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U.S. National Science Foundation

The EHR Core Research (ECR) program establishes a mechanism in the Directorate for Education and Human Resources to provide funding in foundational research areas that are broad, essential and enduring. EHR seeks proposals that will help synthesize, build and/or expand research foundations in the following core areas: STEM learning, STEM learning environments, workforce development, and broadening participation in STEM. We invite researchers to identify and conduct research on questions or issues in order to advance the improvement of STEM learning in general, or to address specific challenges of great importance. Two types of proposals are invited: Core Research Proposals (maximum 5 years, $1.5 million) that propose to study a foundational research question/issue designed to inform the transformation of STEM learning and education and Capacity Building Proposals (maximum 3 years, $300,000) intended to support groundwork necessary for advancing research within the four core areas.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary / Abstract This is a R01 application from Dr. Daniel Lingwood (PI) an Associate Professor of Medicine at the Ragon Institute of MGH, MIT and Harvard and Dr. Batista (co-PI), a Professor of Medicine at the same institution. These investigators define immunological decision making by B cells to inform vaccine design and have built humanized mouse systems that recapitulate human antibody responses. Dr. Andrew Ward (co-I) is Professor in the Department of Structural and Computational Biology at Scripps and specializes in high resolution cryoEM of antibody:antigen complexes. The application goal is universal influenza vaccine development, centered on their discovery of a human broadly neutralizing antibody (bnAb) pathway that the investigators can vaccine-elicit to protect against all influenza A viruses (IAV), the major source influenza disease and all pandemic events. Differences in N-glycosylation generally prevents antibodies from engaging the otherwise conserved hemagglutinin (HA) stem of group 1 versus group 2 IAV. To solve this issue, the investigators engineered nanoparticle immunogens that elicit cross-group IAV immunity by selectively triggering and maturing germline B cell receptors (BCRs) encoding VH1-18 QxxV class bnAbs, a rare but genetically reproducible or ‘public’ category of human pan-IAV bnAbs that accommodates N-glycan diversity on the HA stem. The investigators successfully elicit this cross-group bnAb response using a single shot within a humanized vaccine model containing the VH1-18 QxxV bnAb precursors at physiologically relevant human frequency within the naïve B cell pool. The immunogens select for key affinity enhancing mutations, including N55T in the CDRH2, a hallmark of VH1-18 QxxV bnAbs. The investigators show that N55T alone provides cross-group IAV protection by a novel antibody tilting mechanism that accommodates N-glycan diversity on the HA stem. The investigators will now test the central hypothesis that this ‘molecular switch’ endows humans with an exceptionally simple vaccine- expandable pathway for eliciting broad spectrum IAV immunity. In Aim 1, the investigators will apply their modular human vaccine model to define the number of naive VH1-18 QxxV B cells needed for pan-IAV vaccine protection; if these bnAb precursors are absent, the germline stimulating nanoparticles no longer elicit pan-IAV bnAbs, revealing a human B cell repertoire effect encoding for unprecedently broad IAV immunity. In Aim 2, the investigators will define whether their nanoparticle immunogens can co-expand multiple classes of cross-group IAV bnAbs within their human vaccine model. Critically, their engineered nanoparticle immunogens also bear germline stimulating affinity for the naïve BCRs encoding the other known classes of genetically reproducible pan-IAV bnAbs produced by humans, potentiating multiclass bnAb elicitation via pan-germline stimulation. In the Aim 3, the investigators will define how prior exposure to IAV modulates (and enhances) germline stimulation and vaccine elicitation of pan-IAV bnAbs via imprinting effects. Collectively, this proposal will exploit novel genetically hardcoded templates for eliciting cross-group IAV immunity in humans.

NHLBI - National Heart Lung and Blood Institute

Project Summary Respiratory viruses can acutely lead to high mortality from respiratory failure. However, respiratory viral infections also cause permanent morbidity long after recovery from the acute infection. Such chronic debilitation was best exemplified during the COVID-19 pandemic that left many patients with post-acute sequelae of SARS-CoV-2 (PASC; aka long COVID). One unfortunate consequence after a severe COVID-19 infection is the development of persistent pulmonary fibrosis (PASC-PF). However, this fibrotic remodeling of the lungs is not unique to infections by SARS-CoV-2 and has also been found with other viral infections such as influenza, MERS-CoV, SARS-CoV-1. Investigations into PASC-PF have revealed common features when compared to idiopathic pulmonary fibrosis (IPF), the archetype of progressive lung fibrosis. A fundamental feature in PASC-PF, IPF, and other types of lung fibrosis is the maladaptive repair of the lung epithelium that is an upstream driver of fibroproliferation. In particular, the type 2 alveolar epithelial cell (AT2) cells, which are the facultative epithelial stem cells of the alveoli, are exhausted and fail to properly repair the lungs after injury. Accordingly, damaged alveoli have a repair deficiency with a failure of AT2 differentiation into alveolar type 1 (AT1) cells that are necessary for lining the alveoli to facilitate gas exchange in the lungs. To that end, recent studies have found IFN-γ to be a key signaling node that is enriched in PASC-PF and IPF. Moreover, studies by our group and others have demonstrated IFN-γ blockade to reduce fibrosis and augment epithelial repair in a viral-induced lung fibrosis model. Accordingly, we have developed a central hypothesis that anti-viral immunity by CD8+ T cells directly (via IFN-γ) and indirectly (via MDM secretion of IL-1β) stalls alveolar regeneration thereby shifting the injury response toward maladaptive repair thereby activating fibroproliferative pathways. We will test the hypothesis in three aims: Aim 1. Evaluate viral-mediated maladaptive reprogramming of AT2 cell. Aim 2. Uncover the cellular and molecular mechanisms that mediate the abnormal immune-epithelial interactions contributing to chronic lung fibrosis following viral infection. Aim 3. Identify abnormal immune-epithelial interactions in human post-viral lung fibrosis . The successful completion of this study promises to elucidate the cellular and molecular etiology of lung fibrosis and have a broad impact on understanding fibroproliferative mechanisms in PASC-PF, IPF, and other forms of lung fibrosis.

NIGMS - National Institute of General Medical Sciences

Project Summary The broad, long-term objectives of this application are to characterize mechanisms that allow a competitive germline stem cell (GSC) and its descendants to dominate the GSC population and cause super-Mendelian inheritance. The proposal will determine how a GSC in the Drosophila testis remodels its niche and causes the selective loss of WT neighbor GSCs. To accomplish this, the proposal will utilize immunofluorescence, genetics, RNA interference, extended ex vivo live-cell imaging, transcriptomics, chromatin labelling, and innovative assays of GSC competition and allele inheritance in F1 offspring. We will capitalize upon the powerful genetics available in Drosophila, as well as the ability to unequivocally identify the niche, GSCs, differentiating germline cells, and somatic stem cells (CySCs) and their lineage in the Drosophila testes. This proposal is supported by our published results demonstrating that (1) loss of the transcription factor Chinmo in a GSC causes the ectopic secretion of the extracellular matrix (ECM) protein Perlecan (Pcan), (2) this Pcan accumulates around the endogenous niche resulting in an ectopic ECM termed the moat within the testis lumen; (3) the moat causes the selective loss of WT neighbor GSCs, which no longer have strong adhesion with niche cells; (4) chinmo-/- GSCs remain in the resculpted niche because they upregulate ECM-binding proteins. This proposal is also supported by our unpublished results showing that Chinmo protein expression is promoted by an RNA-binding protein (RBP) in GSCs and that a ZAD-ZNF protein likely acts as a Chinmo co-factor in GSCs. In the first goal, we will determine whether clonal loss of the RBP that promotes Chinmo expression imparts that GSC with a competitive advantage. We will also determine what regulates that RBP in GSCs and test whether loss of any regulators of the RBP imparts a competitive advantage to a mutant GSC. In the second goal, we will determine whether Chinmo and the ZAD-ZNF protein work together to repress Pcan by recruiting histone methyltransferases. We will also determine how niche cells promote the ectopic Pcan produced by chinmo-/- GSCs. In the third goal, we will test the role of somatic stem cells (CySCs) in GSC competition and assess whether they push out WT neighbors GSCs. We will also use live-cell imaging to determine the types of GSC division that occur in chinmo- /- GSCs. The studies in this proposal will increase the knowledge base about GSC competition and will foster new avenues of research into mechanisms and possible treatments for human paternal age effect disorders caused by competitive spermatogonial stem cells and for tumor cells which remodel their microenvironment to benefit themselves and disadvantage WT neighboring cells.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Understanding the fundamental processes that underscore hematopoiesis is necessary to generate therapeutics that can correct hematopoiesis during disease or support hematopoietic regeneration during stress. The bone marrow microenvironment, or niche, supports hematopoiesis by providing cellular and acellular cues that influence hematopoietic differentiation, hematopoietic stem cell self-renewal, and the functional integrity of the bone marrow niche itself. Inadequate or inappropriate hematopoietic differentiation can lead to disease or death by depleting hematopoietic cell populations needed for organismal survival. Additionally, commonly used therapeutics like radiation and chemotherapy cause hematopoietic cell death and damage the bone marrow niche, leaving patients in a vulnerable state of hematopoietic insufficiency. The inability to restore hematopoietic homeostasis after radiation exposure puts patients at a heightened risk for developing deadly complications, such as infection or hemorrhage. Therefore, understanding how hematopoiesis is maintained and restored is of the utmost importance. Our previous studies showed that syndecan-2 (a specific heparan sulfate proteoglycan) expressed by hematopoietic stem cells promotes long-term hematopoietic stem cell self-renewal ability by supporting quiescence. The bone marrow niche is also a rich source of proteoglycans. Our preliminary data indicate that bone marrow mesenchymal stromal cells (MSCs) also highly express syndecan-2. Genetic depletion of syndecan-2 in MSCs using transgenic mouse models caused significant hematopoietic system imbalances in the peripheral blood at steady-state and after hematologic stress. In this application, we propose to elucidate how MSC-derived syndecan-2 regulates hematopoiesis in vivo. We will use a multi-scale approach to test the function of syndecan-2 at the molecular, cellular, and systemic scales by combining transgenic knockout mouse models and in vivo injury models with high-resolution bone marrow imaging and super- resolution imaging of MSCs. We will test the role of MSC syndecan-2 in hematopoietic differentiation, growth factor organization, and signaling. Because hematopoietic demands increase during states of hematopoietic stress, we will also test the function of syndecan-2 from MSCs in hematologic and niche regeneration from radiation injury. Successful completion of these aims will define the role of MSC-derived syndecan-2 in hematopoietic homeostasis and regeneration, providing foundational knowledge needed to leverage proteoglycans to correct or boost hematopoiesis during states of imbalance or stress.

NIGMS - National Institute of General Medical Sciences

Abstract The canonical Wnt signaling pathway is a master regulator of embryonic development, stem cell renewal, and tissue regeneration. Its dysregulation drives diverse human diseases, including cancer, osteoporosis, and cardiovascular and metabolic disorders. At the center of this pathway are the co-receptors LRP5 and LRP6, which act as dynamic molecular hubs that integrate stimulatory and inhibitory cues. Despite their fundamental roles, how LRP5/6 transition between distinct conformational states and translate ligand binding into precise signaling outcomes remains poorly understood, limiting the development of targeted therapeutic strategies. This proposal aims to decode the structural and mechanistic logic of LRP5/6-mediated Wnt signaling. Leveraging cutting-edge cryo-electron microscopy, biochemistry, and cell-based functional assays, we will define how LRP5/6 dynamically remodel in response to Wnt ligands and antagonists, and how cellular factors such as pH and post-translational modifications fine-tune their activity. By linking receptor structure to function and disease-associated mutations, this work will generate a unifying framework for understanding Wnt pathway regulation and open new avenues for precisely modulating Wnt signaling in tissue regeneration and disease intervention.

NHLBI - National Heart Lung and Blood Institute

Project Summary Smoking remains a leading cause of cardiovascular disease (CVD), responsible for millions of deaths worldwide. Despite its widespread impact, the precise mechanisms linking smoking to CVD risk, particularly in relation to genetic factors, remain incompletely understood. One such genetic variation, ALDH2*2, affects approximately 540 million individuals globally and may interact with tobacco smoke to exacerbate CVD risk. However, the addictive nature of smoking, compounded by social and cultural influences, complicates efforts to reduce smoking prevalence in this population. Consequently, understanding the role of ALDH2*2 in smoking-induced CVD is crucial for advancing precision medicine for individuals affected by this genetic variation. Our previous research, which utilized induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) from individuals carrying the ALDH2*2 variant, revealed significant endothelial dysfunction. These cells demonstrated elevated oxidative stress and inflammation, along with reduced nitric oxide production and tube formation capacity (Guo et al., Science Translational Medicine, 2023). Our recent findings further suggest that the endothelial dysfunction associated with the ALDH2*2 variant is exacerbated by exposure to cigarette smoke in both human iPSC and transgenic mouse models. Despite these findings, the specific mechanisms by which tobacco consumption exacerbates CVD risk in individuals with the ALDH2*2 variant remain unclear, impeding the development of tailored approaches for ALDH2*2 smokers. The overarching goal of our proposal is to utilize a multidisciplinary approach that integrates stem cell biology, molecular biology, toxicology, vascular physiology, and endothelial mechanobiology to elucidate the molecular mechanisms underlying ALDH2*2- and smoking- induced endothelial dysfunction. We will pursue two specific aims. In Aim 1, we will examine the ROS-FOXO1- KLF5→IL-18/IL-1β signaling axis in modulating endothelial dysfunction in both human iPSC and mouse models carrying the ALDH2*2 variant. Additionally, we will screen small molecules targeting the ROS-FOXO1-KLF5 axis in cigarette smoke-exposed ALDH2*2 iPSC-ECs to evaluate their effects on endothelial function. In Aim 2, we will examine NUP210’s interaction with the LINC complex in mediating the shear stress response in ALDH2*2- and smoking-induced endothelial dysfunction. We will utilize RNA-seq, ATAC-seq, ChIP-seq, and single-cell RNA-seq to gain mechanistic insights into how NUP210 interacts with LINC complex and regulates the H3K27me3 modification of extracellular matrix genes in response to mechanical forces. Our proposal is supported by robust preliminary data, and the successful completion of this research will identify two novel molecular mechanisms—KLF5-mediated inflammation and NUP210-mediated shear stress response—through which smoking exacerbates CVD risk in the ALDH2*2 carriers. Additionally, the study will provide insights into potential prognostic biomarkers and therapeutic targets to mitigate CVD in smokers with the ALDH2*2 allele.

NHLBI - National Heart Lung and Blood Institute

The human body produces hundreds of billions of blood cells daily, replenished by hematopoietic stem cells (HSCs) in the bone marrow. Over time, HSC clones—populations derived from a single HSC—fluctuate in size, with some clones expanding while others dwindle. Clonal dynamics have been studied by reconstructing HSC phylogenic trees from somatic mutations they have accrued using whole-genome sequencing of single-cell- derived colonies. Our group pioneered the study of clonal dynamics in myeloproliferative neoplasms (MPNs), showing that driver mutations, such as JAK2, arise decades before diagnosis and confer a fitness advantage, enabling mutant clones to dominate the population. Strikingly, similar clonal dominance is observed in aging healthy individuals, though only ~20% of expansions can be attributed to known driver mutations. Understanding why certain HSC clones expand, especially in the absence of clear genetic causes, remains a fundamental unanswered question in hematopoiesis. Clonal expansion of HSCs may result from cell-intrinsic factors, as not all HSCs are equivalent. We would like to understand how during development a heterogenous population of HSCs is generated. Extrinsic factors, such as signals from the niche or systemic inflammation, may also drive clonal expansion. However, we lack basic knowledge of the drivers of clonal dynamics in native hematopoiesis because (1) reconstructing clonal history using single-cell phylogenies is not scalable—whole-genome sequencing of colonies is invasive, slow, and costly; and (2) mouse models, while useful for perturbing clonal dynamics, fail to recapitulate human clonal dynamics. This is because, despite the fitness advantages of certain HSC clones, the short lifespan of mice does not allow sufficient time for these clones to expand and dominate the stem cell population. To resolve clonal expansions in mice, we need scalable methods to reconstruct the phylogenetic history of all HSCs, not just a subset. We propose a comprehensive research program for developing new technologies to address these challenges and uncover the drivers of HSC clonal dynamics. First, we will create a non-invasive, rapid, and cost-effective method to reconstruct HSC clonal histories using long-read bulk sequencing of methylation patterns in blood cells, reducing the cost per sample from $100,000 to $1,000 and enabling large-scale human studies. Second, we will engineer mice to record lineage and key signaling histories of HSCs directly in their own DNA by extending lineage-recording mouse models we previously developed. Phylogenetic trees of all HSCs can then be reconstructed efficiently by sequencing specific target regions instead of entire genomes. By integrating signaling activity with lineage history, we will decorate tree branches with molecular events that drive clonal expansion. These engineered mice will enable mapping the developmental origins of HSC heterogeneity and quantifying the impact of extrinsic factors on clonal dynamics. Together, these approaches will address fundamental questions in stem cell regulation and aging, improve prognosis and treatment of hematological disorders, and provide transformative tools for studying blood.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Hirschsprung disease associated enterocolitis (HAEC) is the leading cause of death in children who lack enteric neurons in distal bowel, a birth defect called Hirschsprung disease. The etiology of HAEC is not well understood, but hypothesized disease mechanisms include altered gut microbes (“dysbiosis”), abnormal mucosal immune system and epithelial barrier defects. To date, there are no immune-targeted therapies to treat or prevent HAEC, but new treatments are needed. This proposal builds on the candidate’s preliminary data suggesting interleukin 22 (IL22) critically modulates HAEC risk and HAEC severity. The central hypothesis is that enteric nervous system (ENS) signaling induces IL22 release and facilitates IL22 epithelial responses to enhance mucosal immunity and strengthen epithelial barrier functions that prevent enterocolitis. The Piebald lethal (sl/sl) Hirschsprung disease mouse model of HAEC will be used, as survival of sl/sl mice is dramatically (> 3-fold) altered by diet (Tjaden et al, in BioRxiv and submitted) and IL22 mRNA is much higher in sl/sl fed a Protective diet that extends median survival (“late onset HAEC”). Aim 1 will define the cellular source(s) of IL22 from bowel regions of sl/sl model mice that develop early or late onset HAEC. In parallel, this aim tests the hypothesis that IL22 prevents HAEC, by using genetic and pharmacologic strategies to alter IL22 levels. Aim 2 will precisely define the role of IL22 on epithelial integrity, stem cell renewal and differentiation in organoids derived from sl/sl mice with early or late onset HAEC and from children with Hirschsprung disease with or without HAEC. Organoids facilitate studies of epithelial stem cell biology and IL22-epithelium interactions in the absence of microbes, neurons, or diffusible small molecules such as neurotransmitters. Collectively, these studies will determine cellular sources of IL22, the effect of ENS cells on IL22 secretion, the role of IL22 in enterocolitis, and the impact of Hirschsprung disease associated aganglionosis on epithelial cell biology. These studies build on the candidate’s training as a pediatric gastroenterologist, who has clinical exposure to the diagnosis and treatment of children with Hirschsprung disease and HAEC, as well as her basic science training in enteric nervous system biology. As the work proceeds, she will become an expert in mucosal immunology and epithelial biology with a focus on neuro-immune and neuro-epithelial interactions. The mentors, Dr. Robert Heuckeroth, and Dr. Kathryn Hamilton are experts in ENS biology and epithelial biology respectively. Both mentors have a strong commitment to mentorship and NIH funding track records. Experiments will be conducted at the Children’s Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania, a collegial, collaborative and state-of-the art institution. The professional development and training plan will position the candidate as a successful pediatrician-scientist, who is focused on the prevention and treatment of Hirschsprung associated enterocolitis. These studies should determine if IL22-based therapies would likely be successful in HAEC, and if a human clinical trial is appropriate.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Vascular calcification serves as the primary risk factor for predicting cardiovascular events. It involves arterial calcification, an actively regulated process mediated by cells, resembling physiological biomineralization but with impaired resorption. The vessel wall contains “calcifying vascular progenitor cells” that react to pro- calcific signals such as inflammation or infection. They then differentiate into osteoblast-like cells, which produce minerals and matrix in the vessel wall. The bulk of progenitor cells within the vasculature reside in the microanatomic progenitor niche, the tunica adventitia. Under normal conditions, these progenitors regulate vascular homeostasis and remodeling. The identity, location, function, and regulatory mechanism of adventitial progenitor cells in vascular calcification remain poorly studied. Further, uncovering the niche- specific role of adventitial progenitors in vessel calcification will guide the development of targeted therapeutic strategies. Recently, our integrated transcriptomic study on normal human blood vessel adventitia discovered a cell surface marker to typify adventitial progenitor cells. This previously undescribed marker in adventitial cell biology is Endothelial Protein C Receptor (CD201). CD201-expressing progenitor cells are spatially localized in the outer layer of the adventitia, and their expression level dictates the osteogenic potential of these cells. These recent observations raised questions regarding their role in the progression of vascular calcification and are comprehensively investigated in the present K99/R00 proposal. To achieve this, integrated spatial transcriptomics and single-cell RNA sequencing-based transcriptomic mapping of the human calcified vessel with implications of CD201+CD34+ adventitial cells in calcification will be initially performed. Additionally, reporter mice with nephrectomy-induced calcification will be also examined (Aim 1, K99 Phase). Secondly, a detailed in vitro functional characterization of FACS purified CD201High/Low cells from human calcified and healthy vessels will be performed, along with CD201 cell ablation in mouse calcification model to determine the functional role of the cell in calcification (Aim 2, K99 Phase). Lastly, the underlying signaling mechanism regulating CD201-expressing cells to be pro-calcific is investigated by CRISPR/Cas9 gene knockdowns, genetic mice calcification models, and integrated transcriptomics (Aim 3, R00 phase). Completing the proposal will greatly improve the knowledge of adventitial progenitor cell-mediated vessel calcification. The proposed research and training plan aligns with my long-term research objective. It will also significantly contribute to my scientific and career goals of establishing an independent research career in blood vessel resident stem cells and their role in vascular pathology.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cells (HSCs) maintain blood production throughout an organism’s lifespan. Thus, HSCs must balance differentiation with self-renewal to protect stemness. To this end, HSCs are largely quiescent. Quiescence protects HSCs from genotoxic insults and functional exhaustion. Understanding the molecular mechanisms controlling HSC quiescence will yield valuable insights into mechanisms underlying hematopoietic disorders. While post-transcriptional regulation is increasingly recognized as important for hematopoietic cell fate specification, its role in HSC quiescence remains poorly understood. Preliminary data indicate that the post- transcriptional regulator DDX6 is important to maintain HSC quiescence. DDX6, an RNA helicase, orchestrates translational suppression and mRNA sequestration in cytoplasmic condensates known as P-bodies. Notably, Ddx6 knockout mice have normal mature blood cell populations but exhibit loss of HSC quiescence. Accordingly, Ddx6−⁄− HSCs exhibit increased proliferation and mitochondrial numbers, which results in diminished fitness during serial, competitive transplants. Mechanistically, initial analysis of DDX6-targeted transcripts in P-bodies revealed an enrichment for untranslated mRNAs encoding crucial regulators involved in exiting quiescence. Together, these data lead to our central hypothesis that Ddx6-mediated RNA processing is pivotal in protecting HSC quiescence and function. Aim 1 will test the hypothesis that Ddx6 is required for in situ stress hematopoiesis by challenging Ddx6−⁄− HSCs in vivo using regenerative and infectious stressors. Aim 2 will elucidate the mRNAs translationally suppressed by Ddx6 in HSCs and characterize the HSC translatome in situ both with and without Ddx6 deletion. Additionally, we will investigate the functional role of Ddx6 targeted transcripts in vivo, specifically Myc. The overall goal of this project is to elucidate a new mechanism controlling HSC function at the molecular and cellular levels and to advance strategies for treating hematologic diseases. This fellowship application is sponsored by Bruno Di Stefano, Ph.D., an expert in post-transcriptional gene regulation in stem cells, and Katherine King, M.D., Ph.D., a physician-scientist and expert in hematopoietic stem cells, who will provide close guidance throughout the fellowship period. The training plan includes strategies to 1) Learn from accomplished scientists and physician-scientists that will advise the applicant through her training goals; 2) Undergo rigorous scientific training in hematopoiesis and gene regulation; 3) Experience opportunities to improve scientific communication skills and expand professional networks; 4) Advance the applicant’s clinical training, especially in hematology. The clinical and scientific training environment at Baylor College of Medicine is within the Texas Medical Center, the largest medical research complex in the world. This environment is ideal to foster the applicant’s scientific and clinical growth toward a career as a physician-scientist investigating the role of post-transcriptional regulation in hemopoietic stem cell function and dysfunction.

Emerson Foundation

Supports STEM education, community building, and basic human needs in Emerson plant communities.

New York State Education Department

STEM education initiative grants for NY to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Liver transplantation is the only curative treatment for end-stage and acute liver disease; however, the scarcity of donor organs underscores the urgent need for alternative therapies. Transplantation of human induced pluripotent stem cell (iPSC)-derived liver cells offer a promising approach to treat liver disease. The most desired cell type to transplant is the hepatocyte, the main functional cell of the liver, however, one limitation is the poor maturation of hiPSC-derived hepatocytes that prevent their clinical benefit especially in the case of acute liver diseases that require rapid replenishment of functional hepatocytes. An alternative is to transplant supportive cells that promote proliferation and function of the remaining hepatocytes. We will specifically leverage the known supportive role of liver endothelial cells in animal models for hepatocyte proliferation and function through paracrine signaling during liver repair. The goal of this proposal is therefore to evaluate the therapeutic potential of iPSC-derived endothelial cell (iEC) therapy for promoting liver regeneration in acute liver injury and elucidate the cellular and molecular mechanisms by which iECs support hepatocyte proliferation, repair and function. Using immunocompromised NSG mice, we will apply two complementary acute liver injury models: monocrotaline (MCT)-induced EC-specific damage and acetaminophen (APAP)-induced injury affecting both ECs and hepatocytes. Additionally, the master regulator of EC biology, VEGFA ligand, will be delivered via nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) to activate in vivo the KDR receptor on iECs, enhancing their engraftment, proliferation, and supportive function. Our preliminary data strongly support this approach: (1) We have established a protocol that efficiently generates a pure population of iECs that are able to engraft after delivery of biologically active VEGFA165 in vivo, and (2) our lab has optimized the mRNA-LNP platform for efficient and transient in vivo VEGFA165 delivery. Based on these data, our central hypothesis is that KDR activation with VEGFA mRNA-LNP in engrafted iECs will enhance their engraftment and restoration of liver function (Aim 1), through supportive endothelial factors that will promote the hepatocyte-iEC niche activation to induce hepatocyte proliferation and function, and hence to promote liver repair (Aim 2). This project will use single-cell RNA sequencing data analyses to identify and further validate critical paracrine factors secreted by iECs that mediate liver regeneration. Altogether, this study will establish the preclinical potential of iEC therapy for acute liver diseases, providing cellular and molecular mechanistic insights in this process and laying the groundwork for potential future applications in chronic liver injury. This research will be conducted at the Center for Regenerative Medicine (CReM) at Boston University, which offers cutting-edge resources for stem cell biology, advanced genomics, and in vivo disease modeling. The training plan integrates rigorous experimental work, mentorship, formal coursework, and professional development activities that will prepare the candidate for a successful academic career.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY This proposal establishes a partnership between the California State Polytechnic University, Pomona (CPP) and the University of California, Los Angeles (UCLA) to create a pipeline for a resource limited institution (RLI) serving many first-generation college students to pursue PhD programs in neuroscience. This career development program provides these students with valuable research training and exposure to an R1 research environment, addressing a critical need for highly skilled labor force in neuroscience and STEM fields. Students will begin their training at CPP, where they will engage in foundational research skills, including techniques in adeno-associated virus (AAV) applications at Cal Poly’s Armamentarium Vector Core (ArmVC). Over the course of a year (summer + academic year), they will gain hands-on experience in AAV production, purification, and capsid characterization, building skills and confidence in a familiar and supportive setting. In the 2nd summer in the program, the students will transition to UCLA to participate in intensive research experiences, applying their training to cutting-edge neuroscience projects—ideally bringing gene tools that they have prepared at the CPP ArmVC. While at UCLA, they will collaboratively use AAVs and other tools to address questions critical to PIs at UCLA, thereby benefiting UCLA faculty and projects. Upon returning to Cal Poly Pomona, students will continue their UCLA-related projects in a supportive role, ensuring continuity in their research and strengthening ties between the institutions. This may include additional AAV production, characterization of tissue samples from ongoing projects, or design and validation of novel AAVs to continue to delve deeper into a gene therapy related project. This program equips students with technical expertise, mentorship, and the experience of working in a high-level research environment, empowering them to pursue advanced degrees. By fostering a well-trained cohort of neuroscience researchers, this partnership creates a sustainable model for promoting excellence in STEM education and research.

NHLBI - National Heart Lung and Blood Institute

Project Summary / Abstract Sympathetic neurons (SNs) are critical regulators of cardiac output in response to hemodynamic stress and are known to contribute to heart development and cardiomyocyte (CM) maturation. SNs are also required for cardiac regeneration after myocardial injury in neonatal mammals but become dysregulated with adult heart injury, leading to increased risk of fatal arrhythmias. Despite documented importance, human neuro-cardiac interactions have been understudied by researchers due to the lack of high-fidelity in vitro models that both accurately recapitulate the physiology of cardiac innervation and enable mechanistic studies using cell-specific functional assessments. The Bursac Lab has previously generated highly functional, three-dimensional (3D) engineered cardiac tissues (“cardiobundles”) made from neonatal rat ventricular myocytes or human induced pluripotent stem-cell derived CMs (hiPSC-CMs). Building on these results, I have recently developed and characterized a compartmentalized 3D coculture model of human cardiobundles innervated with hiPSC-derived SNs (hiPSC- SNs). Over the 4-week coculture, this novel neuro-cardiac model supports robust axonal ingrowth into cardiobundle compartment without detriment to cardiac contractile or electrophysiological function. Importantly, CMs within cardiobundles reliably respond to stimulation of SNs by presynaptic neurotransmitters, indicating the development of functional sympathetic innervation. For the proposed project, I will utilize this human in vitro model to test two hypotheses that functional innervation of cardiobundles will: 1) promote developmental maturation of hiPSC-CMs and neurocardiac junctions and 2) stimulate the regenerative response of cardiobundles to injury, thus allowing for detailed studies of underlying mechanisms. To test these hypotheses, in Specific Aim 1 I will utilize gene and protein expression profiling, functional assays, and genetically encoded sensors of membrane voltage, calcium, and neurotransmitter release to assess the time-course of structural and functional CM maturation and robustness of formed neurocardiac junctions. In Specific Aim 2, I will apply cryoablation and CM-specific injury protocols in multicellular cardiobundle models to assess the effects of SN innervation on CM and non-CM proliferation, matrix remodeling, neurocardiac functional recovery, and molecular mechanisms underlying the resulting regenerative or pathophysiological responses. Overall, this work will establish the first tissue-engineered model of anatomically representative, highly functional innervated myocardium for in-depth studies of the human neurocardiac development and diseases of the brain-heart axis.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Rotator cuff (RC) tears are a leading cause of musculoskeletal impairment, affecting over 250,000 individuals annually in the United States. Despite surgical repair being the standard of care, failure rates remain alarmingly high, with up to 90% of cases experiencing re-tears or poor functional recovery. This failure is largely driven by irreversible muscle degeneration, including atrophy and fatty infiltration, which impair healing and compromise surgical outcomes. Current therapies primarily target the bone-tendon interface while neglecting muscle pathology, underscoring a critical need for regenerative strategies that directly address RC muscle degeneration. This proposal aims to develop a therapeutic approach leveraging lipid nanoparticles (LNPs) encapsulating WNT7a mRNA (W7a-LNP) to promote muscle regeneration and prevent degeneration following RC injury. WNT7a has been shown to increase muscle mass, enhance muscle stem cell (MuSC) expansion, and reduce fatty infiltration, but its recombinant protein form is limited by poor bioavailability and high production costs. W7a- LNP circumvents these limitations by enabling localized, sustained WNT7a production at the injury site, transforming muscle cells into `in vivo protein factories.' Our preliminary data demonstrate that W7a-LNP reduces fibro/adipogenic progenitor (FAP) adipogenesis and fatty infiltration in both in vitro and in vivo models. We will test the hypothesis that intramuscular delivery of W7a-LNP prevents and reverses RC muscle degeneration through three specific aims. Aim 1 will engineer and validate W7a-LNP as a targeted muscle regeneration platform by optimizing delivery, evaluating WNT7a expression kinetics, and assessing its effects on MuSC expansion, myofiber hypertrophy, and FAP adipogenesis in vitro and in vivo. Aim 2 will determine the efficacy of W7a-LNP in preventing RC muscle degeneration when administered at the time of injury using a clinically relevant delayed tendon repair model. Aim 3 will evaluate W7a-LNP's ability to reverse established muscle degeneration when delivered at later stages of injury, with or without mechanical loading, to assess potential synergistic effects. The impact of this research is the development of a scalable, translatable mRNA-LNP therapy that preserves and restores muscle quality in RC injuries. By addressing a critical gap in current treatment paradigms, this strategy has the potential to improve surgical outcomes, enhance functional recovery, and reduce the need for invasive salvage procedures, ultimately transforming the management of RC muscle degeneration.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Chronic idiopathic constipation is a functional gastrointestinal disorder that affects up to 1 in 5 children. As a pediatric surgeon, often the only effective treatments I can offer are intestinal resection or diversion. Thus, I strive to better understand the mechanisms that result in this cryptic disease. The K08 program has been an ideal foundation to develop the technical and scientific skills I need to make a translational impact for my patients. Through my career development award, I have been able to explore the effect of biomechanical forces on the enteric nervous system (ENS) as a driver of persistent bowel dysfunction in Hirschsprung disease. In the last few years, we have uncovered that the chronically distended, ganglionic intestine in Hirschsprung disease has altered tissue mechanical properties. We have shown that the biomechanical forces of stretch and stiffness impact enteric neuronal phenotype and function and we have tied these changes to the mechanoreceptor, Piezo1. Taken together, our preliminary findings suggest that bowel dysfunction may be driven by mechanically- induced changes in the ENS microenvironment. The concept that biomechanical forces regulate enteric neuronal behavior and intestinal function is applicable to many gastrointestinal diseases. However, it is difficult to discern the contribution of individual biomechanical forces and the response of different cell types to biomechanical force using in vivo models alone. Thus, we seek to leverage this R03 mechanism to develop new methods and new technology to study the effect of biomechanical forces on enteric neurons and glia in vitro. This proposal lays out a two-year plan to optimize a novel engineered three-dimensional (3-D) microfluidic, co-culture system to determine the impact of biomechanical forces on enteric neurons and glia. The current proposal builds on the premise of my K08, that biomechanical forces influence ENS behavior to affect intestinal function, with two aims: (1) to test the role of biomechanical forces (stretch and shear stress) on enteric neuronal morphology, neuroglial differentiation, and neuronal function; (2) to determine how neuroglial interactions shape the ENS response to biomechanical forces. This project will leverage an established organ- on-chip platform, enteric neuronal stem cell culture, in vitro modeling, and cutting-edge cellular and molecular biology techniques to achieve these goals. Completion of these aims will further my transition to independence as an investigator and lead to a new mechanistic understanding of gastrointestinal function with the potential to improve the quality of life for millions of adult and pediatric patients.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Invasive fungal infection by Aspergillus species, most commonly by Aspergillus fumigatus, remains one of the leading causes of mortality in immunocompromised patients undergoing solid organ or HSC transplantation because antifungal agents currently used in clinics are poorly effective in treating Aspergillus infection in patients with impaired immunity and neutropenia due to drug toxicities, drug-drug interactions, and the emergence of drug-resistant strains. Neutrophils provide the first line of defense against fungal infection. They effectively kill fungi by fungicidal oxidative bursts, presentation of fungal antigens to T cells, and by increasing fungal clearance by folding their hyphae and decreasing the spreading of infection with extracellular traps (NETs). However, the efficacy of neutrophil transfusions for A. fumigatus has been limited. Therefore, enhancing the antifungal properties of neutrophils is essential for advancing adoptive neutrophil transfer for treating Aspergillus infections. In this application, we aim to develop a novel therapeutic approach to effectively target A. fumigatus infections using induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) armed with anti-Aspergillus CARs. The major goal of the R21 phase is to provide proof of principle that iNeutrophils equipped with anti-Aspergillus CARs possess superior fungicidal properties. In Aim 1, we will identify the single-chain variable fragment (scFv) that is most effective in iPSC-derived neutrophils for targeting A. fumigatus. In Aim 2, we will characterize the anti-fungal potential of anti-A. fumigatus CAR-iNeutrophils in vitro. If the R21 milestones are achieved, we will advance the development of CAR-iNeutrophil therapies into the R33 phase by enhancing their antifungal potential and demonstrating their efficacy and safety in vivo. In Aim 3, we will identify the most effective CAR configuration and genetic modifications that enhance the fungicidal properties of iNeutrophils. In Aim 4, we will assess the efficacy and toxicity of CAR-iNeutrophils in vivo using zebrafish larvae and invasive pulmonary Aspergillosis mouse models. Overall, generating CAR-iNeutrophils that directly target fungal species will enable the development of a new class of antifungal therapies. These therapies will employ the adoptive transfer of readily available neutrophils with enhanced antifungal functions to treat life-threatening drug-resistant A. fumigatus infections in patients with neutropenia or dysfunctional neutrophils.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Autoimmune diseases impact ~25 million people in the United States and are increasing in prevalence. Autoimmune diseases are driven by a failure of immunological tolerance that triggers aberrant immune responses against self-antigens that can impart debilitating morbidities and even death. There are no cures for autoimmune disease and current treatments non-specifically blunt immune responses against both self- and non-self-antigen, requiring life-long treatment compliance that leaves patients more susceptible to infection and malignancy. We propose to develop a develop and test a new strategy for targeted treatment of autoimmune diseases that harnesses the intrinsic immunoregulatory properties of extracellular vesicles (EVs). EVs are secreted by all cell types as a mechanism for promoting transfer of molecules between cells and have been implicated in the maintenance or induction of immunological tolerance through their ability to deliver diverse immunoregulatory cargo. We hypothesize that EVs derived from immunosuppressive cell sources and engineered to deliver autoantigens can be employed as a tolerogenic vaccine (i.e., inverse vaccine) that promotes antigen-specific T cell tolerance that abrogates autoimmune disease. Towards this end, we have devised strategies for exogenous loading of peptide antigens onto EV surfaces, thereby enabling coordinated delivery of antigens and immunosuppressive EV cargo to antigen presenting cells (APCs), resulting in the presentation of autoantigen in a potently tolerogenic context. While our EV-based tolerogenic vaccine platform – tolEVax – is amenable to EVs isolated any cell source and can be applied to several autoimmune diseases, we will focus on engineering of EVs derived from mesenchymal stem cells (MSC-EVs) and will test our approach in a model of multiple sclerosis. We propose to establish tolEVax as a promising strategy for promoting immune tolerance and treating autoimmunity through two Specific Aims. In Aim 1, we will load MSC- EVs with peptide antigens, evaluate effects on antigen biodistribution and uptake by APCs, and characterize effects on antigen-specific CD8+ and CD4+ T cell responses to model antigens. In Aim 2, we will evaluate the capacity of tolEVax to inhibit autoreactive T cell responses and self-antigen mediated inflammation and pathology in a model of multiple sclerosis. We expect these studies to identify MSC-EVs as potently tolerogenic antigen nanocarriers, to provide new insight into how EVs modulate adaptive immune responses, and to demonstrate the efficacy of tolEVax as a potential treatment for MS. Overall, this research will result in a platform technology that addresses the unmet need for effective antigen-specific immunotherapies for autoimmune disease by exploiting the inherent and multimodal immunosuppressive functions of EVs.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Chronic inflammation is a central factor in a major fraction of all human disease burden. In chronic inflammation, immune cells mediate a dysregulated inflammatory response that persists despite the removal of the initial insult, suggesting stable, steady-state dynamics between immune and tissue cells. Macrophages are key players in this process, mediated by their status as central effectors of tissue inflammation and their high plasticity for both inflammatory and anti-inflammatory states, as well as tissue-specific functionality. This process of transition from a healthy to a chronically inflamed tissue steady state involves competition between trafficking and invading macrophages from the periphery vs. resident ones, conversion between inflammatory states, and crosstalk with neighboring tissue cells that is hypothesized to be tissue-specific. Existing tools for studying chronic inflammation are limited in their ability to provide a mechanistic understanding of these processes. They use overly high doses of inflammatory agents, which cause non- specific responses with little predictive value. The macrophages used are not authentic resident cells; they lack the correct epigenetic, immune effector, and metabolic characteristics, and their polarized states are poorly defined. Furthermore, these models fail to account for tissue-specific inflammatory responses, leading to inaccurate, non-physiological results that misrepresent the therapeutic window for chronic diseases. The overarching goal of this project is to develop tools that allow us to manipulate and study inflammatory state space in complex human tissues with a high degree of control. Aim 1 focuses on assembling immunocompetent human brain, liver, and adipose microtissues from pluripotent stem cell-derived progenitors. We will quantify resident macrophage expansion versus monocyte infiltration during inflammation initiation while benchmarking tissue structure and immune responsiveness. Aim 2 will establish advanced optical and genetic tools to continuously track transitions between basal, inflammatory, and reparative tissue states. We will combine fluorescence and label-free imaging techniques (e.g., fluorescence-lifetime and second harmonic generation) with engineered genetic reporters to generate high-density time-series data, identifying links between macrophage functions and inflammation divergence. Aim 3 will solve the problem of nonphysiological inflammatory cues by creating stable and inducible genetic switches to control macrophage activation states orthogonally and in situ within human microtissues. We will assess how these cells influence tissue structure, signaling, and the trajectory of inflammatory state, as well as test whether chronic tissue remodeling can be driven or reversed through specific pathway regulations. The successful development of this platform will provide the first human system to distinguish the roles of resident and infiltrating macrophages, track their real-time impact on tissue trajectories, and define the requisite physiological parameters needed to stabilize or reverse chronic inflammation.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Dysregulated inflammation is a central driver of a significant fraction of all human diseases, including chronic metabolic conditions, tumor metastasis, autoimmune disorders, and aging. Despite numerous advancements in tissue engineering and disease modeling, our ability to accurately capture cell-mediated tissue inflammation in vitro that faithfully mimics in vivo human physiology remains limited. Here, we propose to combine human pluripotent stem cell (hPSCs) and genetic engineering tools to create immunocompetent tissue models that replicate the natural behavior of tissue-resident macrophages with on-demand control over inflammatory states. Specifically, we suggest an immunoengineering strategy to generate human microtissues in a dish containing bona fide resting tissue-resident macrophages. We aim to accomplish this using a “progenitor- based assembly” tissue engineering approach, where myeloid progenitors derived from hPSCs are combined with their developmentally matched tissue and vascular counterparts. Target model systems for this research will include skeletal muscle, liver, and adipose microtissues, as these are all known to exhibit dysregulated inflammation in the context of metabolic diseases—a globally pressing clinical need. Along with strategies to generate and characterize these tissues, we propose to establish bioprocessing procedures to enhance the scale and ability to cryopreserve key progenitors that will enable the dissemination of these tools to labs focused on tissue inflammation research but lack expertise in tissue engineering or stem cell biology. With the development of these microtissues, we then aim to develop and employ genetic engineering tools to overcome known limitations in conventional controllable gene induction systems that are not functionally compatible with hPSCs and their derivatives. Using these tools and a novel lineage tracing and retrieval approach, we further propose to develop multi-lineage CRISPR/Cas9 screens that will identify cell-mediated inflammatory mechanisms that modulate neighboring metabolic tissue cells – the core essence of immunoregulation. Additionally, we will employ these approaches to drive cell-mediated inflammatory and anti-inflammatory states within tissues, overcoming challenges associated with the limited efficacy of in vitro macrophage via recombinant cytokines. Lastly, we will explore using our tool sets to evaluate adoptive hPSC-macrophage transfer strategies as a test bed for immunotherapy development. As part of this effort, we will test hypotheses regarding the “open niche” dependency for successful transfer, the long-term fate and durability of macrophage phenotypes after transfer, and develop additional, more therapeutically relevant genetic tools to modulate these processes. This research will ultimately deepen our understanding of tissue–macrophage biology, create new tools for studying immunoregulatory processes, and develop putative therapeutic strategies for metabolic and inflammatory diseases. Collectively, we aim to greatly expand our understanding of macrophage biology and establish transformative new tools for stem cell-derived tissue modeling and regenerative immunotherapy.

NHLBI - National Heart Lung and Blood Institute

Project Summary: Cardiovascular diseases (CVDs) such as myocardial ischemia/reperfusion (I/R) injury lead to extensive cardiomyocyte death and subsequent reduced cardiac function. Due to the human adult heart’s limited regenerative capacity, there is a significant need for exogenous cardiac function restoration post-I/R. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a favorable cell source to restore myocardial function. While promising, the therapeutic potential of hPSC-CM transplantation is hampered by low cell survival and inadequate integration with host myocardium. Thus, our lab has developed nanowired human cardiac organoids composed hPSC-CM, primary human adult cardiac fibroblasts, endothelial cells, stromal cells, and electrically conductive silicon nanowires (e-SiNWs). Our in vivo data shows that organoids are capable of robustly engrafting and providing functional recovery (69% Fractional Shortening recovery) in I/R injured rat hearts, while using 10% of the cell dose (1E6, 1x106 cells/rat) that other hPSC-CM transplantation studies used (1E7, 1x107 cells/rat). Additionally, to alleviate major histocompatibility class (MHC) mismatching to provide a translational platform for hPSC-CM delivery, our lab developed isogenic cardiac organoids, composed of hPSC-CMs, -cardiac fibroblasts (hPSC-cFBs), and -endothelial cells (hPSC-ECs) from a single hiPSC cell line. The goals of this proposal are to 1) investigate the impact of e-SiNW geometry on isogenic organoid function, and 2) demonstrate the therapeutic efficacy of optimized nanowired isogenic cardiac organoids in a rat I/R injury model. The central hypothesis of this proposal is that engineered nanowire surface geometry will enhance interactions between host myocardium and transplanted hPSC-CMs within isogenic cardiac organoids, resulting in efficient engraftment and significant functional recovery. The innovation of this proposal is engineering e-SiNW surface roughness to optimize the efficiency of myocardial engraftment, and thus the functional recovery of I/R-injured hearts. My long-term goal is to leverage translational engineering and informatics to develop a clinically viable cardiac cell therapy heart repair. Accordingly, we will pursue the following two aims: 1) investigate the effects of e-SiNW geometry on isogenic cardiac organoid function, and 2) determine the therapeutic efficacy of optimized nanowired isogenic cardiac organoids to treat I/R-injured rat hearts and investigate e-SiNW graft-host interactions using spatial transcriptomics. The proposed research would provide a translational platform for cardiac repair with efficient engraftment.

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

Project summary Infertility affects approximately 15% of the global population, and current assisted reproductive technologies fail to address the needs of several populations, including individuals who cannot produce viable gametes and same-sex couples seeking biologically related children. In vitro gametogenesis (IVG), and specifically, producing egg cells (oocytes) from stem cells, offers a promising solution for these underserved groups. The reconstituted ovary (rOvary) system, currently only possible in mice, combines embryonic day (E) 12.5 ovarian somatic cells with developmentally younger E9.5 stem cell-derived primordial germ cell-like cells (PGCLCs). However, only 1-3% of PGCLC-derived oocytes achieve developmental competence, presenting a significant barrier to clinical translation. This developmental mismatch between PGCLCs and their somatic environment may contribute to this low efficiency. My preliminary data shows that E12.5 primordial germ cells (PGCs), which comprise 5% of germ cells in the rOvary, achieve key developmental milestones more effectively than PGCLCs. Specifically, PGCs complete meiotic prophase I earlier, establish timely interactions with supporting granulosa cells and produce significantly larger oocytes and follicles than PGCLCs. To address these limitations, I will: 1) Compare the progression of PGCs and PGCLCs through meiotic prophase I using immunofluorescence and single-cell RNA sequencing; 2) Assess oocyte-granulosa cell communication during follicle formation through protein expression dynamics and transcriptional profiling; and 3) Implement an established protocol using retinoic acid and BMP2 to mature PGCLCs to an E12.5-like state before rOvary formation, potentially enhancing their developmental potential and reducing chromosomal asynapsis. This research will be conducted at UCLA under the mentorship of Dr. Amander Clark, a world-renowned expert in stem cell biology and in vitro gametogenesis. Success in this project could significantly improve the efficiency of in vitro oocyte generation, advancing our understanding of germline development and the potential clinical applications of IVG technology. This work represents a crucial step toward expanding reproductive options for individuals facing infertility and broadening access to biological parenthood for currently underserved populations.