Reduction in Cord Blood Graft Potency Due to Processing Delay Can be Prevented by Trehalose Supplementation
Background and objectives: Maximizing the potency of cord blood (CB) units (CBU) is a goal of all CB banks. Low transplant cell dose in CB transplantation is one of several contributors to the slower engraftment. Processing delays could also be at play since CBU can be stored at room temperature (RT) up to 48 hours before cryopreservation. We hypothesized that prolonged storage at RT reduces the potency and engraftment activities of CBU due to a loss in hematopoietic stem cell (HSC) and progenitor numbers. Materials and Methods: CBU were split in half, with one half processed shortly after collection and the other half after 40 hours of storage at RT. Viabilities and potencies were compared post-thaw, and engraftment activity tested in xenotransplants. Bioinformatic analysis was carried out to identify pathways modulated by prolonged storage at RT. Results: Prolonged storage at RT reduced the viability of CB cells and potency of grafts based on in vitro and in vivo assays. Transcriptomic analyses revealed that paracrine factors released in CBU induce several changes in gene expression programs in CD34+ cells. These included the activation of the senescence gene CDKN1B and down-regulation of cell cycle and autophagy genes. Supplementation of CBU with trehalose prevented the loss in cell viability and potency. The latter was associated with restoration of autophagy and suppression of the senescence gene CDKN1B. Conclusion: These results stress the importance of rapid processing of CBU and support further investigations on trehalose as a natural supplement to protect CBU potency during storage. Overall design: To identify the molecular programs driven by paracrine factors present in plasma from stored cord blood unit, cord blood CD34+ enriched cells were incubated with plasma derived from baseline (i.e. fresh plasma) and cord blood halves stored for 40 hours at room temperature (i.e., stored plasma) for 0, 20 minutes and 4 hours (n=2). RNA was extracted and the changes in transcriptome were profiled via RNA sequencing. CD34+ cells not exposed to plasma were used as controls to account for baseline gene expression. RNA was isolated using miRNeasy Micro Kit (Qiagen, Toronto, ON, Canada).  cDNA libraries were constructed using TruSeq stranded mRNA protocol (Illumina, San Diego, CA, USA) and sequencing was performed by StemCore Laboratories (Ottawa, ON, Canada) using the Illumina HiSeq 2000, following their standard operating procedures. Reads were assigned to transcripts from the GENCODE v32 assembly (GRCh38) using Salmon v1.0 (https://combine-lab.github.io/salmon/).