-cycle arrest. Quiescent neural stem cells are found during development and in the adult mammalian brain, reactivating in response to certain cures such as injury or exercise. The regulation of neural stem cell (NSC) quiescence maintains stem cell populations and provides opportunity for brain repair. On the other hand, quiescence can also be pathological, theorized to contribute to tumor therapy resistance and recurrence. Understanding the regulation of quiescence in stem cells is therefore crucial to understanding development, disease and can serve as a starting point for new regenerative therapies. In Drosophila, quiescent neural stem cells (qNSCs) enter quiescence and reactivate in response to the secretion of insulin-like peptides. The transcription factor Worniu (wor) is translated early during NSC reactivation, even though the transcript is detectable during quiescence, indicating post-transcriptional regulation. Using single cell transcriptomics, we have identified differentially expressed genes during quiescence and reactivation of NSCs. Tis11, an RNA-binding protein was found to involved in regulating NSC reactivation and mediated both the transcript and protein levels of wor. Knockdown of Tis11 causes precocious reactivation, while misexpression delays reactivation, indicating a role in quiescence regulation. However, the precise molecular mechanisms by which Tis11 regulates quiescence and reactivation in NSCs is not known. The main aims of this project are to elucidate the precise molecular mechanisms of Tis11 in the regulation of quiescence. As Tis11 protein is shuttled between the nucleus and cytoplasm, this project will investigate its post-transcriptional and transcriptional roles. Using RNA immunoprecipitation-sequencing and Targeted DamID, the mRNA and genome-wide targeting targets of Tis11 will be profiled. The binding of Tis11 to wor will also be tested via luciferase reporter assay. Finally, the interaction between Tis11 and insulin signaling in qNSC reactivation will be examined via epistasis experiments. Interestingly, Tis11 also has a role in the maintenance of quiescence in intestinal stem cells. This project will therefore identify a potentially conserved molecular mechanism of quiescence regulation for stem cells in different tissues. Furthermore, Tis11 may be a potential therapeutic target, enabling the regulation of reactivation of stem cells for rejuvenation therapy. On the other hand, Tis11 may also be a target of interest, whereby inhibiting its activity may prevent therapy-resistant quiescent cancer cells from persisting in tumors. This training project will be performed at the vibrant Regenerative Medicine Institute at the NYU Grossman School of Medicine. The candidate will be working in a collaborative, interdisciplinary environment. The candidate’s work will pre presented at both local and international conferences, to increase exposure and opportunities to network. Career development training will take the form of workshops and personalized mentoring, preparing the candidate for a career as an independent investigator.