A New Strategy to Overcome the Cell Dose Barrier to Umbilical Cord Blood Transplants: A Proof of Early Hematopoietic Reconstitution By Combined Multiple Units of Allogeneic Stem/Progenitor Cells
Authors: Ishida Takashi , Otsu Makoto , Takahashi Satoshi , Higashihara Masaaki , Nakauchi Hiromitsu 
Published: October 2019

Abstract
Introduction: Umbilical cord blood (UCB) serves as a suitable donor source in hematopoietic stem cell transplantation (HSCT). However, UCB has the major drawback that is delayed engraftment due to its low graft cell numbers, which often limits its use in HSCT. To overcome this cell dose barrier, double units UCB transplantation (UCBT) has been attempted, but the time to engraftment is still relatively delayed. Based on the report by Japan Red Cross Society, the majority of UCB units remain unused clinically because of their insufficient graft cell doses. Overall, these facts prompted us to seek for a new strategy to improve UCBT by using multiple (more than three) units. We here provide a proof of feasibility of such an approach using mouse transplantation models.
Methods: To mimic a clinical setting of UCBT, we first established an "insufficient cell dose" model by using mouse BM KSL (c-Kit+, Sca-1+, lineage-marker-negative) cells. In this model, C57BL/6 (B6-Ly5.2, H2b) mice were the recipients, and congenic B6-Ly5.1 mice (H2b) were the primary donors of cell grafts. We used KSL cells as a unit of grafts, as they contained hematopoietic stem and progenitor cells (HSPC) that were able to restore BM reconstitution. KSL cells were used also to avoid inclusion of mature immune cells, which could compromise the outcomes in HSCT through unpredictable immune reactions. As the donors of combined multiple units, four allogeneic mouse strains were used: BDF1 (DBA2 x B6 F1, H2b/d), B6D1F1 (DBA1 x B6 F1, H2b/q), B6C3F1 (C3H x B6 F1, H2b/k), and CBF1 (Balb/c x B6 F1, H2b/d). The use of F1 strains was to prevent GvHD, which would significantly affect donor cell engraftment kinetics. The recipient mice were lethally irradiated, and thus could not survive with only the "insufficient cell dose" of B6-Ly5.1 graft. The outcomes in HSCT were tested by the addition of mixed allogeneic KSL cells (multi-allo HSPCs) in comparison with the addition of B6-Ly5.1 KSL cells (congenic HSPCs) with the equivalent doses. The effects of multi-allo HSPC transplants were evaluated by recipients' survival rate and complete blood counts over time. Detailed donor cell contribution in peripheral blood and BM was also determined by flow cytometry analysis. The transgenic mouse line expressing a fluorescent marker Kusabira Orange was utilized where needed to test donor cell contribution in erythrocytes and platelets.
Results: Titration studies revealed 500 B6-Ly5.1 KSL cells as optimal for the "insufficient cell dose" model because the recipients were all lethal, whereas ~100% mice survived upon receipt of 2,500 B6-Ly5.1 KSL cells. Interestingly, addition of multi-allo HSPCs rescued otherwise lethal recipients as effectively as congenic-HSPCs with the equivalent acceleration of hematopoietic recovery. Chimerism analysis, however, revealed that this "KSL cells alone" transplantation led to long-term existence of multi-donor hematopoiesis, which was not ideal for a clinical setting. We then replaced B6-Ly5.1 KSL grafts with the whole BM (WBM) grafts. Titration experiments determined 50,000 WBM cells as a single unit, mimicking an "insufficient dose" of unmanipulated UCB unit. Addition of multi-allo HSPCs in this model also showed complete protection of recipients from lethality and enhanced early hematopoietic recovery (figures). Remarkably, dominant B6-Ly5.1 chimerism was established and maintained in this modified model. Experiments using subfractionation of the B6-Ly5.1 grafts demonstrated that small numbers of T cells were responsible to single-donor chimerism formation possibly through graft versus graft reactions. Finally, to maximize transient early hematopoietic reconstitution by multi-allo HSPCs (we call this "bridging effect"), we tested whether cultured hematopoietic stem cells (HSC, defined as CD34negatvie/low KSL cells) worked better than uncultured KSL cells. Using our defined protocol compatible with stem cell amplification, we demonstrated that a mixture of cultured allo-HSCs exhibited the bridging effect even superior to that of an uncultured KSL cell mixture.
Conclusions: We here provide a proof that combined multiple units of allogeneic HSC/HSPC are capable of exhibiting "bridging effect" when manipulated appropriately and added to an otherwise "insufficient dose" setting of transplantation. Development of a new strategy of multiple units transplantation may open a door for wider application of UCBT.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.