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NEI - National Eye Institute

Scarring of the stroma, the central layer of the cornea, due to Injury or infection, and severe stromal thinning due to keratoconus, cause corneal blindness worldwide. Cornea transplantation is often the only cure for many of these conditions, but limited supply of donor tissue is a major concern. Cell therapy for the stroma, still under development, primarily uses limbal stem cells from donor corneas. Therefore, there is a critical need for treatment of the stroma that will be independent of donor tissues. To address this need, we recently developed human cornea organoids (HCOs) that mimic the fetal cornea. Generated from induced pluripotent stem cells (iPSC) the HCOs present a breakthrough model system of co-differentiating stromal keratocyte, epithelial and endothelial cells in a more cornea-like 3-dimensional extracellular matrix (ECM). Our single cell RNA sequence (scRNA-seq) study shows that HCOs harbor stromal progenitor and fetal corneal keratocyte-like cells which may have the desired long-lasting functionality in the cornea. In addition, we found that HCOs express ECM genes and produce a stroma rich in collagen type III, fibronectin, and other ECM components known to make up the immature corneal stroma. Additionally, as the TGF-𝛽 network is the master regulator of healthy and fibrotic ECM, this network must be specifically regulated in HCOs to maintain its regenerative ECM quality. Therefore, we hypothesize that the HCO is a novel stromal therapy resource and a corneal surrogate for identifying TGF-𝜷 signals that promote healthy but limit fibrotic ECM production. Our preliminary data shows that a mix of stem, progenitor and differentiating cells can be rapidly extracted from the HCO stroma without further monolayer culturing. Injected into decellularized donor corneas the HCO-stromal cells can integrate in the tissue. Second, the extracted ECM from HCOs promote migration in cell culture and corneal wound healing in the mouse. Importantly, as the HCO expresses all major receptors and transcription factors, it is feasible to investigate the TGF-𝛽 network in this model. Aim 1 will determine if extracted HCO-stromal cells will integrate and function in a decellularized donor cornea. Aim 2 will test whether the HCO-stromal cells or the extracted HCO-ECM promote scarless wound healing in the mouse. Aim 3 will identify specific TGF-𝛽 signals that underpin the immature ECM of HCOs. Our findings will develop the HCO as a promising donor tissue-free source of regenerative biomaterial and cells and identify regenerative TGF-𝛽 signals for future treatments of corneal scarring.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract Epilepsy is a severe neurological disease affecting more than 65 million people worldwide and is characterized by unpredictable abnormal electrical discharges resulting in recurrent seizures. About one third of patients with epilepsy suffer from intractable seizures that do not respond to anti-seizure medications (ASMs). Neurosurgical interventions and neurostimulator devices are useful options for only a fraction of patients with drug-refractory seizures, underscoring the urgent need to develop new therapies. One strategy with considerable promise is to engraft new neurons to provide enhanced GABAergic inhibition in an activity-dependent manner. However, use of fetal neurons for cell therapy is associated with practical and ethical issues. Therefore, to overcome such hurdles, in our previous studies, we pioneered the transplantation of human pluripotent stem cell (hPSC)- derived medial ganglionic eminence (MGE)-type human cortical interneurons (cINs) into epileptic mouse brains and demonstrated their integration into dysfunctional circuitry, accompanied by the suppression of seizures and comorbid behavioral abnormalities. Furthermore, more recently, we have determined the optimal stage of human cIN differentiation to ensure maximal integration into host circuitry as well as safety without risk of tumor formation, and developed a method to efficiently generate these safe and highly migratory populations of synchronized early postmitotic cINs from hPSCs in large quantities, bringing cell therapy for epilepsy one step closer to reality. Furthermore, we have successfully tested the efficacy of human early postmitotic cINs in 2 different models of temporal lobe epilepsy (TLE), observing >80% of seizure reduction. With these strong previous studies, now we are ready to embark clinical translation of this novel and restorative therapy for epilepsy patients with limited options. Thus, in this proposed study, we will scale up production of synchronized early postmitotic cINs that are optimal for grafting under cGMP condition. For added safety, we will utilize well- characterized HLA-edited hypoimmunogenic iPSCs to minimize the need for immunosuppression for off-the- shelf use of human cINs. We will also extensively analyze the produced early postmitotic cINs’ phenotype, efficacy, safety, tumorigenesis and biodistribution to seek IND approval. Once we obtain IND approval, we will do a first-in-human clinical trial of early postmitotic cIN grafting with a primary goal of safety analysis, while also checking efficacy as a secondary measure. This will be done in patients with intractable TLE who are candidates for resection while they undergo intracranial EEG to identify the seizure focus without additional invasive steps. Completion of these studies is pivotal for translating this experimental therapy into a viable therapeutic strategy for intractable epilepsy.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

PROJECT SUMMARY The inability to build physiologically relevant in vitro tissue models greatly limits both research capabilities and regenerative therapies. Hydrogels are frequently used to mimic the extracellular matrix (ECM) which surrounds cells in tissue, and the physical properties of these scaffolds can be tailored to individual cell types. These scaffolds are often made from polymers crosslinked by peptides that are substrates for cell-secreted proteases to enable the encapsulated cells to spread and migrate within the matrices. A challenge for these systems is that each cell type within a tissue can have a unique set of ideal matrix parameters. For instance, most tissues are highly vascularized, but endothelial network formation is optimized within matrices that are very soft, while other physiological processes, such as osteogenic differentiation, are typically optimized within stiffer, more highly crosslinked hydrogel matrices. This highlights the need for making hydrogels with specific niches for each cell type. To address this need, we propose fabricating scaffolds in which cell-specific protease activity creates tailored microenvironments around individual cell types. Each cell type expresses a unique combination of proteases, and we have developed novel methods to identify peptides that are specifically cleaved by individual cell types. We are also able to determine whether these peptides are cleaved near the surface of the cell or by soluble proteases that induce bulk matrix degradation. Using a "split-and-pool" peptide synthesis technique, we can generate more than 300 variants of protease-substrate peptides to tune the degradation rates to desired values. We hypothesize that hydrogels crosslinked with peptides with optimal spatiotemporal degradation kinetics will have increased biological performance over existing crosslinking peptides. We will test this hypothesis in two Aims: In Aim 1, we will use a split-and-pool synthesis technique to identify hydrogel crosslinking peptides whose degradation kinetics are optimized for either osteogenic differentiation of human mesenchymal stem cells (hMSCs) or vasculogenesis of human umbilical vein endothelial cells (hUVECs). We will also make peptides which are conjugated with chemically-labile bonds that will enable quantification of the fraction of crosslinks cleaved during culture, which will couple physiological behavior in gels to dynamic changes in hydrogel structure. In Aim 2, we will develop co-culture hydrogels that contain both hMSCs and hUVECs to identify a single peptide that supports both osteogenesis and vasculogenesis within hydrogels. This will pioneer the use of hydrogel crosslinking peptides to simultaneously promote multiple physiological processes within a single system. The proposed research plan combines biomaterial synthesis, analytical chemistry, and cell culture to develop a versatile platform that can be used across tissue systems to improve our ability to model tissues in vitro and regenerate them in vivo.

NIA - National Institute on Aging

PROJECT SUMMARY Dementia, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD), are major contributors to mortality, morbidity, and worldwide healthcare expenditure. FTD is fatal and incurable and represents 10-20% of all dementia cases. Approximately 9% of all FTD cases are caused by MAPT mutations (FTD-tau). Given that there are no effective treatments for FTD (an Alzheimer’s-related dementia), novel therapeutic strategies are urgently needed. Targeting the MAPT gene itself by CRISPR/Cas9 genome editing may provide a curative intervention. We have established a novel dual sgRNA strategy, which can excise the mutant MAPT allele in patient-derived induced pluripotent stem cells (iPSCs). The excision preserves expression from the non-diseased allele. In Aim 1, we will maximize efficiency of our gRNA strategy by identifying sgRNA pairs that excise the MAPT transcription and translation start sites on the mutant allele with high efficiency and no side effects in patient iPSCs and post- mitotic patient-derived neurons in vitro. We will then deliver our optimized editing reagents to an FTD mouse model (PS19) via AAV PhPeB, which cross the blood brain barrier and achieve brain-wide distribution. In Aim 2 we will optimize AAV dosing and determine whether CRISPR editing can reverse pathologic hallmarks of FTD- tau or only prevent their onset. In Aim 3 we will determine how three genes embedded in MAPT affect normal and pathologic tau expression, potentially providing new therapeutic targets, and in any case a useful context for any therapy that aims to alter tau expression or the MAPT locus. With the successful completion of these studies, we will have optimized a candidate gene editing strategy that targets the MAPT mutation, and reaches the highest therapeutic efficacy in human neurons in vitro. We will also determine the therapeutic window in vivo. Our editing strategy will then be ready to pair with human-specific delivery reagents that we and others are developing as they become available. We will have additionally addressed a number of open questions in the field, including whether editing efficiencies in post-mitotic neurons differ from mitotic cells, how to deliver CRISPR/Cas9 with multiple sgRNAs widely throughout the mouse brain, whether it is possible to reverse or arrest clinical phenotypes in symptomatic mice, and the impact of embedded genes on MAPT physiologic and pathologic function. This work will inform our understanding of normal MAPT function and provide proof-of-concept and IND-enabling studies for a novel MAPT CRISPR therapeutic. Our approach is likely also applicable to sporadic FTD-tau and other tauopathies, including progressive supranuclear palsy (PSP), Alzheimer’s disease (AD) and corticobasal degeneration (CBD). Our overarching goal is to accelerate genome editing for neurodegenerative diseases toward the clinic.

NHGRI - National Human Genome Research Institute

Project Abstract Understanding the evolution of regulatory DNA has been impeded both by the lack of functional genomics data available outside of a select group of organisms, as well as by the barriers to implementing genome engineering in diverse organisms for functional analysis. Given the low conservation of the genomic regulatory landscape, this presents a serious impediment to studying genome evolution and limits the value of a phylogenetic approach to understanding human genomic function. We recently developed the Big-IN genome engineering technology to rewrite large genomic loci in-place through delivery of DNA payloads upwards of 160 kb. Here we propose a new synthetic regulatory genomics approach for characterization of the regulatory function of genomic sequence across a phylogenetic tree. We will analyze the activity of a set of model loci in mouse embryonic stem cells by delivering orthologous sequences from 5 vertebrate species. We will employ two strategies to: (i) deliver larger regions (up to 160 kb) including the full gene replacing full orthologous mouse locus; and (ii) combinatorially assess pairs of shorter (~1 kb) candidate enhancer sequences. These data will open a novel approach to understanding genomic regulatory syntax through phylogenetic analysis, as well as a bridge to direct functional assessment of human disease-relevant loci.

NCATS - National Center for Advancing Translational Sciences

Abstract The UNC Readiness And Preparation for Informatics and Data Science Careers, or RAPID program, is a 15- week program designed to inspire undergraduates to integrate data science tools into translational science that is rigorous and promotes health outcome optimization across populations. The program components include: 1) didactic training in data science and differential health outcomes in populations 2) mentorship to complete a research practicum, 3) interactions with established investigators who are applying data science tools across the spectrum of translational science and 4) development of a mentoring network comprised of peers, near-peers, staff and faculty. The program is delivered in a hybrid fashion with 2 weeks of preparatory work done by the participant before they arrive at UNC, followed by a 10-week residential summer intensive program. During the 10 weeks, participants are guided through each phase of a data science project from idea formulation to dissemination and are taught choices they can make to incorporate principles of population conscious analysis and rigor into each phase. Participants have the option of 3-weeks of support after they leave UNC to complete and present the research project. Eligible participants are rising sophomore and junior STEM majors. Innovative aspects of the program include use of a conceptual model that gives participants agency to choose their research topic, the application of data science skills in project-based learning, and interactions with community advisory boards to further reinforce communication skills and health outcome optimization across populations.

U.S. National Science Foundation

Cultivating Cultures for Ethical STEM (CCE STEM)funds research projects that identify (1) factors that are effective in the formation of ethical STEM researchers and (2) approaches to developing those factors in all the fields of science and engineering that NSF supports. CCE STEM solicits proposals for research that explores the following: What constitutesresponsible conduct for research (RCR), and which cultural and institutional contexts promote ethical STEM research and practice and why?' Factors one might consider include: honor codes, professional ethics codes and licensing requirements, an ethic of service and/or service learning, life-long learning requirements, curriculaor memberships in organizations (e.g.Engineers without Borders)that stress responsible conduct for research, institutions that serve under-represented groups, institutions where academic and research integrity are cultivated at multiple levels, institutions thatcultivate ethics across the curriculum, or programs that promote group work, or do not grade. Do certain labs have a culture of academic integrity'? What practices contribute to the establishment and maintenance of ethical cultures and how can these practices be transferred, extended to, and integrated into other research and learning settings? Successful proposalstypicallyhavea comparative dimension, either between or within institutional settings that differ along these or among other factors, and they specify plans for developing interventions that promote the effectiveness of identified factors. CCE STEM research projects will use basic research to produce knowledge about what constitutes or promotes responsible or irresponsible conduct of research, and how to best instill studentswith this knowledge. In some cases, projects will include the development of interventions to ensure responsible research conduct. Proposals for awards from minority-serving institutions (e.g. Tribal Colleges and Universities, Historically Black Colleges and Universities, Hispanic-Serving Institutions, Alaska Native or Native Hawaiian Serving Institutions), women's colleges, and institutions primarily serving persons with disabilities are strongly encouraged. Proposals including international collaborations are encouraged when those efforts enhance the merit of the proposed work by incorporating unique resources, expertise, facilities or sites of international partners. The U.S. team's international counterparts generally should have support or obtain funding through other sources.

U.S. Mission to Zimbabwe

Culture of Research and University Exchanges Prep program (CRUX) in One Health and Agricultural STEM

U.S. National Science Foundation

Cyber-Enabled Discovery and Innovation (CDI) is NSF s bold five-year initiative to create revolutionary science and engineering research outcomes made possible by innovations and advances in computational thinking. Computational thinking is defined comprehensively to encompass computational concepts, methods, models, algorithms, and tools. Applied in challenging science and engineering research and education contexts, computational thinking promises a profound impact on the Nation s ability to generate and apply new knowledge. Collectively, CDI research outcomes are expected to produce paradigm shifts in our understanding of a wide range of science and engineering phenomena and socio-technical innovations that create new wealth and enhance the national quality of life. CDI seeks ambitious, transformative, multidisciplinary research proposals within or across the following three thematic areas: From Data to Knowledge: enhancing human cognition and generating new knowledge from a wealth of heterogeneous digital data;Understanding Complexity in Natural, Built, and Social Systems: deriving fundamental insights on systems comprising multiple interacting elements; andVirtual Organizations: enhancing discovery and innovation by bringing people and resources together across institutional, geographical and cultural boundaries. With an emphasis on bold multidisciplinary activities that, through computational thinking, promise radical, paradigm-changing research findings, CDI promotes transformative research within NSF. Accordingly, investigators are encouraged to come together in the development of far-reaching, high-risk science and engineering research and education agendas that capitalize on innovations in, and/or innovative use of, computational thinking. Research and education efforts around the world are beginning to address various aspects of the CDI themes, and CDI projects are expected to build upon productive intellectual partnerships involving investigators from academe, industry and/or other types of organizations, including international entities, that advance CDI objectives within the rapidly evolving global context. Congruent with the three thematic areas, CDI projects will enable transformative discovery to identify patterns and structures in massive datasets; exploit computation as a means of achieving deeper understanding in the natural and social sciences and engineering; abstract, model, simulate and predict complex stochastic or chaotic systems; explore and model nature s interactions, connections, complex relations, and interdependencies, scaling from sub-particles to galactic, from subcellular to biosphere, and from the individual to the societal; train future generations of scientists and engineers to enhance and use cyber resources; and facilitate creative, cyber-enabled boundary-crossing collaborations, including those with industrial and international dimensions, to advance the frontiers of science and engineering and broaden participation in STEM fields. Two types of CDI awards will be supported as a result of the FY 2010 CDI competition:Type I awards will require efforts up to a level roughly comparable to: summer support for two investigators with complementary expertise; two graduate students; and their collective research needs (e.g. materials, supplies, travel) for three years.Type II awards will require larger (than Type I) efforts up to a level roughly comparable to: summer support for three investigators with complementary expertise; three graduate students; one or two senior personnel (including post-doctoral researchers and staff); and their collective research needs (e.g. materials, supplies, travel) for four years. The integrative contributions of the Type II team should clearly be greater than the sum of the contributions of each individual member of the team.In subsequent years, subject to availability of funds, funding opportunities will be provided for three classes of awards, Types I and II as defined above, and Type III as defined below:Type III awards will require the engagement of larger (than Type II) multidisciplinary teams, roughly comparable to multiple senior investigators with complementary expertise, multiple graduate students, several senior personnel, and their collective research needs (e.g. materials, supplies, travel) for up to five years. As for Type II awards, the integrative contributions of the Type III team should be clearly greater than the sum of the contributions of each individual member of the team.

U.S. National Science Foundation

Cyberspace has transformed the daily lives of people. Society's overwhelming reliance on cyberspace, however, has exposed the system's fragility and vulnerabilities: corporations, agencies, national infrastructure, and individuals continue to suffer cyber-attacks. Achieving a truly secure cyberspace requires addressing both challenging scientific and engineering problems involving many components of a system, and vulnerabilities that stem from human behaviors and choices. Examining the fundamentals of security and privacy as a multidisciplinary subject can lead to fundamentally new ways to design, build, and operate cyber systems, protect existing infrastructure, and motivate individuals to learn about cybersecurity. The Cybersecurity Enhancement Act of 2014, as amended by the National Defense Authorization Acts for 2018 and 2021, and the CHIPS and Science Act of 2022, authorizes the National Science Foundation (NSF), in coordination with the Office of Personnel Management (OPM) and the Department of Homeland Security (DHS), to offer a scholarship program to recruit and train the next generation of cybersecurity professionals to meet the needs of the cybersecurity mission of federal, state, local, and tribal governments. The goals of the CyberCorps Scholarship for Service (SFS) program are aligned with the U.S. strategy to develop a superior cybersecurity workforce. The program goals are to: (1) increase the number of qualified and diverse cybersecurity candidates for government cybersecurity positions; (2) improve the national capacity for the education of cybersecurity professionals and research and development workforce; (3) hire, monitor, and retain high-quality CyberCorps graduates in the cybersecurity mission of the Federal Government; and (4) strengthen partnerships between institutions of higher education and federal, state, local, and tribal governments. While all three agencies work together on all four goals, NSF s strength is in the first two goals; OPM s in goal (3); and DHS in goal (4). The SFS Program welcomes proposals to establish or to continue scholarship programs in cybersecurity. A proposing institution must provide clearly documented evidence of a strong existing academic program in cybersecurity. In addition to information provided in the proposal narrative, such evidence can include ABET accreditation in cybersecurity; a designation by the National Security Agency and the Department of Homeland Security as a Center of Academic Excellence in Cyber Defense Education(CAE-CDE), in Cyber Operations (CAE-CO) or in Research (CAE-R); or equivalent evidence documenting a strong program in cybersecurity. Service Obligation: All scholarship recipients must work after graduation in the cybersecurity mission of a federal, state, local, or tribal government organization, or certain other qualifying entities, for a period equal to at least the length of the scholarship. The SFS Program also supports efforts leading to an increase in the ability of the United States higher education enterprise to produce cybersecurity professionals. Funding opportunities in this area are provided via the Secure and Trustworthy Cyberspace - Education Designation (SaTC-EDU) and other programs (see the section "Increasing National Capacity in Cybersecurity Education" for more details.)

Danaher Foundation

Funds STEM education, health, and community development through targeted philanthropy.

U.S. National Science Foundation

The goal of this activity is to foster novel, transformative, multidisciplinary approaches that address the use of large data sets to create actionable knowledge for improving STEM teaching and learning environments (formal and informal) in the medium term, and to revolutionize learning in the longer term. These approaches will involve the work of learning scientists, STEM disciplinary experts, computer scientists, statisticians, database experts and educational researchers who design and study learning environments. Among the potential benefits of integrating approaches from these disciplines are improving student learning and engagement, optimizing personalized instruction, and supporting rapid decision making to help educators respond more effectively to the learning needs of individuals and groups of learners in multiple settings. These approaches may be risky but should have the potential to rapidly advance the field. The scope of this activity does not include infrastructure development focused on data base design and development for education domains. The new approaches envisioned in this solicitation will require the generation and use of data that range from micro-level data on individual learners, to data from online learning sources (such as massively open online courses), to meso-level data from the classroom that provide information to students and teachers about how learning is progressing, to macro-level data such as school, district, state, and national data, including data from federal science and policy agencies. Participants in the Ideas Lab, selected through an open application process, will engage in an intensive five-day residential workshop, the development of multidisciplinary collaborative proposals through a real-time and iterative review process, and, for the participant teams invited to submit full proposals, the subsequent submission of full proposals.

Datadog Foundation

Funds STEM education and tech career pathways for youth in underserved communities.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

(PLEASE KEEP IN WORD, DO NOT PDF) Terminal differentiation of erythroid cells occurs in the erythroid-specific niches. The most studied erythroid niche is the erythroblastic island (EBI), which comprises a central macrophage surrounded by developing erythroblasts. While studies over the past decades have identified many genes that are functionally important for EBI, the field faces major caveats. Current knowledge of EBI is predominantly derived from studies of in vitro reconstitution of mixed cell populations that do not recapitulate in vivo niches. In addition, the EBI compositions in human hematopoietic tissues are unknown. In this project, we aim to uncover the anatomy, composition, and functions of EBIs in mice and humans using unbiased approaches through multiple spatial mapping technologies. Through spatial transcriptomic studies, we revealed a higher positive spatial correlation between erythroid cells and C1q+ macrophages than with other macrophages, suggesting that C1q+ macrophages are likely the EBI macrophages in mice. This strong positive correlation between C1q+ macrophages and erythroid cells was also observed in newborn bone marrow and adult spleen under physiologic and stress conditions. We applied the same technologies to human hematopoietic tissues. In contrast to mice, we did not observe a strong positive correlation between erythroid cells and C1q+ or other macrophages in the human hematopoietic tissues. Instead, there is a strong association between erythroid progenitors and maturing erythroid cells. This erythroid self-assembled EBI structure was recapitulated in a human induced pluripotent stem cell (iPSC)-derived bone marrow organoid model. Furthermore, we identified ICAM4 as a critical erythroid surface protein that maintains erythroid-centered EBIs in humans. These preliminary studies uncover unique erythroid niches in mice and humans. Based on this evidence, we hypothesize that mouse and human EBIs have distinct structures and molecular features that help sustain terminal erythropoiesis. In this project, we propose to investigate the composition of macrophage-centered EBIs and the mechanisms of C1q in EBI macrophages in hematopoietic tissues in mice. The same approaches will be used to study ICAM4 and erythroid self-assembled EBIs in humans. Furthermore, EBI responses and their molecular mechanisms under stress and disease conditions in mice and humans will also be investigated. The success of this project will not only advance the understanding of red cell biology but also offer invaluable insights into hematopoiesis as a whole.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

The adrenal cortex is a vital endocrine organ that produces steroid hormones essential for the body's homeostasis. Its organization into three concentric zones enables spatially regulated production of distinct adrenal hormones, a process known as functional zonation. Structural or functional defects in the adult cortex cause primary adrenal insufficiency (PAI), a life-threatening condition affecting millions with no permanent cure, while developmental disruptions in adrenal formation or zonation can lead to congenital adrenal disorders and tumors. In rodents, zonation is established perinatally via centripetal migration and stepwise “transdifferentiation” of subcapsular progenitors within the definitive zone (DZ), regulated by opposing actions of WNT-activating signals from outer capsule (Cap) cells and adrenocorticotropic hormone (ACTH). However, species-specific differences in adrenal organogenesis and steroidogenesis limit the translational value of rodent models for human PAI. To bridge this gap, we developed the first human adrenal organoid model from induced pluripotent stem cells (iPSCs) that recapitulates adrenal development both in vitro and in vivo. This proposal aims to exploit this platform to define and assess the therapeutic relevance of cellular and endocrine/paracrine signaling mechanisms regulating adrenocortical development, with the goal of transforming treatment of life-threatening adrenal diseases. In humans, adrenal cortical zonation begins prenatally, forming three zones: the DZ, the glucocorticoid (cortisol)-producing transitional zone (TZ), and the androgen-producing fetal zone (FZ), which correspond to the functional zones of the adult adrenal cortex. Notably, our organoids can be reliably directed to produce CD10/MME⁺ DZ-like cell (DZLC) progenitors that exhibit striking similarity to in vivo human DZ cells. DZLCs can be further differentiated into TZ-like cells (TZLCs) through combined stimulation with RSPO3 (a Wnt- ligand dependent potentiator of Wnt signaling) and ACTH, and subsequently into FZ-like cells (FZLCs) with ACTH alone. Exogenous RSPO3 is dispensable when DZLCs are co-encapsulated with iPSC-derived RSPO3- secreting Cap-like cells (CapLCs), which mimic the native capsule and enable ACTH-driven transdifferentiation of DZLCs into TZLCs. This contrasts with rodents, where ACTH promotes and WNT suppresses TZ fate. Thus, our in vitro directed transdifferentiation data support the central hypothesis that prenatal human adrenocortical homeostasis is orchestrated by DZ progenitors within a capsular niche that, through self-renewal and transdifferentiation, give rise to both TZ and FZ under coordinated control of WNT and ACTH signaling. Supporting this, our preliminary data show that encapsulation of DZLCs with CapLCs restores functional zonation after transplantation into a hemi-adrenalectomized immunodeficient mouse model, resulting in a long-lived adrenal cortex producing both cortisol and androgens in an ACTH responsive manner. Leveraging our adrenal organoid platform, we will determine whether these progenitor-derived populations can integrate into the host endocrine axis to fully restore adrenal function in vivo.

NIA - National Institute on Aging

PROJECT SUMMARY Despite aging being a major risk factor for lung cancer incidence, most preclinical and clinical studies do not take aging into account. Thus, our understanding of how aging affects lung cancer progression, evolution and therapy response remains limited. The diversity of subpopulations of tumor cells, and their therapeutic responses are shaped by their complex interactions with the host. Our published studies support this, revealing a causal link between the systemic metabolic reprogramming driven by aging and lung cancer progression and immune evasion, putting aging and the changes it causes in the host at the center stage coordinating cancer evolution. Recently, we showed that beyond systemic changes, alterations in the composition and functional state of old lung tumor microenvironment (TME) shape niche signaling and drive divergent lines of lung cancer evolution resulting in age-dependent intrinsic differences in prognosis upon treatment with standard of care therapies. Yet, the age-driven niche-signaling mechanisms that fuel this divergent evolution remain unknown. Importantly, we have identified a damage-associated state of alveolar epithelial cells (alveolar differentiation intermediates, or ADIs) that accumulate within the NSCLC TME and the adjacent lung as a key difference between NSCLC in young versus old animals and patients. Strikingly, ADIs’ preferential accumulation within the old TME dominates the communication between NSCLC cells and the TME through ADI-produced ligands linked to stemness induction. Thus, in this research program we propose that the emergence of ADIs in the old lung TME shapes the niche signaling that NSCLC cells and the other TME cellular components are exposed to. We posit that these changes steer NSCLC evolution towards a primitive stem-like state with higher grade tumors that confer resistance to chemotherapies. We will leverage multiple model systems (human and mouse cancer cells lines, AT2-derived organoids and in vivo mouse models) combined with high throughput single cell technologies and mass spectrometry to characterize the mechanism that underlies the age-induced ADI accumulation within NSCLC TME (Aim 1) and determine the contribution of ADIs to the evolution of the primitive stem-like state of NSCLC (Aim 2). Consequently, the proposed studies will put forward defined roles for aging in NSCLC biology and inducing the evolution of chemoresistance states that confer poor prognosis, thereby bringing awareness to the need of tailoring treatments to the specific age and biology of the patient. Our studies will also unveil for the first-time normal alveolar cells as major regulators of the NSCLC progression. Moreover, they will also put forward age-specific targets and pathways for subsequent studies with the ultimate goal of leveraging this information therapeutically to improve outcomes of the most common and vulnerable NSCLC patient population: the elderly.

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

Project Summary/Abstract Epigenetic modifiers govern cell fate transition during animal development and their mutations drive multiple human congenital disorders; however, the molecular mechanisms underlying the roles of epigenetic modifiers in these normal and pathological processes remain poorly understood. It is widely believed that epigenetic modifiers function through the epigenetic marks they catalyze. Nevertheless, the discoveries of catalytic- independent role of epigenetic modifiers challenge this view, raising the question about the biological function of epigenetic marks. Mono-methylation of histone H3 at lysine 4 (H3K4me1) is a reliable mark of enhancers that shape cell identity, and its reconfiguration accompanies the differentiation of pluripotent stem cells, suggesting that the regulation of H3K4me1 plays an instructive role in cell fate transition. To examine this hypothesis, we investigated the catalytic function of LSD1 and LSD2, two paralogous histone demethylases targeting H3K4me1, in regulating gene expression during cell fate transition. Using state-of-the-art approaches such as precise genome engineering, epigenetic and transcriptomic profiling, and stem cell differentiation, we demonstrate functional synergism between the demethylase activity of LSD1 and LSD2 in regulating cellular differentiation. Based on these compelling preliminary data, here we propose to dissect the molecular mechanisms underlying how the demethylase activity of LSD1/2 regulates cell fate transition. The results generated from our proposed studies will not only reveal novel molecular mechanisms underlying the roles of H3K4me1 in gene regulation and cell fate transition, but also provide insights into understanding the pathogenesis of diseases driven by LSD1/2 loss-of-function. This research aligns with the NIH mission to advance our understanding of fundamental biological processes and contribute to knowledge relevant to developmental disorders and regenerative medicine.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Hematopoietic stem and progenitor cells (HSPCs), which include hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), give rise to all blood and immune cells across the lifespan. HSCs first emerge during gestation and ultimately becoming the adult hematopoietic compartment. Human studies have associated events during pregnancy such as maternal infection, diet, or exposure to microbes with increased risk of immune dysfunction in offspring; however, the mechanisms behind this are unknown. Since hematopoietic progenitors arise during gestation and seed the adult hematopoietic compartment, we hypothesize that prenatal inflammation reprograms the cellular output of distinct fetal HSPCs, thus influencing postnatal immune function. Recent data has suggested that fetal HSCs and fetal MPPs emerge independently from the intra-embryonic aorta during development. This suggests that fetal MPPs and fetal HSCs of distinct origin have specific functions and relative contributions to postnatal blood production at homeostasis and during inflammatory insults, but this has not been directly investigated. Our working hypothesis is that fetal MPPs drive the response to prenatal inflammation in order to preserve the HSC pool, shaping the postnatal hematopoietic compartment. We will test our hypothesis by integrating genetic fate mapping experiments, transplantation assays, transcriptomics, and early life infection models. Our preliminary data suggests independent emergence of fetal MPPs and fetal HSCs from the developing aorta and has revealed the first evidence of functional differences between fetal MPPs and HSCs. We have found that fetal MPPs are the first responders to Type II-IFN mediated prenatal inflammation and expand the pool of downstream myeloid cells, which remain expanded in the postnatal period. Our findings also support the idea that timing of emergence influences the functional output of fetal progenitors at steady state and in response to inflammation. The objective of this proposed work is to comprehensively examine the specific effects inflammation has on fetal HSCs and MPPs and will define the mechanisms by which prenatal inflammation shapes the adult hematopoietic system. We will determine how inflammation experienced in utero influences offspring immunity and response to postnatal immune challenges at the level of fetal HSPCs. We anticipate that the insights gained from this proposed work will help inform underlying causes of disease that may start during development.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY This proposal aims to elucidate how the dynamic oligomeric transitions of Copper Transporter 1 (CTR1) couple copper (Cu) homeostasis with neuronal developmental pathways. Cu is an essential micronutrient for neuronal function, and a deficiency of Cu in early life can have devastating impacts on development. We recently discovered that CTR1 can reversibly transition between trimeric and monomeric states to regulate Cu uptake. Moreover, a CTR1 mutant that fails to undergo these de-oligomerization events impairs growth factor–activated signaling pathways. These findings challenge the conventional view of CTR1 as a stable trimeric transporter and suggest that Cu-induced allosteric changes in CTR1 oligomerization directly influence neuronal health and development. Our primary objectives are to determine the mechanisms driving CTR1’s oligomeric-state transitions and to clarify how these shifts impact CTR1’s function in Cu regulation and stem cell differentiation. Using innovative single-molecule assays, such as single-molecule localization microscopy and in-cell oligomer stoichiometry assays, we will visualize and quantify CTR1’s oligomeric states in situ. Human embryonic stem cell (hESC)-derived neurons will provide a physiologically relevant model for these studies. In addition, we will conduct comprehensive proteomic and biochemical analyses to uncover key protein interaction networks that modulate CTR1 trafficking and function. This proposal aims to address two main research directions: (1) identifying the triggers behind CTR1’s transition from trimeric to monomeric forms under conditions of excess Cu, and (2) exploring CTR1’s role in stem cell differentiation, with emphasis on its interactions with Laloo and SNT1 to regulate neuronal maturation. This multidisciplinary effort will be supported by collaborations with experts in neurobiology, membrane trafficking, Cu homeostasis, and stem cell research, thereby ensuring a robust and integrative approach. The insights gained will provide significant contributions to understanding CTR1’s role in Cu regulation and cell development, as well as elucidating its broader impact on neuronal health and disease. Additionally, the methodologies developed will have broad applicability for examining the dynamics of other membrane proteins. This project is innovative for several reasons: it introduces novel single-molecule assays for in situ studies, uses a more physiologically relevant hESC model, and addresses a critical gap in our understanding of allosteric regulation mediated by CTR1 in neurons. The insights gained from this research could have far-reaching implications in neurobiology and may inform strategies for treating Cu-related neurodegenerative diseases.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most aggressive and common primary brain tumor, posing significant clinical challenges despite multimodal treatments like surgical resection, chemotherapy, and radiation. The prognosis for GBM patients remains poor, primarily due to its inevitable recurrence and resistance to standard therapies. Therefore, identifying novel molecular drivers of GBM progression is essential for developing more effective treatments. Over the past decade, the isolation of glioblastoma stem cells (GSCs) has provided crucial insights into tumor initiation, maintenance, and recurrence, positioning them as a major therapeutic target. Concurrently, advances in genomic research have revealed a vast landscape of long noncoding RNAs (lncRNAs), offering new opportunities to influence gene regulation and cancer biology. Although tens of thousands of lncRNAs have been discovered, only a small fraction have been functionally characterized. Emerging evidence suggests that lncRNAs exhibit cell-type-specific expression, distinct subcellular localization, and critical roles in key oncogenic processes, including proliferation, invasion, and therapy resistance. These properties highlight their potential as both biomarkers and therapeutic targets in GBM. In this study, we propose to characterize the nuclear-retained oncogenic lncRNA, LINC01896, which is significantly upregulated in GSCs. Our preliminary data show that depletion of LINC01896 impairs GSC proliferation and stemness, emphasizing its critical role in GSC maintenance. Furthermore, higher level of LINC01896 correlates with poor overall and disease-free survival in GBM patients, suggesting its potential as both a prognostic biomarker and a therapeutic target. We will investigate how LINC01896 regulates global gene expression in GSCs and identify the molecular pathways driving GSC proliferation and stemness. Additionally, we will examine the functional impact of LINC01896 knockdown in GSCs using antisense oligonucleotides (ASOs) and evaluate these effects in preclinical 3D cerebral organoid model, which mimic the human brain's cellular complexity and tumor microenvironment. Furthermore, we will map the transcriptomic landscape of GSCs and their tumor microenvironment using single-cell RNA sequencing. Together, these studies aim to elucidate the regulatory role of LINC01896 in GSC biology and pave the way for the development of innovative lncRNA-based therapeutic strategies for GBM patients.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The mammalian heart undergoes profound transcriptional and phenotypical remodeling during postnatal development, a process known as cardiac maturation. However, the molecular mechanisms driving this transition is not fully understood, posing a major challenge in cardiac regenerative medicine, where induced cardiomyocytes from pluripotent stem cell differentiation or non-myocyte reprogramming exhibit an overall immature phenotype that severely limits their application in cell therapy and in vitro disease modeling. In this K99/R00 application, I propose to integrate cutting-edge single cell multiomics with state-of-the-art computational methods to unravel the cell-type-specific gene regulatory networks governing cardiac maturation, and develop a novel dual-reporter system to model and enhance cardiac maturation in vitro and in vivo. During the K99 phase, I will characterize the epigenomic changes of the mouse heart during postnatal development at a single cell resolution using various single cell multiomic technologies (Aim 1), and construct cell-type-resolved gene regulatory networks underlying cardiac maturation using bioinformatic approaches coupled with deep learning (Aim 2). I will also establish cell culture and mouse models with CRISPR-mediated knock-in of dual-fluorescent reporters to track and assess cardiomyocyte maturation (Aim 3a). During the R00 phase, I will experimentally characterize key regulatory elements and novel transcriptional regulators using functional genomic approaches (Aim 3b). I will also leverage these findings to enhance the maturation of in vitro-derived cardiomyocytes for improved therapeutic potential (Aim 3c). The expected outcomes of my proposed research will deepen our understanding of postnatal cardiac development and uncover new therapeutic strategies to improve cardiac function after injury. My career goal is to lead an independent research group that develops and employs innovative technologies to study the regulatory mechanisms underlying cardiac development, regeneration, and disease. In my K99 phase, I will acquire crucial knowledge and skills in advanced single cell genomics and computational biology to complement my previous expertise in developmental biology and cardiac research. My career development will be supported by an exceptional mentoring and advisory committee from UCSD/Salk/HHMI, along with world-class resources, training opportunities, and institutional support at UC San Diego. These elements will provide a strong foundation for my successful transition to an independent tenure- track faculty position.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Post-transcriptional regulation is a critical mechanism controlling cellular differentiation and development, driven by coordinated interactions between RNA-binding proteins (RBPs) and RNA modifications. This proposal investigates the interplay between Quaking (QKI) isoforms and N7-methylguanosine (m7G) RNA modifications. QKI isoforms exhibit distinct subcellular localizations and functions, yet their roles in binding m7G-modified transcripts at 3′ untranslated regions (UTRs) during differentiation remain poorly understood. The central hypothesis is that QKI isoforms regulate differentiation by binding m7G-modified mRNAs at 3′ UTRs, with cytoplasmic QKI6 stabilizing transcripts essential for myeloid differentiation. Furthermore, m7G modifications may independently regulate gene expression in ways yet to be defined. Our preliminary evidence demonstrates that cytoplasmic QKI isoforms mediate myeloid differentiation, highlighting a critical gap in understanding m7G’s functional roles in steady-state cellular processes. Current methods for detecting m7G methylation at single- nucleotide resolution face technical limitations. This project addresses these challenges by employing direct RNA long-read sequencing and orthogonal methods to map m7G modifications with precision and identify QKI isoform-specific mRNA targets. Integrating these approaches will elucidate how QKI-m7G interactions influence myeloid differentiation, with broader implications for RNA modifications in stem cell biology, neurodevelopment, and cancer. Over the next five years, the laboratory’s mission is to (1) develop novel methods for base-resolution m7G detection, (2) define mRNA targets regulated by distinct QKI isoforms, and (3) determine the functional impact of QKI-m7G interactions on myeloid differentiation. These studies will advance understanding of RNA modification-driven gene regulation and may inform therapeutic strategies for diseases such as leukemia. By resolving the interplay between QKI and m7G at 3′UTRs, this work will reveal how their coordination regulates cellular functions across human cell types, directly addressing NIGMS’s mission to support foundational discovery science. The proposed research will provide mechanistic insights into RNA modifications dysregulated in cancer, potentially uncovering new targets for therapeutic intervention in malignancies and developmental disorders.

NHLBI - National Heart Lung and Blood Institute

Project Abstract The alveolar epithelium is composed of two distinct cell types—alveolar epithelial type I (AT1) cells, which facilitate gas exchange, and alveolar epithelial type II (AT2) cells, which act as progenitors for AT1 cells. Successful lung repair following alveolar injuries requires AT2 cell proliferation and differentiation into AT1 cells, a process that involves the restructuring of gene regulatory networks and cell-type specific chromatin landscapes that underly these two cell fates. A dysfunctional regenerative response has been observed in a variety of severe lung diseases, involving AT2 cells acquiring a pathologic, alveolar-basal intermediate (ABI) cell state at the expense of an AT1 fate. The mechanisms that facilitate the cell fate decisions involved in AT2 cell maintenance and differentiation are not well understood, which has resulted in a lack of effective treatments to promote alveolar regeneration. This project aims to identify and characterize regulatory, 3-dimensional “hubs” of chromatin interaction that instruct distinct alveolar epithelial cell fates, and to determine how these hubs and their associated transcription factors regulate the acquisition of healthy and disease-associated states. Using human induced pluripotent stem cell (iPSC) models of AT1- and AT2-like cells (iAT1s and iAT2s), we will apply advanced chromatin mapping techniques to identify cell-type specific enhancer-promoter interactions and to characterize chromatin hubs that potentially regulate normal AT1 and AT2 cell identity. In Aim 1, we will map these interactions in healthy iAT1 and iAT2 cells, comparing their chromatin landscapes to pinpoint regulatory hubs that we hypothesize are responsible for cell-type specific gene expression. In Aim 2, we will explore the effects of haploinsufficiency of the lung lineage transcription factor, NKX2-1, on chromatin topology of iAT2 cells, hypothesizing that reduced NKX2-1 expression disrupts normal AT2 cell identity and favors a pathological ABI state. The findings from this research will enhance our understanding of the chromatin-based mechanisms that control lung cell fate decisions and provide insights into how disruptions of chromatin organization contribute to pulmonary disease.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Skeletal muscle contains a population of adult stem cells called satellite cells or muscle stem cells (MuSCs) that are responsible for regeneration after injury. MuSCs utilize gene expression programs to maintain quiescence and differentiate after injury and a key regulator of gene expression is splicing, which uniquely changes when transcripts interact with nuclear speckles. Nuclear speckles are membrane-less biomolecular condensates that phase separate proteins, RNAs and chromatin, but how these organelles regulate molecular processes in MuSCs remains unknown. Key experiments from our laboratory provide rigorous support for a role of nuclear speckles and splicing in MuSC function, which were attenuated in old age. The overarching objective of this program is to establish a systems-based approach to understand how nuclear speckles and alternative splicing contribute to MuSC programs of activation and regeneration across lifespan. In Aim 1, we will demonstrate that the loss of a nuclear speckle scaffolding protein, Srrm2, will reduce regenerative potential of muscle stem cells. In Aim 2, we will establish that increases in oxidative stress from old age attenuate nuclear speckles and RNA splicing that regulate muscle stem cell activation and repair of muscle injury. In both Aims, we will use novel transgenic animal models, sophisticated bioinformatics analysis and highly innovative molecular tools to build a comprehensive and new understanding of nuclear speckles influence on alternative splicing and stem cell activity. Successful completion of this program will advance our knowledge of fundamental cell biology for regenerative medicine, and provide a myriad set of insights across molecular, cellular and tissue scales.