Search Grants — Free, No Account Required

NEI - National Eye Institute

PROJECT SUMMARY IMPG2 is a crucial extracellular matrix protein for maintaining photoreceptor structure and function. Mutations in IMPG2 are linked to two forms of visual impairment: juvenile-onset rod-cone dystrophy and adult-onset vitelliform macular dystrophy. While no treatment currently exists, packaging IMPG2 amino acid sequences into adeno-associated viruses (AAVs) offers promise for a sight-preserving therapy. We recently generated human retinal organoid (RO) and mouse models to enable us to rapidly engineer a gene augmentation therapy for IMPG2-associated retinal dystrophy (RD). The retinal organoids (ROs), grown from either patient-derived (human) induced pluripotent stem cells (hiPSCs) or gene-edited embryonic stem cells (hESCs), recapitulate the lack of photoreceptor outer segments observed in advanced IMPG2-RD. This fully penetrant phenotype provides a biomarker for assessing functional IMPG2 expression after AAV-mediated gene transfer. Although patient-derived ROs are tractable in vitro models of clinical relevance, their use in assessing viral vector designs for gene therapy development is best complemented by in vivo assessment of safety and efficacy in animal models. Accordingly, we will assess therapeutic safety and efficacy using the Impg2-knockout (KO) model mice, as these mice exhibit gliosis, subretinal deposits, photoreceptor degeneration, retinal detachment, and reduced electroretinogram (ERG) responses that are similar to the human condition. Here, we will accelerate a preclinical program to test our central hypothesis that gene augmentation can prevent retinal pathology in an IMPG2-RD mouse model and patient-derived ROs. To lay the groundwork for a clinical IMPG2 gene therapy, we will complete the following Aims: (1) Use Impg2-KO mice to define endpoints for preclinical trials, (2) optimize a gene therapy viral vector design using Impg2-KO mice and IMPG2 patient-derived and gene-edited ROs, and (3) establish preclinical gene therapy safety and efficacy in Impg2-KO mice. Synergistically employing mouse models and human ROs will accelerate the development of a gene therapy that will meaningfully improve the lives of individuals with IMPG2-RD. More broadly, this work will demonstrate the power of a dual-model platform to advance safe and effective therapeutics with high predictive value for treating inherited retinal disorders.

NIA - National Institute on Aging

The epigenome controls cell type-specific gene expression, establishing the diversity of cell types in the human body. However, over time, the epigenome becomes dysregulated, which promotes aging. Despite the tight link between epigenetics and aging, the mechanisms that preserve the epigenome in young cells and why these mechanisms degrade over time remain poorly understood. Polycomb-mediated gene repression maintains cell identity by silencing the genes that specify other cell types. As facultative heterochromatin, Polycomb is highly dynamic during development, enabling differentiating stem cells to rapidly alter gene expression programs. However, Polycomb switches from being flexible during development to becoming a stable mechanism of repression throughout adulthood. Understanding Polycomb regulation is key to advancing our knowledge of aging, as disrupting Polycomb components alters lifespan across various organisms. How Polycomb repression is maintained in terminally differentiated cells remain unknown. However, studies in embryonic stem cells indicate that spatial organization of repressed sites is crucial, with Polycomb-repressed regions forming ultra-long-range loops to sustain silencing. While these loops were thought to be solely mediated by Polycomb complexes, preliminary work from the applicant shows that cohesin and CTCF (which facilitate long-range enhancer-promoter loops) also mediate repressive loops in embryonic stem cells. In the F99 phase of this proposal, performed at MIT, the applicant will use computational methods developed by the Mirny and Dekker labs to determine whether cohesin and CTCF-dependent looping is a broad regulatory mechanism of Polycomb repression. Aim 1.1 will identify Polycomb targets in embryonic stem cells that derepress when cohesin or CTCF is lost and Aim 1.2 will use mechanistic polymer modeling to link cohesin and CTCF’s roles in 3D looping activity to Polycomb repression. In Aim 1.3, machine learning and polymer modeling will predict how gene expression in different cell types, particularly mature hepatocytes, respond to site-directed CTCF perturbations. These insights will propel the applicant’s transition to aging research, where she will test whether enhancing cohesin activity can protect Polycomb repression in aging mouse livers (Aim 2). The K00 phase will also use cutting-edge and single-cell experimental techniques to measure genome re-organization as Polycomb becomes dysregulated during the normal aging process. In addition to training in machine learning and hepatic chromatin, the applicant will gain expertise in aging research during the F99 stage through lab visits, conference attendance, and a course on aging and its diseases. This study will advance our understanding of aging by comprehensively investigating a new mechanism of Polycomb regulation. Rejuvenating the epigenome is a promising strategy for reversing cellular aging, and this work will determine if targeting the 3D genome offers a new approach.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Human mesenchymal stem cells (hMSCs) can be isolated from various adult tissues including bone marrow, fat tissues, dental pulp and synovium, and exhibit therapeutic promise for tissue repair, regeneration, and treatment of chronic immune and degenerative diseases due to their multipotent differentiation ability, trophic factor secretion, and immunomodulatory functions. However, their clinical efficacy remains limited by two barriers: (i) immune-mediated clearance of transplanted cells and (ii) poor retention and engraftment within injured tissues. Even autologous or well-matched allogeneic hMSCs are frequently recognized as “non-self” after their transplantation, leading to rapid cell loss before therapeutic benefits are realized. Overcoming immune rejection and microenvironmental insufficiency is therefore critical to advance hMSC-based therapies. Systemic immunomodulators can partially mitigate host rejection but often cause off-target effects, while conventional scaffolding like hydrogels provides structural support yet rarely influences immune responses. To address these limitations, we propose a light-activated hydrogel platform that integrates local immunomodulation with in situ scaffolding under a single, spatiotemporally defined stimulus. To achieve this objective, we will pursue two aims: 1) design, synthesis and characterization of BEPA-photocaged hydrogel complex for controlled immunomodulator and hydrogel formation, and 2) preclinical evaluation of BEPA- photocaged hydrogel complex for immune-permissive and ECM-mimetic stem cell engraftment. The proposed platform is expected to establish a “regenerative niche” that is both immune-compatible and mechanically supportive, thereby improving hMSC survival, retention, and integration. The approach is innovative in three ways: (i) a light-activated photocage enabling precise, on-demand immunomodulation, (ii) an injectable, light- triggering hydrogelator that surpasses conventional scaffolds, and (iii) a unified near-infrared (NIR) control axis that simultaneously regulates immune modulation and scaffold formation. By converting engraftment into a programmable event, this strategy provides effectively spatiotemporal control over stem cell transplantation. The anticipated outcome is a safer and more effective platform for regenerative medicine with broad translational potential.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Barrier epithelia face continual damage from environmental insults, and successful injury repair is crucial for organismal health. A prime case study is the one-cell-thick intestinal epithelium, which forms a leakproof bar- rier between the gut lumen and the body cavity. To replace damaged cells, the intestine mobilizes stem cells to divide rapidly; to restore intestinal form and function, these new daughter cells must also differentiate rapidly. Indeed, injury-born intestinal cells acquire their mature identity twice as fast as their normal counterparts. Using the Drosophila adult intestine, we recently discovered this injury-accelerated differentiation arises through disruption of Notch-Delta lateral inhibition circuitry that normally specifies stem versus terminal fate. During injury, many newly born cells exhibit >10x faster Notch signaling speed, which propels faster intestinal differentiation to restore the breached epithelial barrier. Yet this strategy comes with risks for long-term tissue health: For stem cells, loss of Notch-Delta feedback during injury skews daughter fates toward dead-end, ter- minal:terminal outcomes, which depletes the organ’s stem cells and culminates in stem cell exhaustion. For terminal progeny, accelerated differentiation yields provisional ‘stopgap’ cells—mature cells with digestive and barrier-forming functions but altered morphology and a supercompetitor-like transcriptomic profile. Here, we will investigate how the organ copes with these two tradeoffs. We combine physiological injury of the fly gut with in vivo live imaging and cutting-edge cell lineage tracing to elucidate how these ‘side effects’ of accelerated differentiation are managed at the organ-scale for post-injury tissue homeostasis. The fly gut com- bines conserved intestinal cell lineages, fate signals, and digestive physiology with supreme experimental trac- tability: A single Notch receptor and Delta ligand, an unparalleled wealth of genetic tools, and long-term in vivo live imaging—pioneered by our lab—provide the technical bases for deep mechanistic investigation. Leveraging these strengths, in Aim 1 we will define how some, ‘escaper’ stem cells persist after injury, de- spite disrupted Notch-Delta feedback that should force all cells to differentiate. We will test if escaper stem cells inherit an intracellular Notch inhibitor, autonomously override how Notch and Delta interact, or lose con- tact with their signaling partners via injury-induced epithelial fluidization. In Aim 2, we will ascertain the time- evolution and function of stopgap cells during and after injury. We will determine their ultimate fates in the tis- sue during recovery, e.g., they may evolve into normal cells, arrest in an abnormal state, or simply be shed. We will parse these scenarios using longitudinal live imaging, whole-population analyses, and single-cell tran- scriptomics. Finally, we will examine how stopgap cells shape the tissue post-injury by ‘purging’ unfit, toxin- exposed cells during injury or by exerting selective pressure on new cells during post-injury recovery. By probing these lineage tradeoffs of accelerated cell differentiation during injury, our work will suggest new strategies to promote intestinal regeneration and combat chronic intestinal disease.

NCI - National Cancer Institute

Abstract Breast cancer immunotherapy efficacy is still very limited. Thus, there is a pressing need to understand molecular determinants enabling clinically distinct breast cancers to suppress antitumor immune response and harness these mechanisms for novel treatments. Emerging evidence suggests that oncogenic mutations can directly adversely affect tumor immune responses. However, such functions for vast majority of breast cancer mutations have not been explored. In this proposal, we focus on aggressive breast tumors carrying combinations of most frequent breast cancer driver mutations, as a proof of principle of a new and customizable platform of clinically relevant cancer models to understand immunosuppressive mechanisms. MLL3 (also known as KMT2C), encoding a histone methyltransferase, is a novel tumor suppressor in various human cancers. MLL3 is frequently inactivated by gene deletions or truncating point mutations, with especially high rate in breast cancers (up to 24%). Additionally, MLL3 mutant cancers have significantly worse outcomes compared to MLL3 wild type cancers. We have developed a novel method for rapidly generating genetically engineered mouse models (GEMMs) by efficiently expanding and “custom genome editing” mouse mammary stem cells (MaSCs) in culture and then using these MaSCs to regenerate genetically engineered mammary glands in syngeneic immunocompetent mice. Using this model of MLL3 deletion in conjunction with constitutive activation of PI3-kinase (PI3KCA, ~60% of MLL3 mutant tumors in patients are also PI3KCA) and inactivation of p53 (these three mutations altogether account for the most frequent combinations of cancer driver mutations in human breast cancers), we found that the loss of Mll3 promotes early infiltration of Foxp3+ regulatory T (Treg) cells and their further expansion and differentiation to a highly suppressive phenotype, leading to faster tumor immune escape in primary tumors and at metastatic sites. Monoclonal antibody targeting of specific immune receptors highly expressed on tumor-infiltrating Treg cells show remarkable efficacy in inhibiting tumor initiation and growth. Based on these findings, we next propose to investigate how MLL3 loss mechanistically activates HIF1 and harness the understanding to develop therapeutic interventions applicable to aggressive human breast cancers. We will explore underlying mechanisms of Treg cell differentiation into highly suppressive effector Treg cells in the tumors, mediated by both extracellular cues and cell-intrinsic regulators. Thus, in addition to uncovering new mechanisms by which major breast cancer drivers favor early immune escape, the power of our approach can be easily extended to test the tumor promoting effect of any breast cancer mutations, singly or in combination. The mechanistic investigations of these complex tumor-immune system interactions in vivo require the use of similarly complex in vivo models that closely recapitulate tumor development in an immunocompetent host environment in which tumors arise. To our knowledge, mice represent the most cost effective and best tractable mammalian models available to us.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Adult stem cells are progenitor cells capable of tissue regeneration during life through the ability to both self- renewal and to produce specialized cells upon division. Stem cells reside in microenvironments called “niches” that integrate systemic cues and provide signals for stem cell maintenance. Over the years, the use of the stem cell systems present in Drosophila melanogaster has revealed mechanisms that control stem cell niches in homeostasis and pathology. Recently, a model has emerged pointing to a strong conserved correlation between lipid accumulation and stem cell loss. Given the power of Drosophila genetics, the readily accessible molecular tools, the well-characterized stem and niche cell populations, and the high degree of evolutionary conservation in metabolic genes, the fly testis niche is an ideal model for the intersectional study of metabolism and stem cell biology in physiological and pathophysiological conditions. Our long-term goal is to understand how changes in lipid metabolism affect stem cell niche homeostasis. The PI’s published works build a model where the ectopic accumulation of lipids in the fly testis niche is detrimental to stem cell function. Excess lipid accumulation in stem cells led to their loss through differentiation. Accordingly, lipid accumulation has been shown to be detrimental to stem cell maintenance across species. The overall objective of this proposal is to understand mechanistically how the stem cell niche is affected by conditions that trigger ectopic accumulation of lipids. Preliminary data in this proposal show that niche (hub) cells are also sensitive to lipid accumulation, and that multiple mechanisms are likely at play to control lipid levels in the testis stem cell niche. Of note, preliminary data in this proposal show for the first time that lipid metabolism controls somatic cell fate in the testis by inducing conversion between niche and somatic stem cells. Hence, our central hypothesis is that lipid accumulation promotes loss of stem cell niche homeostasis. We will test this hypothesis through three specific aims: 1) determining how microenvironmental stiffness impacts lipid anabolism and niche homeostasis; 2) characterizing the role of apolipoproteins in fat-transporting and stem cell maintenance; and 3) investigating the role of lactate transport (a precursor in lipogenesis) in niche and stem cells. The merit of this study relies on its novelty – showing that changes in lipid metabolism can promote the conversion between a niche and a stem cell – and on the generation of a useful paradigm for testing how pathophysiological changes in lipid metabolism yield in loss of stem cell niches. Given the high incidence of metabolic disorders in the population, understanding how lipid accumulation affects stem cell niches is pivotal for the development of novel stem cell-based therapies, especially those targeting metabolic disorders. The proposed studies will also strengthen the research environment at the University of Louisville, providing opportunities for the training of postdoctoral fellows, graduate and undergraduate research assistants in the laboratory.

NIA - National Institute on Aging

Abstract Epigenetic clocks based on DNA methylation have revolutionized aging research, yet they offer only static snapshots of a highly dynamic process. To overcome this limitation, we will develop novel live-cell imaging tools to monitor biological age in real time based on age-associated DNA methylation changes. Two classes of genetically encoded fluorescent sensors will be engineered: (1) a “ticking” global methylation epigenetic clock (T-EpiC) that continuously visualizes intranuclear 5mC patterns, and (2) an “alarm” locus-specific epigenetic clock (A-EpiC) that develops fluorescence only when methylation occurs at specific aging-associated loci. The corresponding specific aims are to develop a method for quantifying the biological age of live cells by visualizing intranuclear DNA methylation patterns and to develop a turn-on fluorescent sensor that reports DNA methylation at specific aging-associated loci in live cells. By integrating real-time imaging with machine learning-based pattern recognition, we will quantify aging trajectories and detect interventions that modulate the biological age of cells. The novel tools will be validated in primary fibroblasts and induced pluripotent stem cells under various aging and rejuvenation conditions. Our approach addresses critical gaps in the field by enabling tracking of epigenetic aging in living cells—an advance that opens unprecedented possibilities for identifying rapid responses to anti-aging interventions, elucidating heterogeneity within cell populations, and studying the reversibility of aging processes. Ultimately, these technologies have the potential to transform our understanding of aging and accelerate the development of therapies for age-related diseases.

NIGMS - National Institute of General Medical Sciences

Abstract The overall theme of my research program is to understand how enhancers and transcription machineries organize in the 3D genome to regulate the target gene expression. We plan to address this problem from two aspects: 1) at the single-cell level, we will characterize the function and dynamics of transcriptional condensates in mouse embryonic stem cells and investigate the specificity of the condensate, the formation and dissolution mechanisms, and their activity regulated by genome architecture; 2) at the developmental level, we will study the mechanism of how transcriptional condensates achieve differential regulation of target genes during development using the beta-globin regulation in mouse erythroleukemia cells as a model. In the past years, we have made significant contributions in the first direction, and we excited to extend our research to a new direction. New methods were developed and insights generated from these studies form the foundation of this proposal. Theme1: In eukaryotes, RNA Pol II, Mediator and transcription activators form transcription-dependent condensates in live mouse embryonic stem cells and enhance super-enhancer controlled gene bursting in a proximity-dependent manner. However, molecular mechanisms of how condensates function on gene bursting within the 3D genome is not fully resolved. The long-term goal of this theme is to characterize the condensate specificity, to investigate their mechanisms of formation and regulation by enhancers/enhancer RNAs, and to develop a full mechanistic understanding of how they interact with their target regulatory elements. In the next five years, we plan to 1) test whether transcriptional condensate can co-activate a reporter gene with different promoter specificity; 2) develop a labeling strategy to image and study the roles of enhancer RNAs in condensate function; 3) selectively manipulate chromosomal interactions and test how condensate-gene interactions and gene bursting change. Theme 2: The dynamics and functions of transcriptional condensate have not been explored extensively due to their close-to-diffraction-limit size. The long-term goal of this theme is to characterize the composition, dynamics and functions of transcriptional condensates and their effect on beta-globin gene switching during erythroid differentiation, aiming to test the function of condensates across different systems. In the next five years, we plan to: 1) test whether and how transcriptional condensate dynamically controls beta-globin gene bursting; 2) develop a method to probe the condensate composition and its changes during erythroid differentiation. We envision that our study will provide significant insights and open new research directions on gene regulation.

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

ABSTRACT Organ development requires precise spatiotemporal regulation of various signaling factors. While much is known about how genetically encoded signaling ligands are regulated, less is known about lipid and small molecule signaling ligands. One such class of signaling molecules critical for development across the animal kingdom is signaling isoprenoids, which include Retinoic acids and Juvenile hormones (JHs). Our lab overcomes challenges in detection, redundancy and pleiotropy prevalent for these developmentally essential isoprenoids by harnessing the wealth of tools to identify and manipulate individual cells in Drosophila melanogaster. We generated various mutants lacking JH-related factors including a JH synthesis enzyme, two classes of degradation enzymes, and transcription factors, as well as fluorescent JH reporters to investigate JH signaling dynamics. Here, I will leverage these tools to understand how local and dynamic regulation of signaling isoprenoids facilitates the development of two organs: the ovary and testis. The Drosophila embryonic gonads provide an ideal model due to a wealth of validated gonadal markers and drivers. Preliminary data by us and others suggest JH ligand availability is differentially regulated in the embryonic ovary and testis, though this has not been experimentally verified, nor are JH functions in gonad development known. I recently found that both JH synthesis and degradation enzymes are required for female and male fertility. Using our JH reporter, we found that JH signaling is dynamic during juvenile ovary development yet highly restricted in the male germline stem cell (GSC) niche throughout juvenile and adult stages. During this F31 training fellowship, I will test the hypothesis that precise, local and sex-specific JH signaling in the embryonic gonads is achieved through active JH degradation in non-niche testicular cell types to facilitate male GSC niche development and male fertility. Through successful completion of experiments outlined in this proposal, I will determine a) when and how JH signaling first becomes differentially regulated in the gonad, b) the function of JH signaling in testis niche development, and c) which genes are regulated by JH signaling in embryonic gonads. To accomplish these goals, I will combine our lab’s genetic tools to detect and manipulate JH in individual cell types with immunohistochemistry, RNA FISH, confocal microscopy, fluorescence-activated cell sorting, CRISPR- mediated homologous repair, qPCR, and single-cell RNA sequencing. Thus, this F31 fellowship will not only provide a rich training platform to prepare me for my ultimate goal of becoming an independent researcher in the field of reproductive development, but it will also reveal fundamental insights into how local regulation of signaling isoprenoids facilitate organ development.

Lockheed Martin Foundation

Funds STEM education, cybersecurity workforce development, and veteran support programs at scale.

U.S. National Science Foundation

The Louis Stokes Alliances for Minority Participation (LSAMP) program invests in the Nation's colleges and universities to aid student success to create a new generation of STEM discoverers for the national STEM enterprise. The program takes a comprehensive approach to the STEM Learning Ecosystem to impact STEM student development and retention. LSAMP is an alliance-based program, whereby a group of institutions of higher education (IHEs) work together to diversify the nation's science, technology, engineering, and mathematics (STEM) workforce by increasing the number of STEM baccalaureate and graduate degrees awarded to persons from LSAMP populations. LSAMP populations are defined as persons from groups underrepresented in the STEM enterprise: Blacks and African-Americans, Hispanic and Latino Americans, American Indians, Alaska Natives, Native Hawaiians, and Pacific Islanders. The LSAMP program provides funding to alliances that implement comprehensive, evidence-based, innovative, and sustained strategies that ultimately result in the graduation of well-prepared, highly competitive students from LSAMP populations who pursue graduate studies or careers in STEM, while also supporting knowledge generation, knowledge utilization, assessment of program impacts, dissemination activities and dissemination of scholarly research into the field. Projects supported by the LSAMP program include: --Alliance Development Grants (ADG) support the conceptualization and development of new B2B and new SPIO alliances. (New) --Bridge-to-the-Baccalaureate (B2B) alliances facilitate the successful transfer of students from LSAMP populations to four-year institutions in pursuit of STEM baccalaureate degrees. --STEM Pathways Implementation-Only (SPIO) alliances are designed for new and reconstituted alliances. These projects focus on building and strengthening strategies and approaches to assist Institutions of Higher Education (IHEs) increase STEM baccalaureate degrees to LSAMP populations and facilitate entry into STEM graduate degree programs. --STEM Pathways Research Alliances (SPRA) are designed for well-established alliances. These projects serve as models of excellence in STEM broadening participation by (1) steadily increasing STEM baccalaureate degrees to LSAMP populations and facilitating entry into STEM graduate degree programs; (2) producing and disseminating new scholarly research on the broadening participation of LSAMP populations (or underrepresented and underserved populations in STEM disciplines and the nation's STEM workforce) and, (3) holistically assess the state of institutionalization and sustainability of the alliance. --Bridge to STEM Graduate Degrees in National Priorities (BD-Master's) projects support cohorts of six graduate students pursuing a M. S. degree in STEM national priority areas, providing financial support (stipends and cost of education) and support to help develop and maintain academic and research skills that enable participants to successfully persist in STEM graduate degree programs at Master's comprehensive-degree producing institutions only. (New) --Bridge to STEM Graduate Degrees in National Priorities (BD-Doctoral) projects support cohorts of twelve graduate students pursuing a Ph.D. degree in STEM national priority areas, providing financial support (stipends and cost of education) and support to help develop and maintain academic and research skills that enable participants to successfully persist in STEM doctoral degree programs. --STEM Networking Incentives and Engagement (NETWORKS) projects provide support to incentivize the creation and participation of LSAMP populations in STEM networks. (New)

NCI - National Cancer Institute

Project Summary/ Abstract Chemotherapy-induced pulmonary fibrosis (PF) represents a major challenge in the treatment of cancer patients. Drugs that are most associated with PF development include bleomycin (BLM), methotrexate and oxaliplatin. Despite the emergence of novel compounds for the symptomatic treatment of PF, lung transplantation remains the only "curative" option, albeit with a low 5-year mean survival rate following transplantation. Furthermore, due to co-morbidities or concern for cancer relapse, many patients do not qualify for lung transplantation, and many that are qualified die while on the waiting list. This highlights the urgent need for novel therapies, with special emphasis on emerging approaches for transplantation of lung stem cells. We propose a simple and robust approach for lung regeneration by lung cell transplantation, based on our extensive proof-of-concept studies in two distinct mouse models of lung fibrosis, including the more relevant BLM mouse model, which demonstrate that a simple i.v infusion of lung single cell suspension, leads to robust engraftment of donor-derived alveolar and endothelial cells, reduces the progression of fibrosis, and improves lung function. Our data have led to the formation of a new company (Lungevia Bio Inc.) which will form a strategic partnership with the PI , aiming at developing the final pre-clinical data required for attaining CMC and an IND from the FDA for a first-in-human clinical trial in cancer patients with chemotherapy-induced lung fibrosis (Aim 1). Considering that this approach is based on transplantation of MHC disparate cadaveric lung cells requiring chronic immune suppression, and based on our Preliminary Results, further experiments in the BLM mouse model will interrogate the possibility that infusion of allogeneic lung cells with bone marrow cells from the same donor could induce immune tolerance towards donor antigens and allow cession of immune suppression (Aim 2). The proposed studies require vertebrate animals because the mechanisms of lung remodeling, pulmonary function, and stem cell engraftment can only be evaluated in the context of an intact living organism. Mice were selected because they are well-characterized vertebrate models that enable precise investigation of gene function and therapeutic interventions. Furthermore, treatment of mice with bleomycin induces pulmonary fibrosis similarly to that found in cancer patients treated with different chemotherapy agents .These studies cannot be adequately performed using in vitro systems, which do not replicate the complex cellular interactions and physiological responses of the whole lung. Once proof-of-concept studies in Aim 2 are completed, the procedure for harvesting a combined cadaveric lung and bone marrow cell product will be optimized and tested in immune deficient mice for toxicity (Aim 3). At project completion, Lungevia Bio, Inc., will support planned subsequent Phase 1 and Phase 2 clinical trials based on the results of the proposed specific aims. If successful, this project may lead to new therapeutic options for cancer patients that develop pulmonary fibrosis as a consequence of chemotherapy.

NIMH - National Institute of Mental Health

Project Summary Human pluripotent stem cells (hPSCs) have emerged as a powerful tool for generating 3D organoids, enabling the study of human development and disease. These organoids can closely mimic in-vivo cellular context, making them valuable for investigating biological mechanisms. However, the extent to which gene regulation and other cellular and molecular mechanisms are preserved between in-vivo and in-vitro systems, especially in specific cell types, remains uncertain. Recent advancements in single-cell technologies offer opportunities to answer this question, there is a need for effective computational tools for comparing organoids and brain data. Our recent machine learning tool, Brain and Organoid Manifold Alignment (BOMA) successfully integrated eight published single-cell RNAseq (scRNAseq) datasets to uncover shared (or specific) developmental trajectories between human organoids and brains. However, BOMA is limited to scRNAseq. Simultaneous profiling of gene expression and chromatin accessibility of the same cell (single-nucleus multiomics or snMultiomics) allows linking putative regulatory elements to genes thus providing deeper insights of cell-type gene regulatory mechanisms in the developing brain compared to scRNA-seq alone. In fact, NIH funded consortia such as BRAIN Initiative is generating a large amount of snMultiomic data of human brains from prenatal development to adults. Although single cell multiomic data of organoids is currently scarce, we anticipate that more snMultiomics data will be generated for organoids in near future. This project aims to develop machine learning methods and tools for comparative snMultiomics analysis between organoids and brains, enabling the interpretation of developmental gene regulatory mechanisms at cellular level across in-vivo and in-vitro systems. Aim 1 will develop a machine learning method, Brain Organoid Manifold Alignment by Multiomics data (BOMAM), to uncover conserved and divergent developmental stages in brain and organoids. Aim 2 will perform gene regulatory network prediction and analysis to evaluate the fidelity of current organoid protocols. Aim 3 will develop open-source tools for comprehensive evaluation of brain organoids, designed for general-purpose use. In sum, our tools will enhance the efficiency and integration of brain-organoid analyses, especially for biologists and neuroscientists, leading to a deeper understanding of brain cells and their functional characteristics in development.

U.S. National Science Foundation

The Major Research Instrumentation (MRI) Program (MRI Program Website) serves to increase access to multi-user scientific and engineering instrumentation for research and research training in our Nation's institutions of higher education and not-for-profit scientific/engineering research organizations. An MRI award supports the acquisition of a multi-user research instrument that is commercially available through direct purchase from a vendor, or for the personnel costs and equipment that are required for the development of an instrument with new capabilities, thereby advancing instrumentation capabilities and enhancing expertise for instrument design and fabrication at academic institutions. MRI instruments are, in general, too costly and/or not appropriate for support through other NSF programs. MRI provides support to acquire critical research instrumentation without which advances in fundamental science and engineering research may not otherwise occur. MRI also provides support to obtain next-generation research instruments by developing instruments with new capabilities that open new opportunities to advance the frontiers in science and engineering research. Additionally, an MRI award is expected to enhance research training of students who will become the next generation of instrument users, designers and builders. An MRI proposal may request from NSF up to $4 million for either acquisition or development of a research instrument. Each performing organization may submit in revised "Tracks" as defined below, with no more than two (2) submissions in Track 1 and no more than one (1) submission in Track 2. For the newly defined Track 3, no more than one (1) submission per competition is permitted. As a result, it is now possible for an institution to submit up to four MRI proposals within the Track limits as described above. Track 1: Track 1 MRI proposals are those that request funds from NSF greater than $100,000[1] and less than $1,400,000. Track 2: Track 2 MRI proposals are those that request funds from NSF greater than or equal to $1,400,000 up to and including $4,000,000. Track 3: Track 3 MRI proposals are those that request funds from NSF greater than or equal to $100,000[1] and less than or equal to $4,000,000 that include the purchase, installation, operation, and maintenance of equipment and instrumentation to conserve or reduce the consumption of helium. Institutions may submit no more than one Track 3 proposal. Submission of a Track 3 proposal does not impact limits that apply for Track 1 and Track 2 proposals. Cost sharing requirements for new awards in the MRI Program are waived for a period of 5 years beginning with the FY 2023 MRI competition. Institutional submission limits for Track 1, Track 2 and Track 3 proposals remain. The MRI Program especially seeks broad representation of groups, institutions, and geographic regions that are underrepresented in STEM disciplines. Proposals from women, underrepresented minorities, persons with disabilities and early-career PIs are encouraged, as are proposals that benefit early-career researchers and proposals with PIs from geographically underserved regions, including EPSCoR jurisdictions. Additionally, proposals are encouraged from under-resourced institutions, including from emerging research institutions, where MRI can significantly build capacity for research. ___________________________ [1]Track 1 proposals requesting funds from NSF less than $100,000 will be accepted only from: a) eligible performing organizations requesting instrumentation supporting research in the disciplines of mathematics or social, behavioral and economic sciences; or b) non-Ph.D.-granting institutions of higher education requesting instrumentation supporting research in any NSF-supported disciplines.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Diabetes is a tremendous healthcare burden, with costs of hundreds of billions a year in the US. Diabetes has 2 major forms, type 1 diabetes (T1D) and type 2 diabetes (T2D). While these diseases have distinct causes (immune dysfunction or metabolic dysfunction), end stage disease in both leads to loss of beta cell mass and glucose homeostasis. Protocols have been developed to generate beta-like cells from human pluripotent stem cells (PSC)s which are currently in clinical trials as a cellular therapy for T1D and T2D. A beta cell therapeutic faces several hurdles to become a long-term cure for diabetes. First, it must survive transplantation which includes a hypoxic environment prior to graft vascularization and survive the inflammatory environment induced by the transplant surgery. For long term survival, the graft needs to be robust and survive diabetic conditions before the graft achieves full functionality and for use in T2D patients, needs to be able to function and overcome insulin resistance by secreting sufficient insulin long term. Lastly, immune attack of the graft by the host immune system needs to be prevented. Immune attack is being addressed by many groups in the scientific community via multiple strategies. This proposal is focused on generating the most robust stem cell derived beta cell that can survive and function long term in challenging conditions present in diabetic patients. To do so, we are leveraging naturally occurring hyperactive mutations that have been found in the HNF1A gene and which protect individuals from T2D. HNF1A is a transcription factor important in beta cell development and function. Loss of function mutations in HNF1A is the most common cause of monogenic diabetes and genome wide association studies have linked this gene to both T1D and T2D. Multiple studies of loss of function HNF1A mutants have demonstrated defects in insulin secretion, glycolysis, mitochondria function, and response to cellular stress. We hypothesize that the HNF1A hyperactive mutants will have superior function and response to cellular stress. We will test this hypothesis by generating a panel of hyperactive HNF1A mutants previously found in the human population in human PSCs. These mutants will be tested both in vitro and in vivo utilizing rodent xenotransplant studies. A second focus of this proposal is to acquire a deeper understanding of the mechanisms in how HNF1A loss of function can influence diabetes pathogenesis. HNF1A can influence both beta cell development but also plays a role directly in beta cell function. To focus on its role in beta cells, we are utilizing genetically engineered human PSC lines where HNF1A can be degraded specifically after development into beta cells. We will use this system to study how acute loss of HNF1A impacts beta cell function, survival, and response to cellular stressors important in diabetes pathogenesis. This proposal will give new insights into beta cell function and responses to cell stress which is critical for diabetes pathogenesis and developing cellular therapies for this disorder.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

PROJECT SUMMARY/ABSTRACT Directly genome editing cells inside the body could treat numerous genetic diseases, including sickle cell disease. However, genome editing of most cell types outside of the liver, such as hematopoietic stem cells, is limited by inefficient delivery. Only a small subset of cells inside of a tissue are accessible to delivery vehicles and editing enzymes. To overcome this delivery problem, my central objective is to enable cells initially receiving delivery vehicles to transiently produce and distribute genome editing enzymes to neighboring cells in vesicles. This allows editing activity to spread beyond initial delivery. My proposal builds on my postdoctoral research studying the delivery mechanisms of Enveloped Delivery Vehicles (EDVs). EDVs are lentivirus-derived lipid vesicles engineered to package CRISPR-Cas9 ribonucleoproteins that can be targeted to specific cell types using surface-displayed fusogens and antibodies. I hypothesize that transient, local production of EDVs in vivo will amplify genome editing efficiency by increasing the concentration of genome editing enzymes and enabling their spread across cells. This hypothesis will be tested through three specific aims: (1) develop single nucleic acid molecules encoding EDVs, (2) establish methods to target EDV production and uptake to specific cell types, and (3) deliver EDV-encoding plasmids to amplify editing in vivo. In preliminary work for this proposal, I showed that hydrodynamic injection of EDV-encoding plasmids into mice amplified genome editing efficiencies compared to Cas9 only plasmid controls. Successful completion of this proposal will generate fundamental insights into propagating genome editing effects beyond cells initially reached by delivery vehicles starting with hematopoietic stem cells as a model therapeutic cell type. This approach could broadly transform biological therapy delivery by overcoming low delivery efficiencies through localized amplification and spread of therapeutic macromolecules. The training acquired through this proposal in primary cell culture, bioinformatics, and next-generation sequencing will bolster my readiness to lead an independent research program. I will be mentored by Dr. Jennifer Doudna, a global leader in genome editing technology, and leading experts and clinicians in primary cell engineering, hematopoietic stem cell biology and virology in the California scientific community. During the mentored phase of this project, I will hone my scientific and professional skills to become a scientific leader. I will engage in structured professional development activities and actively present my research at leading conferences to facilitate a successful transition to an independent academic research position. The vibrant and collaborative environment provided by UC Berkeley and the Innovative Genomics Institute offers an outstanding environment to complete my training and start my independent scientific career.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY/ABSTRACT Janus kinase inhibitors (JAK}---including tofacitinib, baricitinib, and upadacitinib-have emerged as promising oral treatment options for patients with rheumatoid arthritis who experience an inadequate response to conventional therapies. However, major safety concerns, including increased risk of cardiovascular events, venous thromboembolism, and serious infections, have prompted FDA black box warnings and remain major barriers to widespread use. These safety concerns largely stem from potential signals in integrated analyses of premarketing studies and the ORAL Surveillance trial, which reported a dose-dependent elevated risk of cardiovascular and thromboembolic events with tofacitinib compared to tumor necrosis factor (TNF) inhibitors. However, the ORAL trial has left some critical questions unanswered-specifically, whether the observed risk elevation is attributable to a specific JAK inhibitor toxicity, a protective effect of TNF inhibitors, or both; or how the safety profile of specific JAK inhibitors may compare to approved alternatives, such as interleukin-6 inhibitors. Approved JAK inhibitors have differences in selectivity and target binding affinity, which may translate into differences in adverse health outcomes. As a pan-JAK inhibitor, tofacitinib targets multiple JAK isoforms (JAK1, JAK2, JAK3), while newer agents such as upadacitinib and baricitinib exhibit greater selectivity for JAK1. The current FDA black box warning applies to all JAK inhibitor class members, but comparisons among specific class members are lacking. Moreover, all currenUy approved JAK inhibitors are metabolized via cytochrome P450 3A4 (CYP3A4), making them susceptible to clinically significant drug-drug interactions {DDls). Co-administration with moderate or strong CYP3A4 inhibitors, such as diltiazem, can substantially increase plasma JAK inhibitor concentrations, and potentially increasing the risk of adverse events. In this R01, we propose a comprehensive examination of the safety of JAK inhibitors among patients with rheumatoid arthritis. We will use the estimand framework and target trial emulation methods to compare cardiovascular, thrombotic, and infectious risks between JAK inhibitors and other commonly used therapies, overall and by agent. We will also evaluate how co-prescribed CYP3A4 inhibitors modify these risks. Our findings will directly inform clinical guidelines and prescribing decisions for patients with rheumatoid arthritis.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Hormones, cytokines, and growth factors are essential signaling proteins in blood circulation that coordinate multi-organ functions to maintain physiological homeostasis. These “inter-organ” factors (e.g. Insulin) have inspired widely used therapeutics and biomarkers for diabetes, obesity (e.g. semaglutide), and cancer, owing to their systemic effects and accessibility in the blood. Despite decades of research, our understanding of the body’s inter-organ communication network remains limited. In contrast to systematic efforts to map the brain connectome, comprehensive mapping of inter-organ communication under both normal and disease conditions has lagged behind. This slow progress stems from the challenging qualities and complex roles of inter-organ factors, including their low abundance in the blood, regulated secretion, non-cell-autonomous functions, ligand- receptor binding, and distinct origin and target tissues. High-throughput approaches such as proteomics and transcriptomics struggle to capture this complexity, so most of our knowledge comes from laborious and unsystematic discovery efforts. This project will overcome these barriers by integrating state-of-the-art experimental and computational techniques to map inter-organ proteins in the blood. We will (1) identify and study inter-organ factors from our prior dataset of organ-secreted proteins, (2) identify disease-induced secreted proteins that influence pathology, (3) develop “receive-ome” tools to identify the tissue targets of circulating proteins, and (4) adapt AI-based tools to predict hormones and their receptors. These independent projects allow discoveries to be cross-validated across aims, increasing confidence in the results. In vivo experiments will be initially performed using Drosophila, an ideal model due to its conserved organ and endocrine systems, and then followed up in zebrafish and mice. Computational analysis will directly utilize human datasets. Our preliminary results demonstrate the feasibility of the proposed aims, leading our group to study novel circulating factors influencing body size and synaptic function. As technologies in the field evolve, we will expand to other signaling molecules including lipids and exosomes, as well as extracellular fluids such as cerebrospinal fluid (CSF). We will disseminate a variety of resources and datasets, including plasmids, transgenic animals, and detailed visualizations of inter-organ signaling networks. These resources will broadly benefit researchers investigating inter-organ communication, as well as those studying local cell-to-cell signaling and protein-protein interactions. Through these holistic efforts, we expect to find novel inter-organ ligands and receptors, unexpected tissue sources of circulating proteins, and potential targets for diagnostic markers and therapeutics.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Broadly neutralizing antibodies (bnAbs) that target the CD4 binding site (CD4bs) on the HIV envelope glycoprotein (Env) are an attractive target for an HIV vaccine and antibody-based therapy for HIV protection. However, eliciting CD4bs bnAbs in a vaccine is challenging, and current CD4bs bnAbs are insufficient to provide long-term HIV suppression. This difficulty in CD4bs bnAb-mediated treatment stems from Env mutations near the CD4bs referred to as “loop D,” including the gain of a glycan at N276 that has yet to be dependably overcome in vaccine regimens. Although improvements in antibody isolation have provided mechanistic and evolutionary insight into some CD4bs bnAbs, it remains poorly understood how CD4bs bnAbs evolve breadth and potency from their naïve precursors, complicating attempts to elicit them via vaccination. To address this issue, high-throughput deep mutational scanning (DMS) techniques will be used to characterize the effect of all possible single-amino acid mutations at each position in the variable domains of the VRC01 and IOMA CD4bs bnAbs that represent two stereotyped mechanisms of Env CD4bs recognition. VRC01 and IOMA variant libraries will be expressed on the cell surface of yeast. Using this yeast-surface display (YSD) platform, both VRC01 and IOMA variant binding affinities will be measured against various Env proteins using fluorescence-activated cell sorting and high-throughput sequencing (FACS-seq), facilitating the measurement of the binding properties of these bnAb libraries in parallel. Aim 1 will determine the mutations in CD4bs bnAbs that enable HIV Env N276 glycan accommodation. DMS, YSD, and FACS-Seq will be performed on libraries of mature VRC01 and its naïve precursor to determine their binding affinities to two closely related donor-autologous Env proteins that contain and lack the N276 glycan. These experiments will reveal routes to overcome common barriers in CD4bs bnAb development and illuminate how the next generation of immunogens will better elicit broad and potent bnAbs. Aim 2 will map the mutational landscape that confers CD4bs bnAbs breadth and potency. DMS, YSD, and FACS-seq will be performed on mature VRC01 and IOMA to measure the impact of all single mutations on binding to a previously established panel of 12 heterologous Env strains that comprise a reference of global Env diversity. These results will illustrate how mutations in these antibody scaffolds confer binding to divergent Env strains versus promoting strain-specific binding, fully defining the functional constraints and flexibilities of these CD4bs bnAb classes. The results of this proposal will illustrate a new approach to investigate the evolution and engineering of HIV CD4bs bnAbs, setting the stage for the development of improved immunogens and therapeutic antibodies against HIV. More broadly, the methodological innovation herein will provide a new platform for antibody engineering for the treatment and prevention of various infectious diseases.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY In this application, we seek to continue and enhance the MARC U*STAR program at the University of Rhode Island (URI). This program has had a major positive impact on the career development of our high-achieving underrepresented (UR) STEM students, with 70% (11 of 16) of our trainees matriculating into competitive graduate programs in the biomedical sciences to date. The program has also been instrumental in creating a diverse community of student biomedical scientists at URI, united by a strong sense of belonging and an enthusiasm for scientific discovery. Our institutional self-assessment provides compelling justification for an undergraduate research training program for underrepresented students at URI: 4-year graduation rates for underrepresented (UR) students (33%) continue to lag behind that for well-represented (WR) students (49%). Furthermore, rates of matriculation into advanced biomedical degree programs for UR students (15%) also continue to lag behind that for WR students (20%). Moreover, we have a robust pool of UR STEM students and a diverse pool of actively funded faculty committed to supporting the career development of UR students. Our program goals are to; 1) Develop students who are knowledgeable and well trained in the biomedical sciences, who can readily employ rigorous research methods and critical thinking to solve important scientific problems, and 2) Create a community of student scholars who enjoy a strong sense of belonging, thereby promoting a sustainable culture of undergraduate research excellence at URI. Our approach to achieving these goals will be informed by lessons learned over the course of the previous funding period; we will continue to cultivate a culture of inclusive mentoring excellence across campus by promoting and facilitating mentoring training for faculty and graduate student research mentors; we will use evidence-based approaches to monitor and support trainee mental health and wellness; we will emphasize strong foundations in quantitative and computational competencies and instill a pervasive culture of rigor and reproducibility in experimental design among our trainees. With these enhanced strategies, we expect that 1) 90% of our trainees will graduate with a baccalaureate degree in a STEM field within a 4-year period, and 2) 75% of our trainees will matriculate into PhD or MD-PhD degree programs in biomedical research fields within three years of graduating from the program, ultimately advancing the diversification of the nation's scientific workforce.

Massachusetts Executive Office of Education

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

NHLBI - National Heart Lung and Blood Institute

Project Summary/Abstract: Human-induced pluripotent stem cell-derived vascularized cardiac organoids (VCOs) represent a promising in vitro model for studying human heart development, disease, and drug responses. A robust differentiation protocol has been established for generating VCOs containing cardiomyocytes (CMs), endothelial cells (ECs), and cardiac fibroblasts (CFs). However, VCOs remain structurally and functionally immature, limiting their utility for modeling late-onset cardiomyopathies and preclinical drug testing. Current maturation strategies enhance contractility and metabolic activity but often disrupt multicellular composition and fail to achieve key hallmarks of adult cardiac maturity, such as a compact myocardium-like structure, aligned sarcomeres and longer sarcomere length, fatty acid-dependent metabolism, hallmarks of marker gene expressions, and cardiac functions. To address these challenges, this study aims to enhance VCO maturity while preserving cellular heterogeneity by implementing a dual-modulation strategy targeting metabolism and mechanical stiffness. The first aim focuses on developing a glucose administration protocol that facilitates the metabolic transition from glycolysis to fatty acid oxidation while maintaining ECs and CFs within VCOs. Optimizing glucose concentration, timing, and duration of administration is expected to enhance contractility, calcium kinetics, and mitochondrial function without compromising VCO structural integrity. The second aim introduces a stiffness-tunable biocompatible hydrogel system that mimics the mechanical transition from fetal to adult myocardium. Gradually increasing extracellular matrix stiffness during maturation is expected to improve sarcomere organization, enhance myocardial compaction, and upregulate key maturation genetic markers. Successful completion of this project will establish a physiologically relevant maturation framework for VCOs, enabling advanced cardiac disease modeling and drug screening. This research aligns with the NIH’s mission to develop innovative in vitro human heart models for understanding cardiovascular diseases and improving therapeutic development.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Mesenchymal stem/stromal cells (MSCs) are potent regulators of immune cells, and their immunosuppressive function is being actively investigated for a number of therapeutic applications. In particular, it has been demonstrated that MSCs can inhibit the proliferation of effector T cells and induce regulatory T-cell differentiation for treating graft versus host disease (GvHD). A few MSC products have been approved by regulatory agencies in countries outside of the U.S. However, MSCs have not always shown consistent efficacy in GvHD clinical trials. This is in part due to the challenges of generating MSCs with high therapeutic potency. The overarching goal of this project is to develop MSC therapies with enhanced immunosuppressive efficacy for GvHD treatment by identifying and providing optimal microenvironment mechanical cues in MSC production. Mechanical cues from cell microenvironment play important roles in regulating cell behavior. For example, studies have shown that matrix stiffness directs cell activity and fate such as migration, proliferation, and differentiation. However, matrix or material stiffness only describes their static, elastic mechanical property. Instead of being simply elastic, natural extracellular matrix (ECM) and living tissues are viscoelastic, exhibiting stress relaxation over different characteristic time scales (stress relaxes at different rates). We have developed a hydrogel system that can recapitulate the stiffness and viscoelastic behavior of different types of tissues. Using the hydrogels as culture substrates, we discovered that matrix stress relaxation, in addition to stiffness, is an important mechanical factor regulating cell–ECM interactions and directing MSC activities including spreading, proliferation, differentiation, and in vivo bone regeneration. In collaboration with Dr. Kyung Sung at FDA, we recently found that substrate stress relaxation also regulates MSC's immunosuppressive capacity and their ability to inhibit T cell proliferation; Interestingly, MSCs retained their mechanical “memory” even after being extracted from the hydrogels (preliminary data). In light of these new findings, we hypothesize that biomaterials with tailored stress relaxation properties can provide inducing mechanical cues in MSC production to enhance MSC's immunosuppressive efficacy for GvHD treatment. We will test this hypothesis in the following specific aims: Aim 1: Elucidate the molecular mechanisms by which matrix stress relaxation regulates the immunosuppressive capacity of human MSCs (hMSC) derived from bone marrow. Aim 2: Compare the effect of matrix stress relaxation on hMSCs derived from different donors and tissues, and examine the influence of stiffness in the fast stress relaxing environment. Aim 3: Evaluate the efficacy of hMSCs primed by viscoelastic hydrogels with different stress relaxation properties for GvHD treatment in an animal model. Successful completion of these aims will have significant impact in understanding how matrix mechanical cues regulates the immunosuppressive capacity of hMSCs, with the findings potentially leading to better treatment for GvHD.

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

Project Summary Infertility is a complex disease that impacts approximately 1 in 10 couples of reproductive age in the United States. However, the pathogenesis underlying many of these cases is unknown. Clinically, this proposal aims to gain molecular insights into infertilities associated with defective formation of amnion, a component of the fetal membrane, and primordial germ cells (PGC, precursors of gametes). Formation of human amnion is first seen in the pluripotent epiblast cells as implantation is initiated (within two weeks after fertilization). Thus, couples with pregnancies repeatedly accompanied by defective amnion formation would be diagnosed with infertility. Recent data indicate that, in implanting primate embryos, PGC are specified in the nascent amnion and a growing body of evidence supports the notion that a subset of the pluripotent epiblast cells form a bi- potential amnion/PGC progenitor cell population at the amnion-epiblast boundary of developing embryos. In published work using a human pluripotent stem cell (hPSC)-based Gel-3D amniogenic system, we showed that a mechanical cue initiates amnion specification by triggering a Bone Morphogenetic Protein (BMP) signaling cascade in individual cells of pluripotent cysts. Moreover, our recent single cell transcriptomic analyses of developing Gel-3D showed that, in fact, PGC-like cells (PGC-LC) are also present in Gel-3D, and that cells that are progressing to amnion and PGC-LC lineages appear to share a common progenitor labeled by Claudin 10 (CLDN10). Indeed, our preliminary results revealed the presence of CLDN10/TFAP2C/SOX17-triple positive cells at the posterior amnion-epiblast boundary of a cynomolgus macaque peri-gastrula, suggesting that this progenitor formation as well as the amnion/PGC lineage diversification from the progenitor is an important aspect of successful amniogenesis and PGC formation both in vivo and in vitro. These findings present Gel-3D as a robust, transcriptomically well-characterized and mechanosensitive model to investigate these previously unexplored aspects of amniogenesis and PGC-LC formation. Together, using the Gel-3D model, the goal of this proposal is to understand the mechanisms that triggers amniogenic BMP signaling to initiate CLDN10+ progenitor cell specification (Aim 1), as well as the mechanisms that control amnion-PGC lineage decisions in the progenitor (Aim 2). In vitro findings will be grounded using early cynomolgus macaque embryo samples. Our preliminary data lead us to hypothesize that mechanically triggered WNT/?-catenin signaling activates amniogenic BMP signaling, and, in the progenitor population, Yes-associated protein 1 (YAP1) activity determines amnion or PGC fate. Proposed studies will further expose these signaling and transcriptional machineries. Overall, the work proposed here will greatly accelerate the pace of discovery regarding critical but previously inaccessible peri-implantation events and thus will have enormous implications for understanding early processes that impact embryonic development and human fertility.