Effects of cryopreservation on hematopoietic stem and progenitor cells and innate lymphoid cells in healthy adult bone marrow

Background　A thorough understanding of how the cryopreservation process affects the biological characteristics of various bone marrow cell subpopulations is essential for optimizing clinical transplantation strategies.Objective　To investigate the impact of standard cryopreservation procedures on the single-cell transcriptomic features and cellular composition of rare hematopoietic stem and progenitor cells (HSPCs) and innate lymphoid cells (ILCs) in healthy adult bone marrow, thereby providing a quality control basis for single-cell studies utilizing banked samples.Methods　Bone marrow samples from healthy adults were collected and divided into two groups for either fresh processing (fresh control group) or programmed cryopreservation and recovery (cryopreservation group). After enriching Lineage-CD34+ HSPCs and Lineage-CD45+CD127+CD161+ ILCs via fluorescenceactivated cell sorting (FACS), single-cell RNA sequencing was performed using the 10x Genomics platform. Data from this study were integrated with four public healthy bone marrow datasets. Batch effects were corrected using the Harmony algorithm, followed by cell clustering, differential expression, and functional pathway analysis. Results　Cryopreserved samples largely retained the overall cellular lineage composition of bone marrow, yet differences were observed within specific subsets. A cryopreservationspecific HSPC subpopulation emerged, exhibiting upregulation of stress- and inflammation-related genes (e.g., NFKB1, ELMO1), accompanied by cell cycle arrest and suppression of metabolic pathways, suggesting the induction of a stress-associated HSPC phenotype. Within the ILC lineage, cryopreservation led to the selective loss of a terminal effector ILC3 subset characterized by high expression of GNLY and GZMB, while other ILC subsets were relatively preserved. Multiple ILC subsets displayed common transcriptional stress responses, including altered expression of genes related to antigen presentation, inflammatory signaling, and metabolism. Conclusion　Although standard cryopreservation protocols can essentially maintain the overall heterogeneity of healthy adult bone marrow cells, they induce the formation of a stress-associated HSPC subpopulation and lead to the loss of fragile cell subsets such as terminal effector ILC3s. This study delineates cryopreservation-associated transcriptional imprints, offering crucial insights for data quality control, result interpretation, and the identification of true biological signals in single-cell research based on biobank samples.