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 6

Impaired function of cryopreserved, UCB-derived HSPCs can be ameliorated by SF.

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Impaired function of cryopreserved, UCB-derived HSPCs can be ameliorated...
(A) Experimental scheme for analysis of cryopreserved HSPCs treated with SF. CD34+ cells from cryopreserved UCB were cultured with or without SF, followed by FACS for CFU, liquid culturing, and transplantation assays. For transplantation, UCB-derived CD34+ cells (20,000/mouse) were injected into mice via the tail vein. (B) CFCs per 200 CD34+ cells from fresh and cryopreserved UCB stored for 10 years, cultured with or without SF treatment (n = 5 replicates, 3 independent experiments). (C and D) Percentage of hCD45+ and hCD34+ cell engraftment in BM of recipients 12 weeks after transplantation (n = 6–10 recipient mice per group). (E) Lineage output of Mks (CD41a+) from bulk (100 cells) CD34+ cells from fresh and 10-year cryopreserved UCB with or without SF treatment (n = 3–4 pooled UCB, 2 independent experiments). (F) Percentage of human CD41a+ cell engraftment in BM of recipients 12 weeks after transplantation. (G) Representative histogram and normalized TMRE MFI of CD34+ cells from fresh and 10-year cryopreserved UCB cultured with or without SF (n = 3–5 replicates, 2 independent experiments). (H) Representative histogram and normalized MitoSox MFI of CD34+ cells from fresh and 10-year cryopreserved UCB cultured with or without SF (n = 4–6 replicates, 2 independent experiments). (I) Experimental scheme for cryopreservation with or without 10 μM SF added to the freezing solution (control: n = 6 replicates; SF: n = 6 replicates). (J) CFCs per 120 CD34+ cells from control and SF-treated groups (n = 5 replicates). (K) Lineage output of Mks (CD41a+) from bulk (100 cells) CD34+ cells from control and SF-treated groups (n = 3–4 replicates). All data indicate the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA or Kruskal-Wallis test, with a post hoc Tukey’s test applied for multiple comparisons (B–H) and 2-tailed t test (J and K).