MHC-Matching at 5 Loci Results in Higher Levels of Engraftment of Allogeneic Cord Blood (CB) Hematopoietic Stem Cells (HSC) Following Neonatal Transplantation.
Authors: Almeida-Porada Graca , Porada Christopher D. , LeBlanc Christine , Ascensao Joao L. , McNiece Ian K. , Zanjani Esmail D. 
Published: April 2020

Abstract
CB represents a rich source of transplantable HSC and its use in matched unrelated clinical HSC transplants (HSCT) is rapidly increasing, due largely to its potential for decreased GVHD compared to BM. Although CB use in adult recipients is increasing, the majority of CB transplants (Tx) have thus far been done in children, due to limited numbers of cells in single CB units. Over the past 20 years, we have used the human/sheep model to study human HSC from a variety of sources including CB. Although this model represents a biologically and possibly clinically relevant model of human hematopoiesis, some critical aspects of HSCT involving allogeneic donor cells may not be revealed by this xenogeneic model. Therefore, we developed the necessary methods to obtain and bank functionally viable CB from sheep at normal delivery in volumes sufficient to rescue lethally myeloablated neonatal sheep recipients, thus making it possible to begin performing allogeneic sheep-to-sheep CB HSCT. To this end, CB collected at birth (volume: 62±14ml; MNC: 5.2±0.6 x108; n=74) was used to Tx 3 week-old newborn lambs, with either a single or multiple (male plus Female) CB unit. Recipients were myeloablated with high dose chemotherapy (HDC) consisting of Etoposide plus Cytoxan, and Group I (n=3) was transplanted with 26±0.75×107 MNC/kg from a single cord while Group II (n=3) received the same number of cells pooled from 2 cords; Group III remained as a non-transplanted control. Prednisone was given in all transplanted animals until 5 weeks post-Tx. All the animals in Group III and one recipient in Group I died at about 23 days post-HDC. Engraftment of neutrophils and platelets occurred at approximately 35 days and 45 days respectively for Group I and 29 and 40 for Group II. Karyotypic analysis of donor cell engraftment in Group II revealed that when CB were pooled both were able to engraft but there was a gradual decline in the contribution to blood production of one of the transplanted cords with time post-Tx. Mild chronic GVHD occurred in all of the transplanted animals after withdrawal of the prednisone at day 210 after Tx. All the symptoms subsided after re-administration of prednisone. We therefore felt it was necessary for HSCT to develop reagents that would allow us to employ donor/recipient matched CB cells. After nearly 3 years of work, we now have the necessary MHC class I and II reagents for this purpose. Employing an in utero MHC typing strategy using amniotic fluid cells, we found that transplantation of CB HSC from donors matched at 5 loci into 60 day-old fetal sheep resulted in significantly higher donor cell activity than fetuses transplanted with HSC matched at 2 loci (3.% vs 14%). Long-term observation of these animals revealed that while the donor cell level remained higher in this group than in the group transplanted with HSC matched at 2 loci, by about 1 year post-HSCT, a decline in donor cell activity occurred. We have now performed a head-to-head comparison of human CB as a source of donor HSC for in utero HSCT (IU-HSCT) with matched and unmatched sheep CB. Our results thus far suggest that matched CB represents the more optimal source of HSC for IU-HSCT, and that the development of more specific antibodies that allow better matching may result in clinically useful levels of donor cell engraftment that will persist long-term without the previously observed decrease in donor cell activity. The similarity of our observations in this model to that of humans in kinetics of engraftment and GVHD development in HDC-treated sheep transplanted with allogeneic CB cells suggests that this model may be useful in developing clinically relevant strategies for the use of allogeneic CB in post-natal and IU-HSCT.