TSC22D3-Mediated Quiescence Preservation Boosts HSC Engraftment in Xenografts

Background: Hematopoietic stem cell (HSC) ex vivo culture causes severe loss of repopulation and regenerative capacity without compromising multilineage differentiation, which greatly limits the efficacy of HSC transplantation. The molecular mechanisms underlying culture-triggered HSC dysfunction remain poorly understood. Methods: Human CD34+ HSCs were cultured ex vivo for 96 h to establish a culture-induced HSC dysfunction model. Single-cell RNA sequencing was applied to screen key regulatory genes. TSC22D3 function was verified via overexpression assays, and immunodeficient mice were used to assess HSC engraftment. Transcriptomic profiling were performed to explore downstream molecular mechanisms. Results: Ex vivo culture induced G0 quiescence exit, elevated early apoptosis and impaired in vivo repopulation in human CD34+ HSCs. TSC22D3 was highly enriched in freshly isolated quiescent HSCs and gradually downregulated during culture. TSC22D3 overexpression restored HSC G0 arrest and improved hematopoietic engraftment in mice. Mechanically, TSC22D3 upregulated HSC self-renewal genes, suppressed cell cycle-related genes (CDK2/4), and activated the P53-P21-P27 pathway. Conclusions: This study demonstrates that TSC22D3 preserves HSC function during ex vivo culture by maintaining stem cell quiescence and restricting excessive proliferation. These findings reveal a novel transcriptional mechanism regulating HSC homeostasis and provide a promising target for improving functional HSC ex vivo expansion for clinical transplantation.