IF aged human hematopoietic stem cells (HSCs; donors >65 years, purified Lin⁻CD34⁺CD38⁻CD90⁺ fraction) are subjected to a sequential ex vivo lysosomal reset protocol — first a 16-hour exposure to trehalose (100 mM) to activate TFEB-mediated lysosomal biogenesis and clear accumulated intracellular substrate, followed immediately by a 16-hour exposure to bafilomycin A1 (BafA1; 3 nM) to suppress v-ATPase hyperactivation rebound — prior to xenotransplantation into sub-lethally irradiated NSG mice,

THEN recipients will demonstrate ≥6-fold improvement in long-term human CD45⁺ chimerism at 16 weeks post-transplant relative to untreated aged HSC controls, with balanced myeloid/lymphoid output (CD33⁺:CD19⁺ ratio <2, matching young donor reference), and successful secondary engraftment in ≥60% of serially transplanted hosts,

BECAUSE the following mechanistic chain operates:

1. Aged HSCs accumulate undegraded intracellular substrates (protein aggregates, damaged organelles, lipofuscin-like material) due to insufficient lysosomal biogenesis relative to the rate of damage accrual — this substrate overload drives compensatory v-ATPase upregulation to acidify and process the backlog, producing the hyperactivated lysosomal phenotype observed in aged HSCs. SPECULATIVE link — supported indirectly by the neural stem cell evidence showing that reduced autophagic flux into acidified lysosomes characterizes aged quiescent stem cells, with accumulated GFP-LC3-positive material indicating unresolved substrate burden

2. Trehalose, acting as an mTOR-independent TFEB activator (via direct mTORC1 displacement from the lysosomal surface), upregulates the CLEAR gene network, expanding lysosomal number and biogenesis capacity, increasing cathepsin activity, and driving bulk clearance of the accumulated aggregate substrate — the primary accumulated damage targeted by this REPAIR strategy. The constitutively active TFEB intervention in aged quiescent neural stem cells decreased protein-aggregate abundance and enhanced stem cell activation, establishing TFEB-mediated lysosomal biogenesis as sufficient to remove accumulated intracellular substrate in aged quiescent stem cells

3. Substrate clearance removes the stimulus driving compensatory v-ATPase upregulation; however, v-ATPase subunit expression is transcriptionally elevated in aged HSCs and does not immediately normalize after substrate removal, leaving a residual hyperactivation risk during the transplant window. [SPECULATIVE — based on the known kinetics of v-ATPase complex assembly exceeding CLEAR network-mediated transcriptional downregulation]

4. The subsequent 3 nM BafA1 pulse partially inhibits the residual v-ATPase hyperactivation, restoring lysosomal pH to a range consistent with young HSC physiology, preserving quiescence-associated surface receptor expression (including CD71/transferrin receptor), and preventing t...

SENS category: GlycoSENS

Key references: • doi.org/10.1126/science.aag3048 • doi.org/10.1101/2025.01.14.632900 • doi.org/10.1126/science.aag3048] • doi.org/10.1101/2025.01.14.632900] • doi.org/10.1101/2025.04.11.648417]