Ambient oxygen disrupts amino acid homeostasis and impairs human hematopoietic stem and progenitor cell engraftment

Hematopoietic stem cell transplantation is limited not only by donor cell number and recipient conditioning, but also by loss of stem-cell fitness during collection and processing. Hematopoietic stem and progenitor cells normally reside in a physiologically hypoxic marrow environment, yet routine ex vivo handling exposes them to ambient oxygen outside this niche. Although ambient oxygen exposure during ex vivo handling impairs hematopoietic stem cell (HSC) function, the metabolic basis for this effect remains incompletely defined. Using hypoxic handling as a physiologic reference, we examined whether functionally relevant consequences of ambient oxygen exposure remain modifiable in human CD34+ cells during ex vivo handling. Quantitative LC-MS profiling revealed that ambient oxygen exposure rapidly increased intracellular amino acids, including branched-chain amino acids, together with activation of nutrient-sensing pathways. This metabolic shift was associated with increased mTORC1 signaling, attenuation of lysosomal programs, and impaired primary and secondary engraftment. Complementary single-cell analyses supported a coordinated transition toward nutrient-activated states under ambient oxygen, whereas hypoxia preserved relative metabolic restraint. Transient restriction of essential amino acids or branched-chain amino acids during ex vivo handling restored lysosomal programs and improved engraftment of normoxia-handled human CD34+ cells. Pharmacologic LAT1 inhibition similarly improved engraftment. Together, these findings identify disruption of amino acid homeostasis as a previously underappreciated consequence of ambient oxygen exposure during routine ex vivo handling and demonstrate that a functionally relevant component of this injury remains modifiable after exposure to improve human HSPC engraftment.