Intravascular Route Is Not Superior to an Intraperitoneal Route for In Utero Transplantation of Human Hematopoietic Stem Cells and Engraftment in Sheep

Abstract

Previously, we have reported in utero hematopoietic stem-cell transplantation (IUT) in sheep (1) and nonhuman primates (2) using an intraperitoneal (IP) route, as performed in clinical trials in humans (3,4) to treat congenital disorders, including X-linked severe combined immunodeficiency. Recent studies (5,6) have demonstrated that, for IUT in mice, the intravascular (IV) route facilitates the injection of cell doses higher than those that can be injected IP, thereby resulting in increased levels of donor-derived chimerism after birth. The studies are tremendous in that they have demonstrated the feasibility of such narrow route injection in mouse fetuses and yielded superior outcomes. However, no study has compared the IV and IP approaches with respect to the efficiency of engraftment in large animals. In this study, we compared the efficacy of these approaches for IUT in sheep. Human cord blood CD34+ cells were transplanted into the right ventricular cavity (IV group, n=4) or abdominal cavity (IP group, n=3) of the preimmune fetal sheep (at gestation days 59–61; full term, 147 days). The cells were injected through the uterine wall using a 23-gauge needle under ultrasound guidance. For the IV approach, we transplanted cells into the right ventricular cavity instead of the vitelline vein (5,6), because the vitelline vein was too small to be detected under ultrasound guidance at the gestation days. The number of cells transplanted per fetus ranged from 1.4×105 to 6.3×105; there were no intergroup differences. After birth (4 months after transplant), few transplant-derived cells were detected in the peripheral blood, but the engraftment could be properly examined by clonogenic hematopoietic progenitor assay to detect human hematopoietic colony-forming units in the bone marrow (BM). The fractions of human colony-forming units were 1.3%±0.1% and 1.3%±0.9% in the IP and IV groups, respectively (Table 1), indicating that there was no difference in the engraftment between the two groups.TABLE 1: In utero transplantation and engraftment of human hematopoietic stem cells in sheepInitially, we had hypothesized that the engraftment of the transplanted cells that were injected by the IV route in the BM at approximately day 60 of the gestation period in sheep would be better than that of the cells injected by the IP route because during this period, hematopoietic stem cells IV move from the liver into the BM (7). However, we found no difference in the efficacies of both approaches. Furthermore, bradycardia was often detected in the fetuses that were injected by the IV route, thereby implying that the IV approach might have some risk associated with it. Considering that both approaches are equally efficacious for the engraftment of the transplanted cells and because IP injection is evidently easier and safer than IV injection, we hypothesize that the IP approach is more meritorious than the IV approach for IUT experiments in large animals and clinical studies in humans. Therefore, we believe that further clinical trials of IUT can be conducted using the IP approach. Yujiro Tanaka1 Shigeo Masuda1 Tomoyuki Abe1,2,3 Satoshi Hayashi4 Yoshihiro Kitano4 Yoshikazu Nagao2,3 Yutaka Hanazono1 1 Division of Regenerative Medicine Center for Molecular Medicine Jichi Medical University Tochigi, Japan 2 Department of Agriculture Utsunomiya University Tochigi, Japan 3 Division of Biological Production Science United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology Tokyo, Japan 4 Department of Obstetrics and Gynecology National Center for Child Health and Development Tokyo, Japan