Loss of Autophagy Activity during Storage of Hematopoietic Stem Cell Grafts Is Associated with Reduced Potency
Authors: Maganti Harinad , Jahan Suria , Patel Jaina , Kaushal Richa , Mcgregor Chelsea , Pasha Roya , Pineault Nicolas 
Published: December 2023

Maximizing the potency of hematopoietic stem cell (HSC) grafts including cord blood (CB) units (CBU) is essential to minimize risk of engraftment delays and failures. Low cell dose in CB transplantation is partially responsible for the slower engraftment but 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 such prolong storage reduces graft potencies due to a loss in hematopoietic stem cell and progenitors (HSPC) content.
CBU were used as HSC graft. Units were split in 2 halves; one processed shortly after collection (&lt;15 hrs, fresh) or after storage at RT (~43 hrs, stored). The impact of storage on graft quality was assessed using standard assays and bioinformatic analysis used to identify molecular pathways impacted by storage.
Monitoring assays revealed that all CBU samples passed the minimal FACT-criteria threshold for post-thaw CD34+ cell viability and potency. However, the net number of ISHAGE CD34+ cell counts were reduced in stored samples (-20%, p&lt;0.01). The colony forming-unit (CFU) assay used to measure the impact of storage on potency also revealed a 20% reduction in CFU in stored samples (&lt;0.05, n=7). Losses in potency was confirmed with a second assay, the IL-3-phospho-STAT5 (pSTAT5) assay, which revealed a 10-fold loss in STAT5 signalling in stored vs. fresh CD34+ cells (p&lt;0.0001, n=3).
We hypothesized that factors released by CB could be responsible for the reduction of HSPC. In support of this, plasma isolated from stored CBU induced greater apoptosis of CD34+ cells than fresh plasma (+30% AnnexinV+ cells, p&lt;0.01, n=3). This translated into losses of potency detectable after 30 minutes (CFU assay, p&lt;0.001) and with further reduction over time peaking around 2 hours.
Next, we sought to identify the molecular programs that govern this detrimental effect within CD34+ cells. Towards this we performed RNAseq analysis of CD34+ cells exposed to fresh or stored plasma for 20 minutes and 4 hours. A time course comparative analysis of differentially expressed genes (DEG, Log 2Fold &gt;1.5, and q&lt;0.05) between cells exposed to fresh vs stored plasma identified a total of 810 DEG genes. Gene ontology enrichment analysis identified autophagy, cell cycle, histone and DNA methylation and mRNA regulation as the major molecular programs that might be up regulated by the paracrine factors from fresh plasma but repressed by those from stored plasma.
Autophagy flux assay confirmed that CD34+ cells isolated from stored UCB samples had reduced autophagy activity, and that stored plasma induced a 50% reduction in autophagy flux (p&lt;0.001, n=3). Furthermore, RT-qPCR analysis confirmed that prolonged storage of CBU samples at RT increased the cellular senescence marker p21(CDKN1B), down regulated cell cycle genes ( CDK4 and CDK7) and autophagy genes ( ATG4, ATG12 and BECN1).
We hypothesized that re-activation or prevention of autophagy loss could prevent losses of HSPC within CBUs stored at RT. To test this, CBUs were divided and individually supplemented with either autophagy activators (rapamycin or trehalose), autophagy inhibitor (3-methyladenine, 3-MA) or DMSO control and stored up to 43 hours. As expected, the early autophagy inhibitor 3-MA failed to restore HSPC numbers and potency. However, addition of trehalose prior to storage restored 99% of both net number and potency of HSPCs in HSC grafts as measured by the ISHAGE CD34+ counts, CFU and pSTAT5 assays to baseline level (n=3). Interestingly, the near complete restoration mediated by trehalose coincided with restauration of autophagy activity, repression of senescence gene CDKN1B and activation of cell cycle genes. In contrast, rapamycin only partially restored HSPC numbers and potency which coincided with the activation of autophagy genes but not others mentioned above.
In conclusion, the loss of potency and viability seen in CBU grafts originates in part by paracrine-mediated mechanisms that lead to loss of autophagy, down regulation of cell cycle regulators and induction of senescence in HSPCs. Interestingly, addition of trehalose as a natural supplement precludes these molecular changes and restores CBU potency during storage. Taken together, these results stress the importance of rapid processing of HSC grafts and identify an attractive new solution to maintain high HSC graft potency post-collection during storage at ambient temperature.