Restoring mitochondrial function promotes hematopoietic reconstitution from cord blood following cryopreservation-related functional decline
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
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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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 3

Decreased hematopoietic reconstitution and Mk production of HSPCs during the first 5 years of cryopreservation.

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Decreased hematopoietic reconstitution and Mk production of HSPCs during...
(A) Experimental design for the functional studies of HSPCs from fresh and cryopreserved UCB. Live CD34+ cells were sorted by FACS. For in vitro assays, CFU assays were performed on CD34+ cells from individual UCB samples. For in vivo analysis, 4–6 pooled UCB samples (same storage duration) were transplanted into NOG mice. For the primary transplantation, 36,000 CD34+ cells per mouse (n = 15–24 recipient mice/group) were injected via the tail vein. For the secondary transplantation, 1 × 107 cells from each primary recipient were transplanted into second recipients. CFC, colony-forming cell; 20W, 20 weeks. (B) CFCs per 200 CD34+ cells from fresh and cryopreserved UCB stored for different durations (years) (fresh: n = 8; 1 year, 5 years: n = 5; 10 years: n = 12; 19 years: n = 6). (C–G) Percentages of hCD45+ or hCD34+ cells engrafted in the PB or BM of primary (C–E) and secondary (F and G) recipient mice. (H) Cryopreservation duration predicted neutrophil engraftment. The multivariate model was fitted using the quantile regression method. (I) Lineage output of Mks (CD41a+) from bulk (n = 100 cells) CD34+ cells from fresh UCB and cryopreserved UCB (n = 4–5 replicates, 2 independent experiments). (J) Percentage of Mk versus non-Mk colonies in fresh and cryopreserved UCB (fresh: n = 40 colonies; 5 years: n = 52 colonies; 20 years: n = 34 colonies). (K) Percentage of human CD41a+ Mk cells engrafted in the BM of recipients (NCG-X mice). n = 4 pooled UCB samples; n = 4–7 recipient mice per group. For the transplantation assay, UCB-derived CD34+ cells (40,000 cells/mouse) were injected via the tail vein. (C–G) n = 4–6 pooled UCB samples; n = 15–24 recipient mice per group. Data indicate the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by 1-way ANOVA or Kruskal-Wallis test with post hoc Tukey’s test applied for multiple comparisons (C–G and I) and 2-tailed t test (J and K).