**Background:** Hematopoietic stem cells (HSCs) are long-lived, quiescent cells with unique metabolic requirements. Autophagy, a catabolic process that degrades and recycles intracellular components, is essential for HSC maintenance, but the mechanisms by which autophagy loss leads to HSC depletion remained unclear. Previous studies using pan-hematopoietic deletion of autophagy genes (Atg7, Atg12) could not distinguish cell-intrinsic from cell-extrinsic effects due to peripheral cytopenia and non-hematopoietic targeting. This study aimed to determine whether HSC loss is cell-intrinsic and to identify the underlying molecular pathways.
**Methods:** The authors used multiple mouse models: (1) inducible HSC-specific deletion of Atg16l1 via Fgd5-CreERT2 (tamoxifen-inducible, with tdTomato reporter), (2) mixed bone marrow chimeras (1:1 mix of Mx1-Cre Atg5fl/fl or wild-type CD45.2 cells with wild-type CD45.1 cells) to isolate cell-intrinsic effects, (3) Vav-Cre Atg7fl/fl (pan-hematopoietic deletion), and (4) Rosa26-CreERT2 Atg16l1fl/fl. Autophagy-deficient HSCs were analyzed for amino acid transporter expression (qPCR, flow cytometry), amino acid uptake (kynurenine and cystine assays), MTORC1 activation (phospho-MTOR, phospho-RPS6, phospho-EIF4EBP1 by flow cytometry), cell size (forward scatter), glucose uptake (2-NBDG), protein synthesis (OPP-click assay), proliferation (MKI67), and metabolic gene expression (Fluidigm). Rapamycin (50 µg/ml in drinking water or intraperitoneal) was administered to inhibit MTORC1.
**Key Results:** In Fgd5-CreERT2 Atg16l1fl/fl mice, autophagy-deficient (Tomato+) HSCs showed a 40–50% reduction in absolute numbers compared to controls (p < 0.05). In mixed chimeras, Atg5-deficient HSCs were significantly reduced by week 2 post-poly(I:C) induction, while LSK expansion was not observed, confirming cell-intrinsic HSC loss. Autophagy-deficient HSCs (Atg5 KO, Atg7 KO, Atg16l1 KO) exhibited increased expression of amino acid transporters SLC1A5/ASCT2, SLC38A1/SNAT1, SLC38A2/SNAT2, and SLC26A6/PAT1 (1.5- to 3-fold increase in geometric mean fluorescence, p < 0.05). This was accompanied by increased amino acid uptake (kynurenine uptake increased ~1.5-fold, p < 0.05). MTORC1 activation was elevated: phospho-MTOR, phospho-RPS6, and phospho-EIF4EBP1 were increased 1.5- to 2-fold in autophagy-deficient HSCs (p < 0.05). Downstream consequences included increased cell size (forward scatter ~10% increase, p < 0.05), glucose uptake (2-NBDG increased ~1.5-fold, p < 0.05), and protein synthesis (OPP incorporation increased ~1.5-fold, p < 0.05). Glycolytic enzyme transcripts (e.g., Hk2, Pfkl, Ldha) were upregulated, while TCA cycle genes were unchanged. Proliferation (MKI67) was increased in autophagy-deficient HSCs (p < 0.05). Rapamycin treatment in Vav-Cre Atg7fl/fl mice reversed HSC loss (absolute HSC numbers restored to wild-type levels, p < 0.05), normalized p-RPS6 and protein synthesis (p < 0.05), and alleviated anemia and splenomegaly. However, rapamycin did not reduce amino acid uptake or proliferation. In mixed chimeras, rapamycin rescued HSC frequency and translation without affecting proliferation or amino acid uptake. Transplantation of rapamycin-treated autophagy-deficient HSCs into new hosts resulted in improved multilineage reconstitution (myeloid, B cell, T cell) and maintained HSC frequency in bone marrow comparable to wild-type.
**Clinical Implications:** This study demonstrates that autophagy preserves HSCs by restraining MTORC1-mediated anabolism, and that excessive amino acid influx and MTORC1 activation are maladaptive consequences of autophagy loss. Rapamycin, an FDA-approved drug, can rescue HSC function even in the absence of autophagy, suggesting a potential therapeutic strategy for conditions with impaired autophagy, such as aging or genetic autophagy deficiencies (e.g., ATG7 mutations, Vici syndrome). The findings also highlight the importance of metabolic regulation in stem cell transplantation and regenerative medicine. However, the study is limited to mouse models, and translation to human HSCs requires further investigation.