HSC “aging” of cord blood during cryopreservation and new strategy for prevention

Cord blood (CB) plays an important role as a medical resource containing hematopoietic stem cells (HSCs) and mature immune cells. Cord blood transplantation (CBT) for hematopoietic disorders offers several advantages, including the ability to initiate treatment early, lower rates of severe chronic graft-versus-host disease even in human leukocyte antigen (HLA)-mismatched recipients, resulting in better post-transplant quality of life, and stronger anti-tumor immune responses compared to other transplantation methods, thereby reducing the risk of post-transplant relapse.^1–4 In addition, techniques such as the introduction of chimeric antigen receptor genes have been used to enable immune cells in CB, such as T cells and natural killer (NK) cells, to be used as sources for cell therapies.^5,6

On the other hand, CBT has the problem of a lower number of HSCs in CB units for transplant compared to bone marrow transplantation, which can lead to delayed hematopoietic recovery after transplantation and an increased risk of graft failure. Therefore, research on methods to expand hematopoietic stem/progenitor cells (HSPCs) has been ongoing for decades and some of these methods have already been applied clinically.^7,8 CB used for transplantation is collected at maternity clinics, transported to CB banks, and undergoes cell processing and necessary testing before being cryopreserved. Stored CB is administered to patients after a long period of storage, depending on the unit. To date, results from several studies have reported that the quality of long-term stored CB is stable in terms of recovery rate and functionality.^9,10

In a recent report, Huang et al¹¹ performed a parallel comparison with fresh CB cells to comprehensively evaluate the functions of different cell populations in frozen-preserved CB and found a decrease in the expression levels of HSC/multipotent progenitor (MPP) signature genes in HSPCs and a gradual decrease in long-term engraftment rates. Many previous studies, including those described earlier^9,10 suggesting that prolonged freezing does not affect stem cell function, have shown that frozen CB cells retain the ability to engraft after transplantation, but some studies have not performed parallel comparisons with fresh CB. In contrast, the present report shows that HSC function decreases with prolonged storage compared to fresh CB. On the other hand, Broxmeyer et al¹⁰ reported in a recent study that the number and function of HSCs in long-term cryopreserved CB did not show significant changes compared to those derived from fresh CB. However, they froze and thawed the CB using the fixed protocol and used it for various analyses. In addition, their RNA-seq analysis showed different results from those reported by Huang et al¹¹ indicating that HSC/HPC in frozen CB showed higher stem cell characteristics and lower metabolic activity compared to those in fresh CB. However, they noted that HSC/HPC with lower stem cell function and higher metabolic activity may have lower cryopreservation efficiency, or in other words, may be eliminated due to the stress of cryopreservation. Furthermore, they identified differences in graft engraftment function and several related gene expressions among 3 CB units cryopreserved for the same length of time (27 years), suggesting the existence of inherent differences between CB units. To evaluate the differences between the 2 studies, it is desirable to analyze a larger number of CB units using a standardized cryopreservation and thawing method.

Using a cohort of 171 CBT patients, the researchers confirmed a negative correlation between the time to neutrophil engraftment and the cryopreservation period of the CB unit used for the transplant in this study. However, the effect of cryopreservation on platelet recovery, which could be inferred from the negative effect on megakaryocyte production demonstrated in this report, was not evident in this cohort. To resolve the discrepancy with previous reports indicating that the duration of cryopreservation of CB units used for transplantation does not affect neutrophil engraftment,¹² prospective clinical studies that include evaluations such as the timing of the last platelet transfusion are desirable.

In the study reported by Huang et al,¹¹ single-cell transcriptomic profiles of cells contained in CB were constructed and subpopulations with high levels of mitochondrial gene expression were identified in each cell population. After cryopreservation, an increasing trend in the proportion of these cells was observed. In particular, in the HSPC population, the expression levels of genes associated with oxidative metabolism increased after freezing, and these changes were most pronounced in the first year after freezing, including cell cycle activation, reduced expression levels of HSC/MPP signature genes, and a decrease in the propensity toward megakaryocytic differentiation. CD34-positive cells in CB showed reduced stem cell function after freezing, as evidenced by reduced engraftment rates and colony-forming capacity in transplantation experiments. This change progressed gradually over the first 5 years after freezing, but showed no significant decline thereafter. The mechanism underlying the HSC/MPP-specific functional decline after cryopreservation was elucidated by demonstrating a correlation with increased mitochondrial membrane potential and identifying mitochondrial metabolic dysfunction, including increased oxidative phosphorylation and reactive oxygen species (ROS; decreased oxygen consumption and concomitant decreased ATP production). Based on these experimental results, the researchers screened several antioxidant compounds with the aim of eliminating excessive mitochondrial ROS. The results showed that sulforaphane, through its antioxidant activity, significantly reduced both mitochondrial membrane potential and ROS levels in CD34+ cells from frozen CB unit. This effect of sulforaphane was also shown to have similar protective effects when added to CB before cryopreservation. These findings suggest that sulforaphane may offer a novel approach as a potential clinical intervention to address the risks of delayed hematopoietic recovery and engraftment failure associated with CBT.

In addition, while this study identified functional impairment of HSCs due to cryopreservation, little effect was observed on T cells and NK cells. Given the growing interest in the clinical application of immune cells in CB as a new source of cell therapies in addition to HSCs, the findings of this study have significant implications.

The findings of this study by Huang et al¹¹ provide important insights into CBT and cell therapy and are expected to contribute to the further development of HSCT and cell therapy using CB cells.