Humanized mice (aka, mice with absent or limited immune responses that are transplanted with human immune cells, tissues and stem cells, etc.) are widely used as preclinical models to develop and evaluate novel therapies without putting the patients at risk. Over the past two decades, substantial progress has been made in developing humanized mouse models for modeling various human diseases (e.g., infectious diseases and cancer). Despite all of these successes, replicating human immune responses in humanized mouse models proves to be challenging. A major hurdle is the mouse host's lack of a human thymus gland -- the tissue that is responsible for the generation of T cells, which play such a key role in the human immune system. Human T cells developed in the mouse thymus are defective in engaging other human immune cells and fail to mount proper immune responses. The goal of this research project is to use human pluripotent stem cells (stem cells that can develop into numerous cell types) to generate an artificial thymus that can be used as a substitute to reconstruct the human immune system in humanized mice. Two key technologies will be developed and optimized: 1) novel biomaterial intervention methods to generate thymic cells from human pluripotent stem cells; and 2) innovative bioengineering processes to construct a functional artificial thymus. Successful replication of human immune responses in humanized mice with artificial thymus transplants will have tremendous scientific and translational impacts, including: 1) as a powerful tool to evaluate and optimize the effectiveness of immunotherapy; 2) to further understand disease cause; and 3) to develop novel immune interventions that are ready for clinical translation. This cross-disciplinary project will provide an excellent opportunity for minority women and individuals from underrepresented groups to participate in research and educational activities. The project is a culmination of diverse engineering techniques, encompassing biomaterials, stem cells and immune engineering. Hence, it will provide the participants with well-rounded training opportunities. Collaborative educational and outreach efforts include a joint summer internship program for underrepresented minority undergraduate students and development of course modules in Immune-engineering and Stem Cell Bioengineering that will be incorporated into online educational resources for K-12 students and teachers.

The goal of this project is to generate a humanized mouse model by implanting bioengineered human thymus organoids in CD34+ hematopoietic stem cell (HSC) engrafted NOD-scid.Il2rgnull (NSG) mice in order to promote human T cell development and adaptive immune responses. Preliminary studies have demonstrated the feasibility of establishing normal immune function in athymic mice by reconstructing a decellularized thymus with primary murine thymic epithelial cells (TECs). This project will take the next step by generating human thymus organoids from human pluripotent stem cells (hPSCs) and then using these organoids to produce a humanized mouse model. The Research Plan is organized under three aims. The first aim is to generate functional thymus organoids with hPSC-derived TECs. The hPSCs will be differentiated into TECs while encapsulated in alginate cultures. The derived hPSC-TEPCs will then be used to reconstruct decellularized thymus scaffolds. The functionality of thymus organoid constructs will be characterized in-vitro (to determine gene markers of mature TECS and early T-Cell development) and in-vivo (under the kidney capsules of nude mice). Ex vivo studies will be performed to test the functional efficiency of thymus organoid generated from hPSC-TEPCs to support ex-vivo T cell development. The second aim is to engineer compartmental organization of TECs (cortical (cTECS) and medullary (mTECS) regions) within the bioengineered thymus organoids. The hESC-TEPCs will be segregated into cTEC and mTEC spheroids that will be used to reconstruct the thymus organoid with the goal of reestablishing both cell-cell contact and compartmental organization. The 3D cellular organization, with reestablished cell-cell and cell-ECM communication, is expected to enhance long-term survival and function of the TECs within the thymic scaffold and to improve the selection and differentiation of subpopulations of T-cells. The third aim is to generate humanized mouse models with bioengineered thymus organoids. Humanized mice will be generated by transplanting cord blood CD34+ HSCs into myelosupressive agent preconditioned NSG mice, which are subsequently transplanted with thymus organoids bioengineered from hESC-derived TEPCs. Development of multiple hematopoietic lineages, especially the development of CD4+ and CD8+ T cells, will be characterized and the function of the cells will be examined. The models will be followed for 24 weeks post reconstruction. Successful development of a long lasting, functional human immune system in a mouse model will be advantageous for studying human immune responses and for developing proper therapeutics for cancer, infectious diseases, and autoimmune disorders.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

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Allegheny-Singer Research Institute
United States
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