Retention of In Vivo Functional Potential of Cryopreserved Human HSPC Grafts after 10 Years
Authors: Park Youngmin , Mahmud Dolores , Charan Grace Irene , Ganapathy Amudha , Kassis AL Rabee , Tepak Elena , Rondelli Damiano , Mahmud Nadim 
Published: December 2024

Cryopreservation of hematopoietic stem/progenitor cell (HSPC) grafts for future infusion into patients is a common practice. However, cryopreservation and long-term storage can influence the post-thaw recovery of target cells retaining functional potency. We analyzed 38 graft aliquots stored in liquid nitrogen (auto 10 yrs = 7, auto 1 yr = 12, allo 10 yrs = 5, allo 5 yrs = 5, allo 1 yr = 9). The viable CD34+ cell frequency in allogeneic (allo) HSPC grafts was higher than in autologous (auto) grafts cryopreserved 10 years ago (auto 10 yrs = 0.39 ± 0.24%, allo 10 yrs = 0.96 ± 0.31%, allo 5 yrs = 0.82 ± 0.3%, p = 0.018). The recovery of viable total nucleated cells was 46.83 ± 4.98% for auto 10 years, 43.11 ± 7.76% for allo 10 years, 48.74 ± 4.94% for allo 5 years, and 87.56 ± 6.93% for allo 1 year. The viable CD34+ cell recovery between the groups was comparable. Auto HSPC grafts stored for 10 years displayed 64.05 ± 16%, while auto grafts stored for less than a year yielded 85.4 ± 15.15% viable CD34+ cell recovery. Surprisingly, autografts, in comparison to allografts stored for 5 to 10 years, displayed higher red fraction (JC-1), indicative of higher mitochondrial membrane potential (MMP) and viability (auto 10 yrs = 72.24 ± 12.45%, allo 10 yrs = 40.6 ± 13.92%, allo 5 yrs = 47.2 ± 15.36%, p = 0.011). The higher fraction of cells with intact MMP (red fraction) within CD34+ cells suggest a likely higher resilience of autografts. Grafts stored up to 10 years displayed positive colony-forming unit (CFU) growth. CFU recovery for HSPC grafts stored for less than 1 year displayed 82.93 ± 25.68% for auto while allo grafts displayed 72.46 ± 34.76%. The viable CD34+ cell recovery for autografts was 85.4 ± 15.15%, and for allografts, recovery was 86.85 ± 18.0% for grafts thawed within one year. Furthermore, to validate the in vivo functional potential of post-thaw HSPC grafts from auto and allo donors stored for longer than 10 years, these grafts were transplanted into NSGS mice in a xenotransplantation assay. Mice were sacrificed after 12 weeks, and bone marrow and spleen were harvested to determine human blood cell chimerism by flow cytometry. Human CD45+ blood cells in mice (n = 7) receiving grafts from auto donors had 12.02 ± 1.61%, and mice (n = 7) receiving grafts from allo donors had 13.97 ± 3.19%, which was not statistically significant. However, in the spleen, mice receiving HSPC grafts from auto donors had 4-fold higher human CD45+ cell chimerism than mice receiving grafts from allo donors (38.41 ± 2.71 vs. 9.04 ± 2.04, p &lt; 0.0001). Notably, post-thaw HSPC grafts, even after 10 years of storage, showed comparable in vivo hematopoietic reconstitution, validating the retention of post-thaw functional potency. Higher MMP displayed by post-thaw auto HSPC grafts in comparison to allo grafts, in conjunction with significantly higher hematopoietic chimerism in the spleen after 10 years of storage, may reflect their metabolically activated state or higher resilience, presumably due to past exposure to chemotherapy, unlike HSPCs from allo donors (normal subjects). Post-thaw auto HSPC grafts also displayed relatively higher mitochondrial content than allo grafts stored for 10 years. Taken together, the post-thaw viable CD34+ cell recovery corresponds well with in vivo hematopoietic reconstitution. Most notably, retention of in vivo reconstitution capacity displayed by both auto and allo grafts in NSGS mice validates the stability of cryopreserved HSPCs. The relatively higher resilience of auto HSPC grafts in contrast to allo grafts stored for 10 years, as demonstrated by significantly higher engraftment in the spleen and relatively greater mitochondrial content, will need to be further validated.