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

Abstract
In utero stem cell therapy holds promise for the amelioration of disorders before birth. Futhermore, due to the preimmune state of the developing fetus, recipients do not reject allogeneic cell transplants and postnatal transplantation from the same donor may be possible without the need for anti-rejection drugs. 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 sac of day 25 or day 35 fetuses. Cell tracking was facilitated by labeling male canine cells with micron-sized superparamagnetic and fluorescent polystyrene beads. Both magnetic resonance imaging (MRI) and fluorescence imaging were employed to assess the fetal distribution of transplanted cells.
Total bone marrow was harvested from adult canine donors and processed for bone marrow mononuclear cells (BMMC). Marrow stromal cell (MSC) cultures were initiated from BMMC on the basis of plastic adherence. Both MSC and BMMC were labeled for 16 hours with fluorescent superparamagnetic beads. Five pregnancies have been studied, wherein 1–2 x 106 MSC or 0.1 – 1 x 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 were allowed to develop in vivo for an additional seven to fourteen days at which time ovariohysterectomy and fetal retrieval were performed.
Dispersion of the injected cells within each fetus prevented conclusive detection of labeled cell distribution by MRI (~3mm3 volume elements, 1.5 Tesla GE Signa). 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 noninjected controls. The signal was predominantly localized to the thoracic and abdominal regions, with no apparent fluorescence visible in the yolk sac. To asses donor cell engraftment, sagittal-plane cryosections were analyzed by fluorescence microscopy for detection of the fluorophore as well as Prussian blue staining for detection of superparamagnetic iron particles via light microscopy. The localization of iron particles and fluorescence label was co-registered on images taken from sequential cryosections. These analyses indicated that labeled BMMC and MSC migrated from the yolk sac to the fetal liver. Furthermore, molecular confirmation of donor cell engraftment in the livers of female fetuses was obtained after manual micro-dissection of fetal livers from day 32 fetuses or after laser capture of iron labeled cells from day 43–45 fetuses. Y chromosome positive cells were detected in fetuses receiving either male MSC or BMMC, but not in saline control injected fetuses.
Our studies demonstrated that injection of cells into the yolk sac during early fetal gestation is an effective strategy to deliver cells to the developing fetus. We are currently following in utero transplant recipients to determine whether long-term engraftment and immunotolerance of donor cells during the neonatal period can be achieved.