Spatiotemporal Proteome Remodeling Directs Human Hematopoietic Stem Cells via a Metabolic-Cytoskeletal Axis

Hematopoietic stem cells (HSCs) sustain the lifelong production of all blood and immune cells and are central to transplantation and gene therapies. However, the clinical use of these approaches remains limited due to the functional attrition of HSCs during ex vivo adaptation and expansion in culture. As these transitions are executed at the protein and structural level, the primary drivers of cellular adaptation have remained elusive in transcriptional studies. Here, we overcome the technical resolution limits of rare primary cells by integrating a scalable, ultra-low input proteomics pipeline with 3D super-resolution imaging to map the proteomic and spatial organization of primary human HSCs during ex vivo expansion. Our analysis reveals a previously unrecognized, metabolic-cytoskeletal axis that coordinates organelle reorganization and structural polarity. Perturbation of this axis preserves structural polarity while uncoupling it from biomass expansion, demonstrating that growth and functional deterioration are separable processes in ex vivo HSC culture. These findings establish a framework for understanding ex vivo HSC adaptation and expansion and inform the rational development of next-generation cellular therapies.