Experimental Double Cord Blood Transplantation (DCBT) of An Expanded Unit Together with a Non-Manipulated Unit in NOD/SCID Mice.

Abstract

We found that nicotinamide (NAM), a form of VitB3 and a recognized inhibitor of SIRT1, the human ortholog of the yeast Sir2 class III NAD+-dependent histone deacetylase, inhibits in vitro differentiation and promotes expansion of hematopoietic progenitor cells. Cord blood (CB)-derived CD34+ cells cultured with cytokines (FLT3, TPO, IL-6, SCF; 50ng/ml) and NAM (2.5mM) (Sigma Aldrich, catalog number N5535) display enhanced in vitro migratory activity toward SDF-1 and home to the BM (24hr following infusion in vivo) with higher efficacy than cells cultured with cytokines only. The number of SCID-repopulating cells increased by 9- and 7.6-fold in cultures treated with NAM relative to non-cultured cells and cytokine only cultured cells, respectively. This net increase in repopulation potential was sustained in competitive transplant experiments where cultured cells were infused along with non-cultured competitor cells derived from the same CB unit. Several experimental clinical protocols of CB-derived expanded cells involve co-transplantation of cultured and non-cultured cells in a double CB transplantation (DCBT). We therefore sought to investigate the engraftment potential of NAM-treated cultured cells in a DCBT setting in NOD/SCID mice, using two CBUs marked as “unit-1” and “unit-2”. Two similar experiments were conducted, each experiment had 4 groups of mice (n=10/ experimental group):non-cultured cellscells cultured with NAM transplanted along with the CD34 negative cell fraction from the same unit that was kept frozen till the day of transplantation.groups a + bnon-cultured cells from unit-1 transplanted along with non-cultured-cells from unit-2.Mice were transplanted with similar number of nuclear cells from the cultured or non-cultured units in the single or the DCBT groups. In Experiment-1, non-cultured cells were derived from one unit-1 and NAM-treated cultured cells were derived from another unit-2. In Experiment-2, we switched between the units (such an experiment was possible since our CBUs are frozen in several portions). Level of engraftment was evaluated two weeks post transplantation by FACS analysis of human CD45+ cells while the contribution of each unit to engraftment was measured by quantitative PCR for informative short tandem repeat (qSTR) regions that distinguished the units. The results show that in both experiments, the level of engraftment in the DCBT cohort of a cultured unit transplanted along with a non-cultured unit (12.8±1.8 %) was similar to the level of engraftment of the cultured unit when individually transplanted (15.4 % ±3, p>0.05). This level of engraftment was 9.8-fold higher (p<0.05) than the level of engraftment obtained in the DCBT cohort of two non-cultured units (1.3 % ±0.2). Chimerism analysis indicated that in recipients transplanted with two non-cultured units, engraftment was predominantly derived from unit-1, while in the two experiments of DCBT of a cultured unit transplanted along with a non-cultured unit the engraftment was predominantly derived from the cultured unit. In conclusion, the outstanding engraftment advantage of NAM cultured cells may explain their predominance in a DCBT setting.