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Cleveland High School PTSA

The Cleveland High School PTSA will organize an event to promote community building, health, and wellness among South Seattle youth. The event will be held October 30, 2019 from 4 pm - 7 pm at Cleveland STEM High School located at 5511 15th Ave S. The event will offer demonstrations and activities that support wellness and cultivate a sense of belonging among youth and families.

Heising-Simons Foundation

Supports fundamental physics, astronomy, and mathematics research, and initiatives to increase diversity in STEM fields.

NCI - National Cancer Institute

ABSTRACT Intravesical administration of bacillus Calmette-Guérin (BCG), the first immunotherapy and the only bacterial therapy of cancer, is the most effective treatment for non–muscle invasive bladder cancer (NMIBC), but cancer recurs in approximately 50% of treated patients, many of whom require major surgery and are at risk for metastatic disease. Despite substantial efforts, there are no reliable pretreatment predictors of BCG response, partially due to an incomplete understanding of BCG’s mechanism of action. We discovered that BCG-induced tumor elimination in mice is due to induction of long-term T cell immunity to tumor antigens, and there is evidence that this mechanism plays a role in the efficacy of BCG in treating human disease as well. However, the upstream events stimulated by BCG that ultimately lead to tumor-specific T cell immunity are unknown. It is now recognized that certain stimuli, including BCG, lead to epigenetic changes in hematopoietic stem and progenitor cells (HSPCs) that can confer differentiation bias (eg, increased myeloid and granulocyte output) and the acquisition of epigenetic programs in mature progeny cells, resulting in an adapted capacity of innate immune cells, particularly macrophages and dendritic cells, to react to restimulation (termed innate immune memory). Although there is emerging evidence that the innate immune memory stimulated by BCG can provide heterologous immunity against viral infection, its role in the antitumor effects of BCG is relatively unexplored. Our recently published data in mice demonstrate that intravesical BCG can traffic to the bone marrow, where it alters the phenotypic and epigenetic state of centrally positioned bone marrow HSPCs through interferon gamma. Human bladder cancer patients receiving intravesical BCG have strong evidence of HSPC remodeling through the same IFN gamma stimulated pathways. Reconstitution of the hematopoietic compartment of irradiated mice with Lin-Sca1+c-Kit+ (LSK) HSPCs from BCG-treated mice inhibits tumor growth, enhances myeloid cell infiltration of the tumor, reprograms tumor infiltrating neutrophils, and synergizes with PD1 blockade, demonstrating that HSPC-derived innate immune cells reprogram the myeloid tumor microenvironment and enhance T cell mediated anti-tumor immunity. This proposal will elucidate the IFN dependent mechanisms by which BCG stimulates HSPC reprogramming, the innate immune mechanisms by which HSPC encoded anti-tumor immunity eliminates tumors, and will determine whether measurement of HSPC encoded myeloid reprogramming, detected in peripheral blood, can predict BCG response in NMIBC patients. These studies use complex immunologic models, including bone marrow transplantation, in vertebrate animals as these mechanistic studies are not possible in surrogate model systems. If successful, these studies will provide new mechanistic insights into the oldest immunotherapy of cancer, identify candidate biomarkers to predict the success of this specific therapy for bladder cancer, and give a deeper understanding of how HSPC encoded myeloid reprogramming can be applied to immunotherapy of a wider range of cancers.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract HIV disease remains a considerable public health concern without a practicable cure. Drug-based therapy can control HIV but is costly, has severe side effects, and is not curative. Stem-cell based therapies have provided the only known cures for HIV infection, with only a handful of individuals functionally cured to date. However, replicating these successes has been challenging due to the high toxicities of treatment, need for transplant antigen matching, and require extensive myeloablation. However, these “cures” strongly suggest that immune system modification involving hematopoietic stem/progenitor cell (HSPC) transplantation can play a strong role allowing HIV clearance from the body. We aim to achieve a HIV-1 cure by enhancing and optimizing anti-HIV cellular immune responses through genetic modification of autologous Hematopoietic Stem/Progenitor Cells (HSPCs) with an anti-HIV Chimeric Antigen Receptor (CAR) molecule (CAR-HSPC). Unlike combined antiretroviral treatment (ART), which cannot eradicate HIV due to persistent reservoirs, our approach targets lifelong anti-HIV responses for HIV clearance. We will improve the engraftment of CAR-modified stem cells by using clinically relevant conditioning methods, maintain long-term progenitor phenotype in CAR stem cells for repopulation capability, and improve homing to the bone marrow. Additionally, we will characterize the differentiation and therapeutic effects of HSPC-derived CAR modified immune cells in various tissue reservoirs using humanized mouse models. We will develop an in vivo targeting regimen incorporating stem cell targeted nanocapsules encapsulating CAR lentivirus to generate CAR-modified stem cells in vivo and evaluate for feasibility and efficacy. Our proposed study will provide crucial insights for investigational new drug (IND) development of HSPC-based CAR immunotherapies, potentially leading to ART-free HIV suppression and a functional cure.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY / ABSTRACT The mammalian hematopoietic system develops in the early embryo through a series of spatio-temporally separated programs, each of which harbors different functional potential, culminating in the specification of the hematopoietic stem cell (HSC). The overall goal of our research is to understand the origins and development of each program in the embryonic hematopoietic system. Where does each developmental program originate from? How does it develop? Why is each different? And, is there clinical utility to embryonic cell types that are no longer found in adult donors? Dr. Sturgeon's prior work has focused on these questions, through the lens of human pluripotent stem cell (hPSC) directed differentiation, leading to the pivotal discovery of hematopoietic commitment occurring very early, within nacent mesoderm, referred to as hemogenic mesoderm (HM). The proposed research program builds upon Dr. Sturgeon's productive research track record to delineate the molecular and transcriptional mechanisms by which HM gives rise to the embryonic hematopoietic programs. Dr. Sturgeon has shown that HMs are found in multiple immunophenotypically distinct subsets, each of which are specified in ACTIVIN/NODAL- and/or WNT-dependent processes. Further, Dr. Sturgeon has found that each HM first gives rise to a hemogenic endothelial cell (HEC) population, in VEGF- and RA-dependent processes. HM express genes associated with early gastrulation, yet each HM is highly restricted, ultimately each giving rise to a specific hematopoietic program, such as yolk sac-like erythromyeloid progenitors (EMPs), or intra- embryonic-like definitive multipotent progenitors (MPPs). Finally, Dr. Sturgeon has found that hematopoietic lineages common across multiple HM populations harbor distinct functional properties from one another. Building off these groundbreaking findings, the research program is divided into 3 projects. The first project will delineate the signal, transcriptional, and epigenetic mechanisms underlying how each hematopoietic program is specified and functionally restricted. These studies will improve our ability to obtain progenitors from each program, including the HSC. The second project will define the mechanisms regulating how HECs give rise to different lineages. Finally, the third project will continue our studies on the translational potential of hematopoietic lineages from each developmental program. Collectively, these studies will provide us with a more comprehensive understanding of hematopoietic development. This is of fundamental importance to basic biology, and the insights generated from these studies will have clinical implications, such as the in vitro generation of HSCs or other embryonic hematopoietic lineages for a wide array of regenerative medicine applications.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Though the liver produces most of the lymph in the body, the role of hepatic lymphatics in liver disease is relatively less studied. While it is recognized that alterations in hepatic lymphatics cause ascites formation in chronic liver disease like cirrhosis, its contribution to acute liver failure (ALF), like that caused by drugs such as acetaminophen (APAP) are not well characterized. An APAP overdose is the most common cause of ALF in the United States, partly due to the short therapeutic window of the only FDA approved antidote, N-acetylcysteine (NAC). Excess APAP induces centrilobular necrosis, and a failure of inherent liver regeneration in a significant percentage of patients, especially after a severe overdose, causes ALF. Thus, insight into mechanisms of liver recovery which are compromised in patients with ALF, would allow their targeting to complement NAC treatment. One such beneficial phenomenon is the innate immune response induced by hepatocyte necrosis. Though interaction between the infiltrating immune cells and surviving hepatocytes facilitates their regeneration, repopulation of areas of necrosis also requires an orderly exit of the infiltrating immune cells to allow coordinated reestablishment of liver infrastructure and lymphatics to regain functional homeostasis. The lymphatic system is a central mode of immune cell emigration from tissues, and specific pro-resolving lipid mediators (SPMs) facilitate resolution of inflammation. However, their role in immune cell exit after acute APAP-induced ALF is unknown. Our preliminary data shows transient changes in hepatic lymphatics after APAP overdose with elevations in SPMs, which are known to facilitate lymphangiogenesis and immune cell clearance. Blocking lymphangiogenesis after an APAP overdose also extended hepatic residency of immune cells. Treatment with Wharton's Jelly mesenchymal stem cells (WJMSC) which are cleared for human use, also enhanced liver recovery in the mouse model with elevation in circulating VEGF-D, which activates lymphangiogenesis. This data led to the hypothesis that hepatic lymphatics play a critical role in immune cell clearance during liver recovery from an APAP overdose, a process facilitated by SPMs which could be targeted by WJMSC treatment to enhance liver recovery. This hypothesis will be tested by 1) evaluating mechanisms of immune cell clearance through hepatic lymphatics after acute APAP overdose, and 2) examining the role of SPMs and immune cell clearance as mechanisms facilitating recovery after delayed treatment with WJMSC. Collectively, we will define the molecular mechanisms responsible for efficient immune cell exit through hepatic lymphatics after reconstruction of areas of hepatic necrosis and study the consequence when this exit is compromised such as ALF. We will also evaluate a therapeutic intervention to enhance recovery, which can be rapidly translated to the clinic.

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

PROJECT SUMMARY Congenital hydrocephalus (CH) involves ventricular enlargement and has historically been attributed to impaired cerebrospinal fluid (CSF) flow. Recent evidence, however, reveals that neurodevelopmental defects underlie many CH cases. Indeed, genetic studies frequently implicate neural differentiation and timing factors rather than direct regulators of CSF homeostasis. Our work focuses on the MIR302 family of microRNAs, which orchestrate developmental timing by controlling both post-transcriptional and epigenetic programs. We previously found that complete loss of mir-302 causes severe neural tube defects. More recently, we developed a hypomorphic mir- 302 mouse model that displays classic CH features—dome-shaped skulls, ventriculomegaly, and aqueduct stenosis—and exhibits altered chromatin accessibility in neural stem cells. Preliminary single-nuclei RNA- sequencing indicates a defect in neurogenesis across forebrain and midbrain populations, highlighting a broader timing dysregulation. We hypothesize that miR-302 enforces heterochronic control of neuroepithelial stem cells, preventing precocious differentiation and safeguarding specialized structures like the subcommissural organ (SCO). In Aim 1, we will define how miR-302 functions as a post-transcriptional regulator by mapping direct miRNA:mRNA interactions (via AGO2-chimeric eCLIP) and measuring translational changes (via Ribo-seq), thus linking aberrant gene expression to the loss of miR-302. In Aim 2, we will examine how distinct MIR302 members modulate chromatin accessibility, particularly in dorsal midbrain cells forming the SCO, using single-nuclei RNA+ATAC multiome and Polycomb (PRC2) occupancy assays. By pinpointing the epigenetic mechanisms that fail in CH mutants, we will reveal why the SCO is especially susceptible to timing defects. Together, these studies will yield new insights into how miRNA-driven heterochronic regulation ensures proper neuronal lineage commitment and SCO maintenance—key processes disrupted in CH. Our findings may inform novel therapeutic strategies aimed at restoring developmental timing in congenital brain malformations.

Advanced Research Projects Agency Energy

This is Modification 01 to the NOFO: Inserted certain deadlines and updated dates, including the deadlines for submitting questions and Full Applications (Basic Information, Section IV.C). NOFO Number: DE-FOA-0003623 High-performance Optimized Recycled Nuclear Isotopes for Gen IV reactors (HORNIG) Agency Overview: The Advanced Research Projects Agency Energy (ARPA-E), an organization within the Department of Energy (DOE), is chartered by Congress in the America COMPETES Act of 2007 (Public Law 110 69), as amended by the America COMPETES Reauthorization Act of 2010 (Public Law 111 358), as further amended by the Energy Act of 2020 (Public Law 116 260). ARPA-E issues this Notice of Funding Opportunity (NOFO) under its authorizing statute codified at 42 U.S.C. 16538. The NOFO and any cooperative agreements or grants made under this NOFO are subject to 2 C.F.R. Part 200 as supplemented by 2 C.F.R. Part 910. ARPA-E funds research on, and the development of, transformative science and technology solutions to address the energy and environmental missions of the Department. The agency focuses on technologies that can be meaningfully advanced with a modest investment over a defined period of time in order to catalyze the translation from scientific discovery into early-stage technology. For the latest news and information about ARPA-E, its programs and the research projects currently supported, see: http://arpa-e.energy.gov/. ARPA-E funds transformational research. Existing energy technologies generally progress on established learning curves where refinements to a technology and the economies of scale that accrue as manufacturing and distribution develop drive improvements to the cost/performance metric in a gradual fashion. This continual improvement of a technology is important to its increased commercial deployment and is appropriately the focus of the private sector or the applied technology offices within DOE. In contrast, ARPA-E supports transformative research that has the potential to create fundamentally new learning curves. ARPA-E technology projects typically start with cost/performance estimates well above the level of an incumbent technology. Given the high risk inherent in these projects, many will fail to progress, but some may succeed in generating a new learning curve with a projected cost/performance metric that is significantly better than that of the incumbent technology. ARPA-E will provide support at the highest funding level only for submissions with significant technology risk, aggressive timetables, and careful management of associated risk. ARPA-E funds technology with the potential to be disruptive in the marketplace. The mere creation of a new learning curve does not ensure market penetration. Rather, the ultimate value of a technology is determined by the marketplace, and impactful technologies ultimately become disruptive that is, they are widely adopted and displace existing technologies from the marketplace or create entirely new markets. ARPA-E understands that definitive proof of market disruption takes time, particularly for energy technologies. Therefore, ARPA-E funds the development of technologies that, if technically successful, have clear disruptive potential, e.g., by demonstrating capability for manufacturing at competitive cost and deployment at scale. ARPA-E funds applied research and development (R&D). The Office of Management and Budget defines applied research as an original investigation undertaken in order to acquire new knowledge directed primarily toward a specific practical aim or objective and defines experimental development as creative and systematic work, drawing on knowledge gained from research and practical experience, which is directed at producing new products or processes or improving existing products or processes. Applicants interested in receiving financial assistance for basic research (defined by the Office of Management and Budget as experimental or theoretical work undertaken primarily to acquire new knowledge of the underlying foundations of phenomena and observable facts ) should contact the DOE s Office of Science. Office of Science national scientific user facilities (http://science.energy.gov/user-facilities/) are open to all researchers, including ARPA-E applicants and awardees. These facilities provide advanced tools of modern science including accelerators, colliders, supercomputers, light sources and neutron sources, as well as facilities for studying the nanoworld, the environment, and the atmosphere. Projects focused on early-stage R&D for the improvement of technology along defined roadmaps may be more appropriate for support through the DOE applied energy offices including: the Office of Critical Minerals and Energy Innovation (CMEI), the Office of Hydrocarbon and Geothermal Energy, the Office of Nuclear Energy, and the Office of Electricity. ARPA-E encourages submissions stemming from ideas that still require proof-of-concept R&D efforts as well as those for which some proof-of-concept demonstration already exists. Submissions can propose a project with the end deliverable being an extremely creative, but partial solution. Program Overview: Transuranic (TRU) elements such as plutonium (Pu), neptunium (Np), and americium (Am) are a significant source of fissile materials that are available across existing nuclear inventories and strategic reserves and have the potential to drive advanced reactor deployment. , The technical feasibility of recycling TRU elements into new fuels has been proven at the experimental scale. Advances in fabrication, safeguards, equipment design, and modeling will create the opportunity to transition from experimental success to commercial-scale deployment, enabling higher-throughput and lower-cost fuel production with real-time materials accountancy. The High-performance Optimized Recycled Nuclear Isotopes for Gen IV reactors (HORNIG) program will support research and development projects that deliver commercially deployable TRU fuel technologies, thereby strengthening U.S. energy security, reducing nuclear waste, and enabling long-term energy deployment for public benefit. The HORNIG program seeks to overcome key technical and economic barriers that have historically prevented using TRU fuels in commercial reactors and to create a clear path to domestic nuclear fuel security by supporting the design, fabrication, and qualification of TRU fuels. The program will fund coordinated, multidisciplinary efforts to deliver transformative advances in fuel performance, manufacturability, cost, and regulatory readiness. Technologies developed under this program must have the potential to enable the following program metrics: A domestic TRU fuel supply chain A levelized cost of fuel (LCOF) = 1 /kWh TRU fuel qualification and regulatory acceptance within seven years By enabling production of advanced reactor fuels from domestically sourced fissile materials, the program will reduce dependence on imported uranium and enrichment services, expand U.S. fuel supply options, and support establishment of a closed fuel cycle. These objectives support ARPA-E s statutory goals of improving energy security and resilience, improving the management of radiological waste, and maintaining U.S. technological leadership in energy technologies. If successful, HORNIG will strengthen U.S. energy security and infrastructure resilience and deliver lasting public benefit through reliable nuclear power. To view the NOFO in its entirety, please visit https://arpa-e-foa.energy.gov

Advanced Research Projects Agency Energy

This is Modification 01 to the NOFO: • Inserted certain deadlines and updated dates, including the deadlines for submitting questions and Full Applications (Basic Information, Section IV.C). NOFO Number: DE-FOA-0003623 – High-performance Optimized Recycled Nuclear Isotopes for Gen IV reactors (HORNIG) Agency Overview: The Advanced Research Projects Agency—Energy (ARPA-E), an organization within the Department of Energy (DOE), is chartered by Congress in the America COMPETES Act of 2007 (Public Law 110–69), as amended by the America COMPETES Reauthorization Act of 2010 (Public Law 111–358), as further amended by the Energy Act of 2020 (Public Law 116–260). ARPA-E issues this Notice of Funding Opportunity (NOFO) under its authorizing statute codified at 42 U.S.C. § 16538. The NOFO and any cooperative agreements or grants made under this NOFO are subject to 2 C.F.R. Part 200 as supplemented by 2 C.F.R. Part 910. ARPA-E funds research on, and the development of, transformative science and technology solutions to address the energy and environmental missions of the Department. The agency focuses on technologies that can be meaningfully advanced with a modest investment over a defined period of time in order to catalyze the translation from scientific discovery into early-stage technology. For the latest news and information about ARPA-E, its programs and the research projects currently supported, see: http://arpa-e.energy.gov/. ARPA-E funds transformational research. Existing energy technologies generally progress on established “learning curves” where refinements to a technology and the economies of scale that accrue as manufacturing and distribution develop drive improvements to the cost/performance metric in a gradual fashion. This continual improvement of a technology is important to its increased commercial deployment and is appropriately the focus of the private sector or the applied technology offices within DOE. In contrast, ARPA-E supports transformative research that has the potential to create fundamentally new learning curves. ARPA-E technology projects typically start with cost/performance estimates well above the level of an incumbent technology. Given the high risk inherent in these projects, many will fail to progress, but some may succeed in generating a new learning curve with a projected cost/performance metric that is significantly better than that of the incumbent technology. ARPA-E will provide support at the highest funding level only for submissions with significant technology risk, aggressive timetables, and careful management of associated risk. ARPA-E funds technology with the potential to be disruptive in the marketplace. The mere creation of a new learning curve does not ensure market penetration. Rather, the ultimate value of a technology is determined by the marketplace, and impactful technologies ultimately become disruptive—that is, they are widely adopted and displace existing technologies from the marketplace or create entirely new markets. ARPA-E understands that definitive proof of market disruption takes time, particularly for energy technologies. Therefore, ARPA-E funds the development of technologies that, if technically successful, have clear disruptive potential, e.g., by demonstrating capability for manufacturing at competitive cost and deployment at scale. ARPA-E funds applied research and development (R&D). The Office of Management and Budget defines “applied research” as an “original investigation undertaken in order to acquire new knowledge…directed primarily toward a specific practical aim or objective” and defines “experimental development” as “creative and systematic work, drawing on knowledge gained from research and practical experience, which is directed at producing new products or processes or improving existing products or processes.” Applicants interested in receiving financial assistance for basic research (defined by the Office of Management and Budget as “experimental or theoretical work undertaken primarily to acquire new knowledge of the underlying foundations of phenomena and observable facts”) should contact the DOE’s Office of Science. Office of Science national scientific user facilities (http://science.energy.gov/user-facilities/) are open to all researchers, including ARPA-E applicants and awardees. These facilities provide advanced tools of modern science including accelerators, colliders, supercomputers, light sources and neutron sources, as well as facilities for studying the nanoworld, the environment, and the atmosphere. Projects focused on early-stage R&D for the improvement of technology along defined roadmaps may be more appropriate for support through the DOE applied energy offices including: the Office of Critical Minerals and Energy Innovation (CMEI), the Office of Hydrocarbon and Geothermal Energy, the Office of Nuclear Energy, and the Office of Electricity. ARPA-E encourages submissions stemming from ideas that still require proof-of-concept R&D efforts as well as those for which some proof-of-concept demonstration already exists. Submissions can propose a project with the end deliverable being an extremely creative, but partial solution. Program Overview: Transuranic (TRU) elements—such as plutonium (Pu), neptunium (Np), and americium (Am)—are a significant source of fissile materials that are available across existing nuclear inventories and strategic reserves and have the potential to drive advanced reactor deployment. , The technical feasibility of recycling TRU elements into new fuels has been proven at the experimental scale. Advances in fabrication, safeguards, equipment design, and modeling will create the opportunity to transition from experimental success to commercial-scale deployment, enabling higher-throughput and lower-cost fuel production with real-time materials accountancy. The High-performance Optimized Recycled Nuclear Isotopes for Gen IV reactors (HORNIG) program will support research and development projects that deliver commercially deployable TRU fuel technologies, thereby strengthening U.S. energy security, reducing nuclear waste, and enabling long-term energy deployment for public benefit. The HORNIG program seeks to overcome key technical and economic barriers that have historically prevented using TRU fuels in commercial reactors and to create a clear path to domestic nuclear fuel security by supporting the design, fabrication, and qualification of TRU fuels. The program will fund coordinated, multidisciplinary efforts to deliver transformative advances in fuel performance, manufacturability, cost, and regulatory readiness. Technologies developed under this program must have the potential to enable the following program metrics: • A domestic TRU fuel supply chain • A levelized cost of fuel (LCOF) = 1¢/kWh • TRU fuel qualification and regulatory acceptance within seven years By enabling production of advanced reactor fuels from domestically sourced fissile materials, the program will reduce dependence on imported uranium and enrichment services, expand U.S. fuel supply options, and support establishment of a closed fuel cycle. These objectives support ARPA-E’s statutory goals of improving energy security and resilience, improving the management of radiological waste, and maintaining U.S. technological leadership in energy technologies. If successful, HORNIG will strengthen U.S. energy security and infrastructure resilience and deliver lasting public benefit through reliable nuclear power. To view the NOFO in its entirety, please visit https://arpa-e-foa.energy.gov

NIMH - National Institute of Mental Health

Project Summary Region-specific brain organoids derived from human induced pluripotent stem cells (hiPSCs) provide a tractable platform to study disease mechanisms in a human cellular context. Moreover, organoids have the capacity to generate a diversity of cell types that can be maintained in culture long-term and assembled in vitro to form physiologically relevant connections. However, despite this exciting potential, the high variability of organoid differentiation across and within hiPSC lines have halted their broad utility and significantly hindered biological and technical interrogations of human neural development reproducibly and at scale. To address these challenges, this MPI group has established the Brain Organoid Hub at Emory University. A primary goal for the hub, and for this proposal, is to understand the underlying biology of progenitor cells that contribute to variability in 3D neural cultures. Additionally, we hope to develop metrics of hiPSC biology (molecular and/or morphological) that accurately predict the likelihood of successful organoid differentiation, as well as attributes of young organoids that correlate with robust molecular and functional maturation at later stages of culture. Towards this goal, we have designed three specific aims to test the hypothesis that progenitor cell states contain information about future differentiation potential. First, we will use molecular profiling of hiPSCs prior to their 3D formation to ask whether cell state heterogeneity and/or specific gene programs correlate with successful cortical organoid differentiation. Second, we will use machine learning-based approaches to ask whether morphological features of hiPSC growth dynamics are correlated with organoid success. Finally, we will address questions of organoid maturation, and ask whether molecular readouts of young organoids can predict successful functional maturation in late-stage organoid cultures. Altogether, these complimentary aims will not only (1) help reveal fundamental principles that contribute to variation in neurodevelopmental patterning, and (2) provide new assays for improving brain organoid reproducibility while avoiding costly and uninformative differentiations, but also 3) uncover novel biological insights into the genetic programs underlying neural cell specification and maturation. This knowledge could circumvent wasteful studies and instead lead to inclusion of additional biological replicates (hiPSC lines) in experiments. Importantly, our questions are deliberately crafted to align with the core objectives of the Brain Organoid Hub, to ensure reproducibility, efficient resource management, accessibility, and effective dissemination in the field of brain organoid cultures.

U.S. National Science Foundation

Hispanic Serving Institutions (HSI) are an important component of the nation s higher education ecosystem and play a critical role in realizing the National Science Board Vision Report for a more diverse and capable science and engineering workforce. Aligned with this vision and the NSF Strategic Plan 2022 -2026 the goals of the NSF HSI Program are to: 1. Enhance the quality of undergraduate science, technology, engineering, and mathematics (STEM) education at HSIs. 2. Increase the recruitment, retention, and graduation rates of students pursuing associate s or baccalaureate degrees in STEM at HSIs. Meeting these goals requires institutions to understand and embrace their students strengths, challenges, identities and lived experiences. This can happen in many ways and across many areas of an institution. As such, the IUSE: HSI program provides multiple opportunities to support an institution s goal to become more student centered, including theEquitable Transformation in STEM Education (ETSE) competition. This competition includes the following tracks: Departmental/Division Transformation Track (DDTT) - New Institutional Transformation Track (ITT) Emerging Faculty Research Track (EFRT) - New HSI Program Resource Hubs (Hubs) This solicitation will also accept conference proposals and planning proposals, as defined by the PAPPG. The ETSE competition focuses on (1) institutional transformation projects that support HSIs in their effort to achieve equity in STEM education, and (2) the infrastructure the HSI-Net network of resource hubs which supports the overall program goals. Institutions are encouraged to consider how their HSI designation, and their organizational mission align to better support STEM success of all students. The ETSE competition welcomes proposals that look to implement and evaluate promising practices and/or conduct research related to broadening participation or improving recruitment, retention, graduation, and other successful outcomes in STEM undergraduate education. The ETSE solicitation supports projects designed to catalyze change and help HSIs meet students where they are, accounting for their assets and the challenges they may face. Identities and experiences are not determined solely by membership in a single monolithic population of students (e.g., Hispanic, first-generation, commuter, etc.). Consequently, institutions are expected to use institutional data to identify equity gaps, identify areas of need, and unpack the factors that shape students individual identities and shared experiences. The perspectives gained from this data should be central to the design of the proposed project. Please see below for specific information about each track. While proposals are focused on mechanisms for transforming undergraduate STEM education, projects should also consider student voices and include mechanisms to aggregate and analyze existing student feedback and collect quantitative and qualitative student data throughout the life of the proposed project.

Honda Foundation

Funds youth development, STEM education, and environmental programs through employee-driven community grants.

Honeywell Foundation

Funds STEM education, environmental sustainability, and workforce development in Honeywell communities.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT In the developing embryo, in adult tissues such as the skin and intestine, and in many types of cancer, stem cells exist in close association with a supporting niche. The importance of the niche cannot be overstated. It both regulates stem cells’ ability to self-renew and controls these cells’ survival and differentiation. As a result, niche function influences many aspects of human health and disease. Niche function is often associated with the proximal signals they send to direct stem cell behavior. How a niche coordinates the timing, intensity and duration of the signals controlling stem cell activity remains poorly understood and represents a critical knowledge gap in our understanding of basic stem cell biology. Niches often have a precisely defined spatial organization that includes multiple cellular and extracellular matrix components. This organization is critical for niche function which suggests that niche structure is likely critical for stem cell regulation. My lab’s long-term goal is to discover how the structure and organization of a niche facilitate its ability to precisely control the signaling environment experienced by stem cells. We study the germ line in C. elegans as a model for this process. The germ line’s simple, well-understood developmental program, along with the extensive genetic toolkit and ease of 4D in vivo imaging available in C. elegans, make it an ideal system to investigate the structure and function of a niche. In worms, assembly of a functional niche is essential for controlling germline stem cell quiescence during embryo development and for balancing proliferation and differentiation in larval and adult animals. We focus on two components of niche structure which control these processes and are found in many different types of niches in other animals. First, we are investigating how the organization of the extracellular matrix in the niche contributes to its function. The extracellular matrix provides both biochemical and mechanical signals to adjacent cells. Our research will uncover how this essential niche component is constructed and remodeled during embryonic and larval development, and how it’s mechanical properties determine stem cell quiescence and proliferation. Second, a prominent yet poorly understood feature of many stem cell niches is that niche cells extend membrane protrusions over the surfaces of stem cells. This is called wrapping. We are studying the developmental basis of wrapping in the germline niche to identify fundamental adhesive, signaling and polarity mechanisms driving cellular wrapping. We are also investigating how wrapping functions to modulate the signaling environment experienced by germline stem cells, either by amplifying useful signals from the niche or by excluding signals from surrounding tissues. Our research is uncovering fundamental mechanisms for niche-stem cell regulation, advancing our basic understanding of basement membrane structure and function, and providing new insights into the mechanisms cells use to modulate signal transduction.

NIAID - National Institute of Allergy and Infectious Diseases

Summary The gastrointestinal (GI) tract is the largest mucosal surface in the body. A single cell layer thick intestinal epi- thelium separates the host interior from luminal pathogens while at the same time a series of intercellular junc- tions, including tight junctions, allow for selective movement of nutrients, ions, and water. Maintenance of this barrier is critical for a healthy intestine and its disruption leads to diseases such as diarrhea, a common outcome of enteric viral infections. Notably, the GI tract is not uniform; it exhibits distinct anatomical and functional prop- erties between the small and large intestine, including variations in tight junction protein expression. Enteric pathogens evolved to overcome the intestinal barrier to infect the host. However, how intestinal regionalization impacts pathogenesis of human enteric viruses is largely unknown. Human astroviruses (HAstV) are a good model to address this fundamental question in viral pathogenesis. We have demonstrated that epithelium-only human intestinal organoids (HIO), which are “miniguts” derived from stem cells isolated from human intestinal biopsy tissues or surgical resections, support HAstV infections from all clades and in all segments of the intestine. HAstVs are highly prevalent viruses that infect the entire lengths of the GI tract causing mostly pediatric diarrhea but can also cause disseminated disease in the immunocompromised. They are genetically diverse and classi- fied into classical human astroviruses, serotypes 1 – 8 (HAstV 1- 8), and two non-classical clades, VA and MLB. In vitro work from polarized model colonic epithelial Caco-2 cells suggests that the pathogenic mechanism of classical HAstV-1, but not VA1, occurs when the enterotoxin function of the HAstV-1 capsid disrupts tight junc- tions by downregulating occludin. Our new findings demonstrate that VA1 alters electrical conductance of T84, another model human colonic epithelial cell line, (but not Caco-2) by modulating a different group of tight junction transmembrane proteins, the claudins. Some members of the claudin family but not occludin exhibit intestinal segment specific expression patterns. This raises the fundamental question whether HAstVs may interact with the intestinal epithelial barrier in a segment-specific manner and positions HIO as an ideal non-transformed and physiologically relevant model of the human intestinal epithelium for detailed mechanistic studies of HAstV inter- action with the small and large intestine. The goal of our research is to advance our understanding of HAstV pathogenesis by determining the interaction of HAstVs with the intestinal epithelial barrier. Towards that end, we will use a combination of virological, molecular, genetic, and imaging approaches to pursue the following aims: 1) Investigate barrier properties of the small and large intestinal epithelium infected with HAstVs, and 2) Deter- mine whether the VA1 spike changes claudins and paracellular permeability. These aims are in direct response to NIH Notice of Special Interest (NOSI) AI-23-048, as they will “improve understanding of basic virology of understudied viruses such as HAstV”. This research has high potential for transformative impacts on our under- standing of the pathogenesis of viral gastroenteritis and intestinal epithelial biology.

OD - NIH Office of the Director

PROJECT SUMMARY Gastrointestinal acute radiation syndrome (GI-ARS) is a consequence of exposure to high doses of ionizing radiation and is characterized by extensive damage to the intestinal epithelium that leads to loss of barrier function, sepsis, and in some cases mortality. Currently there is a critical need to develop new physiologically relevant human models to study GI-ARS and to evaluate potential medical countermeasures (MCMs). This project aims to establish human intestinal organoids (HIOs) as a robust in vitro model for GI-ARS using high- content imaging approaches to assess the therapeutic potential of a microbial based MCM. Optimization of radiation dosing will be performed using a large cohort of HIOs that allow assessment of sex, age, and intestinal region on the response to radiation to be interrogated. A library of biomarkers of radiation damage will be assembled using novel screening approaches that combine multi-omics analysis, bioinformatic pipelines, and network analysis. Following biomarker identification, Cell Painting, a high content morphological profiling technique, will be implemented to characterize the cellular response to radiation treatment in a high throughput manner using a scanning disc confocal microscope and customized analyses package. The generation of detailed cellular and subcellular phenotypic profiles that associate with radiation will be used to enable rapid, quantitative assessment of therapeutic efficacy of a microbial based MCM. The MCMs to be tested are Limosilactobacillus reuteri 6475 (LR6475) organisms as a modality to deliver key growth factors necessary to promote regeneration and repair of the intestinal epithelium that target the intestinal stem cell. At the completion of the proposed studies, HIO will be validated as a translatable model for GI-ARS and a novel imaging based phenotypic screening pipeline for radiation injury and MCM evaluation will be established. This work will significantly advance efforts in radiation countermeasure development, facilitate future therapeutic screening and support preparedness for radiological emergencies.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Protective humoral immunity is mediated by both long-lived memory B cells (MBC) and antibody secreting plasma cells (ASC). Recently it has become increasingly clear that MBC and ASC are in fact composed of functionally diverse subpopulations that can be distinguished based on unique surface marker expression, tissue localization, and the B-cell receptor (BCR) isotype expressed. During a humoral immune response, naïve B cells (nB) give rise to differentiated subsets, with the ultimate composition of the MBC and ASC pool primarily influenced by the antigen, the involvement of T cells, and the cytokine environment. Given the evidence for increased heterogeneity, it is surprising that we currently lack the genetic tools to further interrogate the complexity, molecular properties, and immunological importance of the known B cell fates. A precise phenotypic analysis of MBC and ASC subsets cannot currently be performed with current Cre-lox mouse models. Cis- regulatory elements (CEs), or enhancers, are DNA sequences that act to promote cell-type and context-specific gene expression programs. These sequences are regulated by epigenetic mechanisms, which act to control the accessibility of CEs to DNA-binding transcription factors. Over the past several years, we have characterized the epigenetic architecture that controls primary humoral immune responses to T cell dependent and independent antigens, integrated newly published datasets defining MBC subsets, and generated new preliminary data defining the CEs of ASC expressing distinct BCR isotypes. These data have revealed specific CEs that are active in defined stages of B cell differentiation, including IgA ASC and extrafollicular (EF)-MBC that arise independently of a germinal center reaction. Therefore, we hypothesize that cell-type specific CEs can be co-opted to provide precise genetic manipulation that enables functional exploration of B-cell subsets. To address this, we propose two aims designed to 1) develop new Cre recombinase tools for genetic manipulation of IgA ASC and 2) map the CEs for EF-MBC that arise during influenza infection and integrate CEs specific for EF-MBC to allow precise genetic editing of this MBC subset. These aims will utilize novel hematopoietic stem cell engineering to rapidly generate chimeric mice expressing genes of interest; therefore, bypassing the need to generate transgenic animals. Completion of these aims will provide a novel set of DNA sequences that allow for MBC and ASC specific genetic manipulation. These tools are critically important begin to derive the biology, molecular properties, and importance of the entire spectrum of B-cell differentiation to humoral immunity. If successful, it also has the potential to redefine how Cre recombinase vectors are engineered and further our understanding of CE biology in B cells and the immune system.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT Neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), exhibit neuropathological features (e.g., amyloid and p-tau accumulation) that disproportionately affect certain brain regions and subsets of cells. This selective vulnerability to neuropathological features has been recognized for decades, yet its underlying molecular mechanisms remain unclear. Long COVID, a prevalent post-pandemic condition with largely unknown disease mechanisms, is reported to exhibit p-tau pathology, linking it to neurodegeneration. Current methods, including single-nucleus RNA sequencing, are unable to integrate spatial and pathological contexts with transcriptomic profiles, limiting direct molecular characterization of disease pathology at the single cell level. Here, I propose to leverage single-cell spatial multi-omics on postmortem brain tissues to in-situ capture molecular and cellular alterations that directly linked to selective vulnerability to neuropathological features across AD, FTLD, and Long COVID at single-cell resolution—insights previously unattainable. I will also identify potential pathological modulators using functional genomics in human induced pluripotent stem cell (iPSC)-derived neurons. In this proposal, Aim1 will characterize neuropathological and transcriptomic alterations in Long COVID and compare findings to AD and FTLD. Aim2 will leverage same- slide single-cell spatial multi-omics with neuropathology phenotyping (Xenium 5K + Phenocycler-Fusion) and define the molecular-neuropathological signatures specific to amyloid, neuronal p-tau, and glial p-tau pathology in AD, FTLD and Long COVID. Aim3 will employ CRISPRi/a screening in iPSC-derived neurons to identify neuronal p-tau modifiers from signatures uncovered in Aim2. By integrating single-cell spatial multi-omics with functional genomics, this project will bridge the gap between neuropathology and molecular profiling, provide novel insights into selective vulnerability to neuropathological features and identify pathological modulators. My long-term goal is to become a physician(neuropathologist)-scientist leading a NIH-funded research laboratory focused on neurodegenerative diseases and COVID-19. This five-year mentored career development plan will provide the necessary training for my transition to independence, emphasizing expertise in single-cell spatial transcriptomics and proteomics, bioinformatics, imaging analysis, iPSC technology and CRISPR-based functional genomics. Additionally, I will deepen my knowledge of neuropathology, molecular genetics, and clinical aspects of AD, FTLD, and Long COVID. I have assembled a multidisciplinary mentorship team of distinguished physician-scientists that includes Dr. Daniel Geschwind (primary mentor), Drs. Harry Vinters and Shino Magaki (co-mentors), and advisory members Drs. Inma Cobos, Vivek Swarup, and Edward Lee. With UCLA's cutting- edge facilities, exceptional research environment, and strong clinical resources, this award will prepare me to become a competitive neuropathologist-scientist, advancing our understanding of neurodegenerative diseases and COVID-19 while informing therapeutic strategies.

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies targeting adaptive immune checkpoints have improved cancer outcomes, but innate immune checkpoints also play crucial roles in cancer immune evasion and are promising targets for immunotherapy. The innate immune system uses "eat-me" and "don’t-eat-me" signals to regulate phagocytosis, essential for maintaining tissue homeostasis and preventing cancer. Phagocytic cells have also recently emerged as new key actors in the success of immunologically mismatched allograft transplants through human leucocyte antigens (HLA) allorecognition. Thus, identifying the molecular patterns and receptors governing phagocytosis is vital for understanding cancer clearance and transplantation. Recently published studies of the PI revealed novel functions for Vascular Cell Adhesion Molecule-1 (VCAM1) on healthy and malignant hematopoietic stem cells (HSCs). We have found that VCAM1 is highly expressed on healthy HSCs, serving as an innate immune checkpoint for entry into the bone marrow by providing a "don't-eat-me" signal in the context of major histocompatibility complex (MHC) class-I presentation. In addition, we found that leukemia cells exploit this tolerance mechanism to avoid innate immune recognition, suggesting that the VCAM1-receptor axis is a promising target for immunotherapy. However, the specific receptor mediating this interaction remains unknown. In preliminary studies, we employed proteomics and AlphaFold modeling to identify novel VCAM1 receptor candidates on phagocytic cells. Our Specific Aim 1 focuses on identifying the VCAM1 receptor promoting immune tolerance and leukemia evasion and validating its function in vitro and in vivo using mouse and human models of leukemia. Specific Aim 2 will assess the impact of inhibiting or deleting VCAM1 receptor signaling on myeloid and lymphoid leukemia cell clearance, as well as allogeneic transplantation outcomes. Successful completion of this research will advance knowledge of innate immune recognition mechanisms, identify new leukemia immunotherapy targets, and improve outcomes in stem cell transplantation. 1

NCI - National Cancer Institute

PROJECT SUMMARY In the past decade, immunotherapies have revolutionized the way many cancers are treated. By activating the immune system, these treatments enable the body's own defense mechanisms to attack cancer cells and halt tumor growth. Unfortunately, many cancers are refractory to these new therapeutics and there continues to be a desperate need for drugs with novel mechanisms of action to enhance the long-term effectiveness of cancer treatment. Type I interferons (IFN-Is) are attractive candidate drugs to fill this role since these cytokines “interfere” with the growth of tumors; indeed, manufactured IFN-Is have been used in the clinic to treat more than 10 types of cancers but the outcomes were somewhat disappointing. Due to negative feedback regulation of IFN signaling, most interferon stimulated genes (ISGs) are transiently expressed, and these feedback mechanisms ultimately suppress the anticancer effectiveness of administered IFN-Is over time. This process also limits the signaling from intrinsically produced IFN-Is, critical to the success of chemo-, radiation, and immuno-therapy. In pioneering work over many years, we unraveled the details of the negative feedback pathway and identified the ubiquitin protease family member USP18 as the central regulator driving feedback suppression of the IFN-I response. By single-cell RNA-seq analysis, we revealed that cancer stem cells are especially sensitive to USP18 depletion- triggered cell death. These novel and exciting discoveries demonstrate that targeting USP18 represents an effective and promising, yet to-date underutilized, therapeutic option for treating cancer by directly promoting immunogenic cancer stem cell death and by increasing both innate and adaptive immune responses against cancer. With the goal to discover small molecule inhibitors of the USP18-mediated IFN-I feedback loop, we conceptualized an innovative high-throughput screening (HTS) assay based on our novel IFN-I signaling biosensor cell line with CRISPR/Cas9 inserted fluorescent reporters and validated it in a pilot screen of known compounds. Since it is currently unclear which specific mechanisms for USP18 inhibition are druggable by small molecules and provide the best therapeutic window, we opted for a phenotypic discovery approach combined with target deconvolution assays. Additionally, we developed a pipeline of secondary and mechanistic assays that validates the hit compounds and provides initial insight into the targeted components of the feedback pathway. Here we propose to 1) identify inhibitors of the USP18-mediated feedback pathway through a large phenotypic HTS, 2) validate the hits in secondary assays and map their effects to the specific pathway components, and 3) evaluate the therapeutic anti-cancer potential of the final chemical probes. Successful completion of these studies will identify and validate small molecule inhibitors of USP18 that can be used to probe the therapeutic potential of the different mechanisms for inhibiting the USP18-mediated negative feedback regulation of IFN signaling. Additionally, such immunomodulators could be further development toward a new class of cancer therapeutics.

NINDS - National Institute of Neurological Disorders and Stroke

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare inherited neurodegenerative disease caused by expansion of a glutamine-coding CAG repeat in the Atrophin-1 (ATN1) gene. The role of ATN1 in the adult central nervous system (CNS) is currently unknown. However, the clinical manifestations of DRPLA lead to a constellation of symptoms that are inversely correlated to CAG-expansion (CAGEX) size, with younger individuals typically carrying the largest number of CAG repeats, experiencing the most severe disease burden. Importantly, young patients with DRPLA face poor cognitive outcomes, including developmental delay, with a prevalence of high-frequency, drug-resistant seizures and epilepsy diagnosis. The mechanisms leading to increased neuronal hyperexcitability and seizure risk in DRPLA patients are presently unknown. Thus, preclinical humanized in vitro and in vivo models of ATN1 CAGEX represent a novel platform to expediently assess innovative therapies for symptomatic seizure control and disease modification. Further, these models allow exquisite translational fidelity to define how ATN1 mutations lead to neuronal hyperexcitability. Indeed, our labs have recently identified both an increased neuronal excitability using a versatile patient-specific induced pluripotent stem cell (iPSC)-derived in vitro system and an altered seizure threshold in vivo using a novel mouse model expressing a humanized ATN1 CAG repeat expansion. In vitro studies in human patient iPSC-derived cortical neurons using live-cell calcium imaging and multi-electrode array recordings demonstrate altered neuronal network activity by manifesting increased calcium spike amplitude and hypersynchronization, both characteristics simulating epileptiform-like phenotypes. Notably, we have preliminarily demonstrated that neuronal hyperexcitability (in vitro) and seizure threshold (in vivo) can be rescued with exogenous administration of investigational ATN1- silencing antisense oligonucleotides (ASOs). Further, circadian behavior of ATN1 CAGEX mice administered the investigational ASO were normalized, indicating a disease-modifying effect in this clinically relevant rodent model of DRPLA. The present proposal thus aims to expand these pilot studies to address the mechanism behind ATN1 CAGEX-dependent spontaneous seizure risk and neuronal hyperexcitability in vitro and in vivo, and to further define the potential seizure-modifying effects of ASO infusion in the early disease course. Aim 1 will utilize patient iPSC-derived neuro-glial model to characterize the physiological and molecular mechanism of the DRPLA epileptiform and asses ASO efficacy in phenotypic rescue. Aim 2 will then expand on these in vitro studies to establish whether Atn1 CAGEX mice exhibit spontaneous recurrent seizures and define the degree to which an investigational ASO infusion influences the occurrence of these events and improves neuropathological burden. This study will deepen our understanding of the impact of ATN1 CAGEX on pathological neuronal activity and seizure risk while establishing proof-of-principle evidence that ASO intervention is a disease-modifying strategy for DRPLA, a progressive myoclonic epilepsy syndrome with no palliative or curative options.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

As we age, the efficiency of the hematopoietic system declines resulting in bone marrow failure symptoms in some individuals. Myelodysplastic syndromes (MDS) are disorders primarily affecting older adults and impact around 10,000 individuals annually in the United States. MDS arises due to dysfunction in hematopoietic stem and progenitor cells (HSPCs), leading to ineffective blood cell production and an elevated risk of progression to acute myeloid leukemia (AML). The mechanisms driving the onset and progression of MDS remain poorly understood. Studying the germline and somatic genetics of inherited bone marrow failure syndromes (iBMFS) offers valuable insights into the fundamental processes required to maintain healthy hematopoiesis. While the biology of iBMFS is shaped by specific genetic defects, many of the principles uncovered are broadly relevant to HSPC regulation. Individuals with germline heterozygous loss-of-function mutations in the ATPase DEAD-box Helicase 41 (DDX41) gene tend to develop MDS later in life, resembling the sporadic form of the disease. These patients often present with high-risk MDS characteristics, such as elevated blast counts and increased risk of transformation to AML. Paradoxically, they also tend to have a more favorable overall prognosis compared to others with transforming MDS. This suggests that DDX41-mutant MDS follows a uniquely aggressive yet somewhat protective disease trajectory. We propose that studying this atypical, late-onset iBMFS can reveal broader principles governing hematopoietic fitness and dysfunction during aging. Using a germline ddx41 HET mutant zebrafish that develops age-associated MDS-like symptoms, we uncovered elevated inflammation as well as stem cell stress and quiescence gene signatures enriched in HETs. We will test the hypothesis that DDX41 mutations synergize with age-associated inflammatory signaling and/or secondary somatic mutations to exacerbate hematopoietic dysfunction. In Aim 1, we will examine how DDX41 insufficiency cooperates with age- associated cGAS activity promotes HSPC malfunctioning. In Aim 2, we will explore how co-mutations of DDX41 with CUX1 contribute to aberrant differentiation. Gaining a better understanding of this pathway may lead to the discovery of biomarkers and therapeutic strategies applicable to both hereditary and sporadic forms of MDS.

NEI - National Eye Institute

SUMMARY The optic nerve is comprised of Retinal Ganglion Cells (RGCs), which are the only neurons capable of relaying visual information from the retina to the brain. A variety of conditions contribute to ganglion cell loss, which can result in total blindness. Many therapies have been designed to protect the optic nerve, but to date no therapy has been developed that reverses damage that has already occurred. While regeneration of the optic nerve may seem ambitious, there are several species such as zebrafish that can completely regrow a damaged optic nerve, or replace lost RGCs, providing a roadmap to how this process could be reconstituted in humans. Significant progress has been made towards this goal across various mammalian systems, with particular mutations or transgenic overexpression paradigms leading to partial reconstitution of the regenerative process. These preliminary findings provide strong support that thorough characterization of the regenerative process has the potential to translate into effective therapies. However, several methodological barriers have hampered thorough understanding of RGC regeneration in zebrafish. Firstly, multiple stem cell pools are capable of producing RGCs in zebrafish, which has made it difficult to define the precise sequence of events underlying RGC production. Further, it is unclear what signals tune this regenerative response to ensure that the appropriate number and types of cells are replaced. We have generated the hypothesis that the regenerative response must be initiated by a damage signal derived from dying RGCs. We have designed a series of aims to (1) characterize the process of RGC regeneration in zebrafish with unprecedented detail, (2) identify the origins of the cues that regulate RGC regeneration, and (3) identify factors that drive RGC regeneration even in the absence of RGC loss. Together, these findings will highlight a collection of biomolecules that instruct MG to regenerate the optic nerve, which will have important translational ramifications for the treatment of Glaucoma.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT The preservation of organ functions, such as eyesight and mobility, is a significant health concern in the elderly population. Organ frailty and disease progression are associated with the dysregulation of tissue homeostasis, which is typically regulated by regenerative units, consisting of adult stem cells (ASCs) and neighboring niche cells that regulate ASC function. Therefore, to better understand why regenerative functions decrease in aged individuals, uncovering how aging alters niche cell types and expression (mRNA) is critical for discovering regenerative therapeutical approaches. The potential to uncover molecular mechanisms to reverse age-related disorders prompted me to spatially profile microenvironmental niches in young, old, and regenerated tissues. I have recently developed Ex-Scope, which integrates Expansion Microscopy and Seq-Scope, a submicrometer-resolution ST (spatial transcriptomic) technology, to obtain a high-resolution multi-Omic method that represents an order of magnitude improvements over Seq-Scope. With the assistance of Dr. Guo, who has extensively worked on planarian tissues, we optimized Ex-Scope to spatially profile planarian tissue. Planarians are capable of regenerating any lost body part, but most importantly, regenerated tissues have a youthful tissue architecture; thus, making them ideal to study tissue homeostasis and rejuvenation. Using mRNA single-cell data on young, old, and regenerated planarians, as a reference dataset (obtained by Dr. Guo), we will provide spatial insight into rejuvenating mechanisms between microenvironmental niches and stem cells. Concurrent, we will demonstrate the advantageous resolution of Ex-Scope by profiling RNA granules in planarian stem cells and oocytes (young, old, and regenerated), which are compartmentalized biomolecules that regulate transcription in stem cells and the establishment of pluripotency. In aim 1) we propose to characterize RNA granules and soluble transcriptomes in planarian stem cells and oocytes, with a hypothesis that the granular structures in oocytes and ASCs would have transcriptome contents distinct from soluble cytoplasm, and 2) we propose to profile microenvironmental niches and their changes during aging and rejuvenation, with a hypothesis that aging and rejuvenation will affect cellular (single cell), tissue-level (microenvironment) and subcellular level (RNA granule) transcriptome, each of which is important for tissue function and homeostasis. We expect that the current work will give us a systematic understanding of how aging deteriorates tissue function by altering transcriptomic structure at both microscopic and macroscopic levels, and how regeneration can reverse it and rejuvenate tissue homeostasis.