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