The Impact of Cryopreservation on the Function of Hematopoietic Stem and Progenitor Cells

Abstract

Cryopreservation is currently the only method allowing for the long-term preservation of hematopoietic stem and progenitor cell (HSPC) grafts until their use. However, cryoinjuries reduce cell viability and potency of HSPC. New cryoprotectant (CPA) solutions have recently emerged that have not yet been investigated that may improve the cryopreservation of HSPCs. The overarching hypothesis of the work described in this thesis, is that different CPAs have diverse impact on the key biochemical processes essential for HSPC homeostasis which influences post thaw cell viability and potency. To test this hypothesis, 4 CPAs were extensively characterized for their cryoprotective properties on cord blood (CB) HSPCs in comparison to DMSO control. CryoProtectPure (CPP) supported similar post thaw cell viability and engraftment as DMSO control, whereas pentaisomaltose (PIM) and cryonovo (CN) failed as CPAs for HSPCs. Subsequently, the impact of CPAs on key biological pathways was explored to identify potential biochemical pathways implicated in HSPC cryopreservation. The impact of CPAs on cell membrane integrity, oxidative phosphorylation, glycolysis, and autophagy was examined. CPP and DMSO had varying impact on glycolytic and mitochondrial respiratory activities of HSPCs post-thaw, whereas both CPAs as well as PIM and CN had negligible impact on cell membrane parameters prefreeze. Cryopreservation and thawing strongly induced autophagy in HSPCs. Importantly, early inhibition of autophagy with 3-Methyladenine (3-MA) reduced the recovery of functional CB HSPCs post thaw. Together, my findings provide new insights regarding the biological processes impacted by CPAs and cryopreservation of HSPCs and identify potential targets to improve cryopreservation of HSC grafts.