Sean J. Morrison

Summary: Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate–cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC -deficient, but not GPX4 -deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC -deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Stem cell factor (SCF) from Leptin Receptor-expressing (LepR ⁺ ) mesenchymal stromal cells and endothelial cells is required for hematopoietic stem and progenitor cell maintenance in bone marrow. Consistent with this, Scf is highly expressed by LepR ⁺ cells and at lower levels by endothelial cells in bone marrow. However, endothelial cells outside the bone marrow produce circulating SCF, raising the question of whether the SCF that promotes stem and progenitor cell maintenance is produced inside or outside bone marrow. We found that Scf deletion in spleen and liver endothelial cells reduces SCF levels in the blood without affecting stem or progenitor numbers in the bone marrow. Moreover, deletion of Scf from mesenchymal cells in the long bones, but not vertebrae, of Prx1 ^Cre ; Scf ^fl/fl mice depletes stem and progenitor cells in long bones but not vertebrae. SCF thus acts locally within the bones in which it is made to promote stem and progenitor cell maintenance. Key Points: Scf deletion in endothelial cells outside of BM reduces SCF levels in blood without affecting stem or progenitor cell frequencies in BM Scf deletion from mesenchymal cells in long bones, but not vertebrae, depletes stem and progenitor cells in long bones but not vertebrae

(Related to Figure 1). TCA cycle labeling and metabolite abundance in tumors and lungs from NSCLC patents.

Supplementary Figure S7: Clinical variables associated with time to hematopoietic regeneration after allogeneic transplantation at Vanderbilt.

(Related to Figure 3). Overall and recurrence-free survival summary.

Supplementary Figure S9: The inhibitory effect of carvedilol on hematopoietic regeneration after syngeneic transplantation can be overcome by transplanting larger doses of bone marrow cells.

(Related to Figure 2). Transcriptomic data for NSCLC and adjacent lung tissue fragments.

Supplementary Table S1. Characteristics of UTSW autologous transplant patients. Continuous measures are shown as mean (SD) and categorical measures as percentages. A one-way ANOVA was used to compare continuous variables, and a χ2 test was used to compare categorical measures.

Supplementary Table S6. Related to Fig. 1-7. Key Resources Table.

Supplementary Figure S8: Hematopoietic stem and progenitor cell frequencies after allogeneic transplantation.