Stephan A Ramos1, John J Morton2, Prabha Yadav1, Brendan Reed3, Sheila I Alizadeh1, Ali H Shilleh1, Loni Perrenoud2, James Jaggers4, John Kappler5, Antonio Jimeno6, Holger A Russ7. 1. Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, Colo. 2. Division of Medical Oncology, Department of Medicine, University of Colorado School of Medicine, Aurora, Colo. 3. Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, Colo; Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colo. 4. Surgery-Cardiothoracic Department, University of Colorado School of Medicine, Aurora, Colo. 5. Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, Colo; Department of Biomedical Research, National Jewish Health, Denver, Colo. 6. Division of Medical Oncology, Department of Medicine, University of Colorado School of Medicine, Aurora, Colo; Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Aurora, Colo. Electronic address: antonio.jimeno@cuanschutz.edu. 7. Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, Colo; Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Aurora, Colo. Electronic address: holger.russ@cuanschutz.edu.
Abstract
BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.
BACKGROUND: The thymus is a glandular organ that is essential for the formation of the adaptive immune system by educating developing T cells. The thymus is most active during childhood and involutes around the time of adolescence, resulting in a severe reduction or absence of naive T-cell output. The ability to generate a patient-derived human thymus would provide an attractive research platform and enable the development of novel cell therapies. OBJECTIVES: This study sought to systematically evaluate signaling pathways to develop a refined direct differentiation protocol that generates patient-derived thymic epithelial progenitor cells from multiple induced pluripotent stem cells (iPSCs) that can further differentiate into functional patient-derived thymic epithelial cells on transplantation into athymic nude mice. METHODS: Directed differentiation of iPSC generated TEPs that were transplanted into nude mice. Between 14 and 19 weeks posttransplantation, grafts were removed and analyzed by flow cytometry, quantitative PCR, bulk RNA sequencing, and single-cell RNA sequencing for markers of thymic-cell and T-cell development. RESULTS: A direct differentiation protocol that allows the generation of patient-derived thymic epithelial progenitor cells from multiple iPSC lines is described. On transplantation into athymic nude mice, patient-derived thymic epithelial progenitor cells further differentiate into functional patient-derived thymic epithelial cells that can facilitate the development of T cells. Single-cell RNA sequencing analysis of iPSC-derived grafts shows characteristic thymic subpopulations and patient-derived thymic epithelial cell populations that are indistinguishable from TECs present in primary neonatal thymus tissue. CONCLUSIONS: These findings provide important insights and resources for researchers focusing on human thymus biology.
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