Migration of Donor Cells from the Yolk Sac to Fetal Tissues after In Utero Transplantation of Early to Mid-Gestation Canine Fetuses.
Authors: Vaags Andrea K. , Gartley Cathy , Zheng Yanzhen , Dobson Howard , Halling Krista B. , Foltz Warren D. , Dick Alexander J. , Kruth Stephen A. , Hough Margaret R. 
Published: October 2019

Abstract
Transplantation of stem and progenitor cells, under permissive conditions, can result in the long-term engraftment of allogeneic donor cells. In utero transplantation is of particular interest in that mixed chimerism may allow for the amelioration of disorders before birth and the development of immune tolerance towards donor cells prior to the maturation of the immune system. In order to more fully establish the feasibility of in utero cell transplantation, we are developing a canine model through the injection of allogeneic cells to the yolk sacs of day 25 or day 35 fetuses. Cell tracking was facilitated by labeling transplanted male canine cells with micron-sized superparamagnetic, fluorescent, polystyrene beads. Total bone marrow (BMMC) and mesenchymal stromal cells (MSC) were labeled for 16 hours with fluorescent superparamagnetic beads, prior to transplantation. Five pregnancies were studied, wherein 1–2 × 106 MSC or 0.1–1 × 107 BMMC were delivered to individual yolk sacs of day 25 (n=13) or day 35 (n=14) fetuses under ultrasound guidance. Each pregnancy included 1–2 fetuses that received an equal volume saline injection (n=7). Fetuses developed in utero for an additional seven to fourteen days at which time ovariohysterectomy and fetal retrieval were performed. Ex vivo whole body fluorescence imaging of fetuses verified cell migration from the yolk sac injection site to the fetus proper based on increased levels of green fluorescence in injected versus non-injected controls. The signal was predominantly localized to the thoracic and abdominal regions, with no fluorescence visible in the yolk sac. Fluorescence microscopy for detection of the fluorophore and light microscopy of Prussian Blue stained sections for detection of superparamagnetic iron particles was performed to assess donor cell localization. The co-localization of iron particles and fluorescence label was detected on images taken from sequential sections. These analyses indicated that labeled BMMC and MSC migrated from the yolk sac to the fetal liver and to a lesser extent to the developing bone marrow cavity. In some cases, the use of superparamagnetic particles has been confounded by free particles being scavenged by macrophage. To determine if cell labeling was restricted to macrophage, sections were stained with an anti-macrophage antibody and analyzed by fluorescence microscopy. Co-localization of the anti-macrophage stain and the fluorescence of the particle was not detected. Furthermore, molecular confirmation of male donor cell engraftment in the livers of female fetuses was obtained via canine Y chromosome specific Q-PCR. Y chromosome positive cells were detected in female fetuses receiving either male MSC or BMMC, but not in saline injected controls. Our studies demonstrate that injection of cells into the yolk sac during early to mid gestation is an effective strategy to deliver cells to the developing fetus, and in particular to sites of fetal hematopoiesis. We are currently following in utero transplant recipients to determine whether long-term engraftment and immune tolerance of donor cells during the neonatal period can be achieved.