Restoring mitochondrial function promotes hematopoietic reconstitution from cord blood following cryopreservation-related functional decline
Yaojin Huang, … , Yingchi Zhang, Tao Cheng
Yaojin Huang, … , Yingchi Zhang, Tao Cheng
Published March 4, 2025
Citation Information: J Clin Invest. 2025;135(9):e183607. https://doi.org/10.1172/JCI183607.
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Research Article Hematology

Restoring mitochondrial function promotes hematopoietic reconstitution from cord blood following cryopreservation-related functional decline

Abstract

Umbilical cord blood (UCB) plays substantial roles in hematopoietic stem cell (HSC) transplantation and regenerative medicine. UCB is usually cryopreserved for years before use. It remains unclear whether and how cryopreservation affects UCB function. We constructed a single-cell transcriptomics profile of CD34+ hematopoietic stem and progenitor cells (HSPCs) and mononuclear cells (MNCs) from fresh and cryopreserved UCB stored for 1, 5, 10, and 19 years. Compared with fresh UCB, cryopreserved HSCs and multipotent progenitors (MPPs) exhibited more active cell-cycle and lower expression levels of HSC and multipotent progenitor signature genes. Hematopoietic reconstitution of cryopreserved HSPCs gradually decreased during the first 5 years but stabilized thereafter, aligning with the negative correlation between clinical neutrophil engraftment and cryopreservation duration of UCB. Cryopreserved HSPCs also showed reduced megakaryocyte generation. In contrast, cryopreserved NK cells and T cells maintained a capacity for cytokine production and cytotoxicity comparable to that of fresh cells. Mechanistically, cryopreserved HSPCs exhibited elevated ROS, reduced ATP synthesis, and abnormal mitochondrial distribution, which collectively led to attenuated hematopoietic reconstitution. These effects could be ameliorated by sulforaphane (SF). Together, we elucidate the negative effect of cryopreservation on UCB HSPCs and identify SF as a mitigation strategy, broadening the temporal window and scope for clinical applications of cryopreserved UCB.

Authors

Yaojin Huang, Xiaowei Xie, Mengyao Liu, Yawen Zhang, Junye Yang, Wenling Yang, Yu Hu, Saibing Qi, Yahui Feng, Guojun Liu, Shihong Lu, Xuemei Peng, Jinhui Ye, Shihui Ma, Jiali Sun, Lu Wang, Linping Hu, Lin Wang, Xiaofan Zhu, Hui Cheng, Zimin Sun, Junren Chen, Fang Dong, Yingchi Zhang, Tao Cheng

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Figure 1

Characterization of subclusters with high expression levels of mitochondrial genes.

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Characterization of subclusters with high expression levels of mitochond...
(A) Schematic diagram showing the sequencing design. Live CD34+ cells and MNCs from fresh and cryopreserved UCB stored for 1 (Y1), 5 years (Y5), 10 years (Y10), and 19 years (Y19) (n = 4 biological replicates for each storage period) were sorted to perform single-cell RNA-Seq (scRNA-Seq) using the 10x Genomics platform. (B) UMAP plot displaying the distributions of 5 cell populations from all UCB samples. The surrounding UMAP plots display the expression levels of signature genes used to identify each cell population: HSPCs (characterized by CD34 expression), T and NK cells (characterized by CD3D and KLRD1 expression), B cells (characterized by CD79A expression), monocytes (characterized by lysozyme [LYZ] expression), and stromal cells (characterized by secreted protein acidic and rich in cysteine-like 1 [SPARCL1] expression). (C) The proportion changes of the Mitohi subpopulations over time. The proportions of Mitohi cell clusters at 1 year, 5 years, 10 years, and 19 years were compared with those of fresh samples using the Wilcoxon test (*P ≤ 0.05). (D) UMAP plot displaying the distributions of 12 cell clusters of HSPCs. Colors indicate cell clusters determined by unsupervised Leiden clustering. (E) Cellular compositions of 12 cell clusters in HSPCs derived from fresh UCB and UCB cryopreserved for 1, 5, 10, or 19 years (n = 4 replicates; data indicate the mean ± SD). The cellular abundances of different subclusters at 1 year, 5 years, 10 years, and 19 years were respectively compared with that of fresh UCB using the χ2 test (see the Supporting Data Values file).