Project Summary This application is from the Nebraska Research Network in Functional Genomics (NE-INBRE) in response to NOT-GM-24-001 regarding the availability of administrative supplements to IDeA Awards to Fund Team Science Development Projects. We have assembled a team of four Nebraska investigators with diverse and complementary backgrounds and expertise who will use cutting edge bioinformatic and human organoid approaches to investigate the molecular mechanisms regulating human hematopoiesis and bone related disease. Hematopoietic Stem Cells (HSCs) are governed by numerous positive and negative feedback signals within the bone marrow niche. The crosstalk between HSCs and cells in the microenvironment is critical for efficient production of blood cells and when it is perturbed, as with diseases like cancer, it can have serious consequences. However, the molecular mechanism regulating this interaction are not well understood and are difficult to model ex vivo. This project will combine expertise in hematopoiesis and bone marrow biology with state-of-the-art human bone marrow organoids and cutting edge genomic and bioinformatic analysis with a team science approach to address one of the main themes of the NE-INBRE. Goals of the Parent Award: The Nebraska Research Network in Functional Genomics (NE-INBRE) is aimed at developing the biomedical research capacity at institutions of higher education in Nebraska. The NE-INBRE is structured around two major components: Primary Undergraduate Institutions (PUIs) and PhD granting Research Institutions (RIs). Support for each PUI consists of: 1) campus research capacity development through support for faculty research and infrastructure enhancement, and 2) development of the undergraduate research pipeline of students through the NE-INBRE Scholars Program. Two features are associated with expanding research capacity at the RIs: 1) Providing significant support to multi-user core facilities in order to allow investigators from PUIs and RIs access to cutting-edge technology, and 2) providing support in the form of first-year graduate assistantships for NE-INBRE scholars who matriculate into PhD programs at the RI campuses. The three participating RIs in the research network include two campuses of the University of Nebraska system and one privately supported medical center. Cutting edge multi-user core facilities include cores in genomics, bioinformatics, and imaging. The three themes reflect the scientific foci of the NE-INBRE: cancer, cell signaling, and infectious disease. These themes serve to link faculty and students at the separate institutions into productive networks based on their areas of expertise and interest. Research Question to be addressed in the Supplement Proposal: Until recently, the major limitation in dissecting the complex interactions between the HSCs and the microenvironment has been the lack of suitable models. Traditional ex vivo co-culturing approaches do not replicate the complex architecture of the bone marrow. Mouse models have important limitations due to species difference between mice and humans. The primary goal of this proposal is to develop human bone marrow organoids as a tool to dissect the mechanisms regulating hematopoiesis in both the normal and the disease context. Establishing and refining this technique in Nebraska will reveal novel discoveries and lead to new research collaborations. To achieve this goal, we assembled a team of four accomplished investigators with expertise in human organoid biology, hematopoiesis, leukemogenesis, and bioinformatics. AIM 1 (led Dr. A. Black) will produce bone human marrow organoids for use in other Aims and develop tools to enhance use of these structures. AIM 2 (led by Dr. C. Guda) will develop machine learning models to characterize cellular and transcriptional hematopoiesis with human bone marrow organoids. AIM 3 (led by Dr. K. Hewitt) and AIM 4 (led by Dr. K. Hyde) will use the tools developed in AIM 1 to test mechanisms regulating human HSC self-renewal and differentiation under healthy and malignant conditions. Transcriptomic data from these aims will be incorporated into the models developed in AIM 2. Benefit of the Team Science Effort: Each sub-project in this proposal uses cutting-edge techniques and could be performed independently, but will greatly benefit from incorporating the tools, protocols, and advancements developed by the other projects. For instance, the tools developed in AIM 1 will be incorporated into the experimental plans for Aims 3 and 4. The computational characterization from AIM 2 will be used as a basis for comparison with the models of normal and malignant hematopoiesis developed in AIM 3 and 4, but also lead to new ideas for approaches in AIM 1. In addition, integration of the data from AIMs 3 and 4 has the potential to reveal the differences and similarities between different causes of hematopoietic dysfunction. The orthogonal tools and models developed under this award will lead to new insights and advancements that would not be possible as stand-alone projects. This project falls well within the scope of the parent NE-INBRE award, and the research questions proposed here do not duplicate any part of the work funded under the parent award.

Narrative Recently-developed human bone marrow organoid (hBMO) systems can mimic the biology of normal and diseased hematopoietic niches. Our goal is to build robust genetic and computational tools using hBMOs for disease modeling and drug screening. A team science approach is needed to generate reproducible hBMOs in vitro, test physiological and stress hematopoietic models, establish engraftment protocols for human leukemia, and build machine learning tools to map developmental trajectories and disease-induced changes in organoids.