Human immune system humanized modeling of down syndrome immune responses to pluripotent stem cells

The National Institutes of Health Office of the Director awarded University of Wisconsin–Madison a $1.49 million Project Grant under the Trans-NIH Research Support program (CFDA 93.310) on September 16, 2025, with a completion date of August 31, 2027. This research initiative focuses on developing and characterizing humanized mouse models to study Down syndrome immune responses, specifically addressing the immune dysregulation associated with trisomy 21. The project will establish the Thymocyte-HU humanized mouse model using neonatal thymocytes and develop a complementary neothermic model incorporating umbilical cord blood hematopoietic cells and neonatal thymus fragments to investigate T cell maturation, development, and antigen-specific responses in a Down syndrome context.

The deliverables include creation of mosaic Down syndrome hypoimmune gene-edited induced pluripotent stem cell (iPSC) lines for cardiovascular cell therapy development, comprehensive characterization of T cell phenotypes and functions in the humanized models, and establishment of biological materials and data resources for the Down syndrome research community to support clinical translation of iPSC therapies. The Morgridge Institute for Research, Inc., serving as a sub-awardee since January 2026, provides critical bioinformatics and high-throughput analysis support, including bulk and single-cell RNA sequencing analysis, data management, and bioinformatics services to enable the project's scientific objectives.

Abstract

People with down syndrome (DS) have increased susceptibility to immunological pathologies, certain cancers, congenital heart defects, and cognitive decline, all of which involve immune dysregulation. Elevated inflammatory cytokines associated with DS may impact disease treatments, such as immunotherapy for leukemia and organ transplantation. Current mouse models do not fully replicate the unique immunobiology of human trisomy 21, highlighting the need for more robust animal models to study DS-specific immune responses to therapies. Humanized mouse models, created by introducing human hematopoietic cells into immune-deficient mice, offer unique opportunities to evaluate pharmacological and cellular therapies in a DS context. No DS humanized mouse models of this class have been reported until now. We developed a DS humanized mouse model, the thymocyte-HU, using neonatal thymocytes. Preliminary data show long-term human t cell engraftment and reduced graft-VS-host disease susceptibility. This R24 project aims to fully characterize the phenotype and function of engrafted t cells in thymocyte-HU mice, which will provide a better understanding of DS t cell maturation and antigen-specific responses in the context of clinical translation of hypoimmune induced pluripotent stem cell (ipsc) therapies for people with DS. Additionally, we will leverage another new DS neothy model, combining umbilical cord blood hematopoietic cells and neonatal thymus fragments to study DS t cell development and function within a full immune repertoire. In our specific aims, we will establish multiple biological material and data resources for the DS research community, by: 1) creating mosaic DS hypoimmune gene-edited ipsc lines. We will differentiate these cells into cardiovascular cell therapies and assess in vitro DS immune responses; 2) characterizing thymocyte-HU model t cell phenotype and function using bioluminescence imaging, single cell RNA sequencing, luminex cytokine assays, and flow cytometry; 3) assessing DS neothy humanized mouse t cell dynamics and immune responses to hypoimmune ipsc cardiovascular cell therapies using single cell RNA sequencing, epigenomics, bioluminescence imaging, luminex, flow cytometry, and histology; and 4) sharing biological and data resources by distributing humanizing tissues, gene-edited ipsc lines, and experimental data and by providing access to humanized mice through the university of wisconsin humanized mouse core. Successful completion of this project will provide novel DS humanized mouse models and data resources to the research community, fostering high-impact discoveries and potential new therapies for people with DS.