Developing 3D culture systems for thymic epithelial progenitor cell self-renewal and differentiation

The prompt re-establishment of thymopoiesis is paramount following cytoablative therapies such as chemotherapy and irradiation. In paradox to this need, the thymus undergoes progressive involution from early life, with consequent effects on our capacity to generate a repertoire of naïve T lymphocytes. This presents as a major concern in clinical medicine, particularly in the elderly and other immunocompromised patients. A plethora of pre-clinical approaches to thymus regeneration have thus been explored, albeit with very limited success. Given the importance of thymic cross-talk, our lab has focused on identifying and re-activating thymic epithelial progenitor cells. Here we establish and characterize a novel Foxn1^eGFP knock-in mouse line that allows for live Foxn1⁺ TEC purification and visualization studies. We optimize our in vitro 3D culture systems towards clinical translation, investigating the effects of growth factors and extracellular matrix components on TEC progenitors. Lastly, we examined thymic embryogenesis, to advance the knowledge on thymus biology and identify molecules potentially associated with adult TEPC development.
   
   To our knowledge, we have generated the first mouse model that both accurately reflects Foxn1 protein expression and allows for purification of live Foxn1⁺ TECs. Additionally, our knock-in line permits live cell visualisation, and does not demonstrate signs of haploinsufficiency. We subsequently utilize this model to improve in vitro TEPC maintenance, demonstrating that Bmp4 supplementation maintained Foxn1 expression, and improved self-renewal in TECs. In an effort to move towards clinical utilization, we also substituted commercial Matrigel with our defined synthetic hydrogel that allows control over environmental composition, and therefore limited batch-to-batch variation. Since the ECM is one of the key components in thymic cross-talk, we compared native thymic stromal cell populations to the currently used MEFs in our 3D TEC cultures. We found that the former improved both TEC self-renewal and survival, indicating the importance of organ-specific ECM. Furthermore, we demonstrated that our seeded decellularized thymic matrix was at least comparable to the gold standard RTOC technique, and promoted differentiation even in the presence of Bmp4. Given that the identification of other growth factors and/or adult TEPC markers will aid in advancing our studies, we also examined early thymic embryogenesis through transcriptome and miRNome analysis. We detected several genes and molecules of interest, including Igf2, Krt15, and members of the let-7 miRNA family. Taken together, our findings outline the initial steps for a clinically translational approach to in vitro immune regeneration. Further research on our data could ultimately elucidate the true adult TEPC phenotype, or give rise to novel miRNA-based therapies.