IF autologous adult dermal fibroblasts transduced with a doxycycline-inducible FOXN1 lentiviral vector (generating adult FOXN1-reprogrammed fibroblasts; aFREFs) are seeded onto decellularized thymic extracellular matrix (dECM) scaffolds co-populated with postnatal thymic interstitial cells (TICs) at a 5:1 TEC-equivalent:TIC ratio, and the resultant bioengineered organoid is surgically engrafted subcapsularly into the kidney of aged (18–24 month) male C57BL/6J mice,

THEN restoration of functional thymopoiesis — measured as a ≥2-fold increase in peripheral naïve CD4⁺ and CD8⁺ T-cell output (CD44ˡᵒCD62Lʰⁱ), measurable TCR Vβ repertoire re-broadening by deep sequencing, and histological evidence of cortical-medullary zonation with DLL4⁺ cortical and AIRE⁺ medullary domains in the graft — will be observed within 12 weeks of engraftment compared to aged vehicle-control mice receiving dECM scaffold without aFREFs,

BECAUSE the following mechanistic chain operates:

1. FOXN1 is the master transcriptional regulator of TEC fate; its forced expression in fibroblasts is sufficient to activate the TEC gene-regulatory network, including Dll4, Il7, Cxcl12, and Ccl25, as demonstrated by the functional thymic rejuvenation achieved with Foxn1-reprogrammed embryonic fibroblasts (FREFs) transplanted into aged hosts (Thymic rejuvenation via FREF transplantation)[https://doi.org/10.1172/jci.insight.140313].

2. The decellularized thymic ECM provides indispensable architectural and biochemical niche signals — including retained laminin, fibronectin, collagen IV, and growth factor reservoirs — that primary TEC progenitors alone cannot self-organize without; co-seeding with TICs at a 5:1 ratio prevents interstitial overgrowth and promotes cordoned stromal organization phenocopying early fetal thymus architecture within five days (Functional thymus reconstitution via dECM scaffold)[https://doi.org/10.1038/s41467-020-20082-7].

3. The combinatorial niche effect is the novel mechanistic link [SPECULATIVE]: the dECM scaffold matrix provides spatial polarization cues (cortical versus medullary microdomains defined by differential matrix composition and stiffness gradients) that FOXN1 overexpression alone cannot supply, while FOXN1 reprogramming supplies the transcriptional drive that primary TICs alone cannot provide — making this a synergistic, not merely additive, combination.

4. Age-related thymic involution has eliminated the endogenous TEC progenitor pool, meaning the lamin-B1-deficient and FOXN1-depleted residual stroma of the aged thymus cannot self-repair; only exogenous replacement of functional TEC-like cells can restore thymopoiesis, as the accumulated structural damage (loss of EpCAM⁺ epithelial architecture, disappearance of mTEC AIRE⁺ subsets, and interstitial fibrosis) is irreversible without cellular replacement (Age-related TEC compositional collapse and lamin-B1 reduction)[https://doi.org/10.1111/acel.12952].

5. **Autologous adul...

SENS category: RepleniSENS

Key references: • doi.org/10.1172/jci.insight.140313]. • doi.org/10.1038/s41467-020-20082-7]. • doi.org/10.1111/acel.12952]. • doi.org/10.1016/j.jaci.2021.07.021]. • doi.org/10.1101/2025.04.22.649893].