Human Cord Blood Cells with Rapid In Vivo Platelet Regenerating Activity in NOD/SCID-IL-2Rγc-/- Mice Show Variable Aldehyde Dehydrogenase Activity.
Authors: Cheung Alice M.S. , Eaves Connie J. 
Published: October 2019

Abstract 2429

Poster Board II-406

Human cord blood (CB) has emerged as an attractive source of hematopoietic cells for patients lacking a suitable donor. However, marked delays in platelet and immune recovery pose significant challenges to the use of CB cells as transplants for either children or adult patients. These difficulties in the use of CB have been attributed to low absolute numbers of repopulating cells (RCs) in most CB units which is not readily overcome by simply combining multiple units. Xenotransplantation of human hematopoietic cells into highly immunodeficient sublethally irradiated NOD/SCID mice has proven to be a powerful approach to characterize different types of primitive human hematopoietic cells with repopulating potential. However, the residual NK activity intrinsic to NOD/SCID mice poses a significant barrier to the engraftment of intermediate types of repopulating human cells and also to the terminal stages of their differentiation, as shown by recent studies using more immunodeficient mice as hosts. Nevertheless, the cells responsible for early platelet recovery post-transplant and factors that regulate their activity remain largely unknown. To address this issue, we have developed a quantitative and sensitive assay for characterizing the phenotypes of human CB cells that regenerate mature platelets detectable in the blood of NOD/SCID-IL-2Rγc-/- mice 3-6 weeks post-transplant. Lineage marker-negative (Lin-) human CB cells were stained with Aldefluor and then those with low light side-scattering properties were further separated by FACS according to whether they displayed aldehyde dehydrogenase activity above (ALDH+) or below (ALDH-) that detected in the presence of an ALDH inhibitor. Assays of different pooled human CB preparations showed that the most primitive class of in vitro megakaryocyte (Mk) colony-forming cells and cells responsible for rapid human platelet production in NOD/SCID-IL-2Rγc-/- mice were variably and comparably distributed between the small ALDH+ and prevalent ALDH- fractions. From 3 experiments, the following values were obtained for the ALDH+ and ALDH- subsets, respectively; ALDH+ - mature CFU-Mk = 47.8±21.3% of the total Lin- fraction, intermediate CFU-Mk = 66.3±25.4%, primitive CFU-Mk = 75.5±14.0%, 3-week platelet-producing STRC = 63.8±12.3%, 6-week platelet-producing STRC = 81.5±5.8%; and ALDH- - mature CFU-Mk = 52.2±21.3% of the total Lin- fraction, intermediate CFU-Mk = 33.7±25.4%, primitive CFU-Mk = 24.5±14.0%, 3-week platelet-producing STRC = 36.2±12.3%, 6-week platelet-producing STRC = 18.5±5.8%. Limiting dilution assays revealed 1 in 830 Lin-ALDH+ CB cells to be a cell that can produce detectable platelets in vivo within 3 weeks (95% CI = 1 in 534 to 1 in 1290) but only 1 in 1996 (95% CI = 1 in 1226 to 1 in 3247) within 6 weeks. The present study demonstrates the feasibility of using NOD/SCID-IL-2Rγc-/- mice for the sensitive detection of human CB cells with in vivo platelet regenerating activity and suggests that these may be closely related to primitive cells with in vitro Mk clonogenic activity (>50 Mk per colony). Biologically important platelet progenitors may thus be heterogeneous with respect to ALDH+ activity.

Disclosures:

No relevant conflicts of interest to declare.