Cryopreserved Ex-Vivo Expanded Murine Hematopoietic Progenitors Can Engraft Across MHC Barriers and Improve Survival In a Murine Model of Acute Radiation Syndrome.

Practical clinical countermeasures that could enhance survival after accidental or deliberate radiation exposure are lacking. We have previously demonstrated that culture of murine hematopoietic stem/progenitor cells in the presence of immobilized Notch ligand Delta1 results in a multi-log increase in the number of lin^-/sca-1⁺/c-kit⁺ (LSK) cells with short term lymphoid and myeloid repopulating ability. Here we show that Delta1 cultured LSK cells, cryopreserved after expansion, can be an effective therapy in a murine model of the hematopoietic acute radiation syndrome (hARS).

Whole bone marrow (BM) was obtained from Ly5a mice (H-2b, CD45.1). LSK cells were isolated by flow cytometry and placed in culture in the presence of either immobilized Delta1 or control human IgG. Serum free conditions were used, consisting of Iscove's medium supplemented with cytokines mSCF, hFlt-3 ligand, hIL-6, and hIL-11. After 14 days, expanded cells were harvested and cryopreserved in 90% FBS + 10% DMSO. On the day of transplant, frozen cells were thawed, washed, and resuspended in PBS + 1% FBS; manual viable cell counts and LSK phenotyping were performed prior to tail vein infusion with escalating doses of Delta1 or IgG cultured cells at doses of 1, 3, 5, or 10 × 10⁶ (Delta-1 group only at 10×10⁶) into supralethally irradiated (8.5 Gy) MHC-mismatched Balb/c mice (H-2d, CD45.2). Peripheral blood (PB) and BM were collected from mice at 1, 2, 3 and 4 weeks after transplantation for chimerism determination by cytofluorometry.

Culture with Delta1 resulted in significantly greater increases in absolute numbers of LSK cells (7.2 × 10⁴-fold expansion) as compared to growth with control IgG (0.8 × 10⁴-fold expansion). Approximately 90% of viable LSK cells were recovered post thaw. PB samples from recipient Balb/c mice receiving Delta1 cells demonstrated significantly higher Ly5a+ donor cell engraftment as compared to recipients receiving IgG-cultured LSK cells (p=0.0001). Donor IgG cultured cells were detectable only at day 7, whereas cells grown in the presence of Delta1 persisted through day 14 and 21. Only mice transplanted with Delta1-expanded cells showed engraftment in marrow, although by 2 weeks donor cells had decreased substantially. No signs of graft versus host disease (GVHD) were observed. Survival was significantly prolonged among mice that received Delta1-cultured cells, whereas all mice that received IgG cultured cells died within the first 3 weeks after irradiation (p=0.0001). Overall survival at day 30 was 11, 20, 26 and 63 percent after receiving 1, 3, 5 and 10 × 10⁶ Delta1-cultured cells, respectively. Mice that received 10 × 10⁶ cultured cells showed a statistically significant better survival (p=0.02), demonstrating a dose response relationship with the highest survival observed in mice that received the highest dose of expanded cells.

Using the Notch ligand Delta1 for the ex vivo expansion of murine hematopoietic progenitor cells, we have demonstrated that the cultured cells can be efficiently cryopreserved without loss of in vivo function. Infusion of Notch-expanded and cryopreserved cells into lethally irradiated mismatched recipients demonstrated that short-term engraftment without manifestations of GVHD can be achieved across major H-2 barriers and resulted in. significantly enhanced survival in a dose dependent manner. We have previously demonstrated that culture of human cord blood CD34+ cells in the presence of Delta1 also results in a significant increase in the absolute number of hematopoietic progenitor cells that are capable of rapid myeloid reconstitution in vivo. The findings presented herein thus support further development of a parallel human ex vivo expanded and cryopreserved product for clinical application in a non-HLA matched setting.

No relevant conflicts of interest to declare.