P2D389: Epithelial Engraftment Following in Utero Hematopoietic Cell Transplantation Is Limited to Subtherapeutic Levels

Background: In utero hematopoietic cell transplantation (IUHCT) exploits fetal immunologic immaturity to facilitate the engraftment of donor cells without myeloablation or toxic immunosuppression. The intravenous (IV) route of injection has been well-established as an effective method to optimize hematopoietic engraftment in previous studies. We have previously reported that intra-amniotic (IA) delivery of donor cells failed to demonstrate meaningful epithelial or hematopoietic engraftment. We sought to characterize epithelial and GI engraftment after intravenous delivery to determine whether engraftment following IUHCT reaches clinically relevant levels in these normally protected niches.

Methods: Following 24-hour pairings, time-dated BALB/cJ dams underwent laparotomy and intravascular injection of donor bone marrow (BM) harvested from adult B6-GFP mice at E14. Following ficoll separation, 20 x106 GFP+ donor BM-mononuclear cells were injected into the vitelline vein of each fetus. Peripheral blood chimerism was measured monthly using flow cytometry with terminal histological analysis including hepatocytes and lung/GI epithelial cells at six months.

Results: Seventeen injected fetuses were found to have detectable levels of peripheral blood chimerism at one month with a mean of 19.78% (SEM 5.97; Range 1.00 – 86.80, and at terminal harvest the mean peripheral blood chimerism was 23.86% (SEM 12.44; Range 0.00 – 98.90). (Figure 1). At terminal analysis, GFP+ cells were noted in all target tissues including the bowel, liver, and lung, but these cells co-stained for lung epithelial (Epcam) and hepatocyte (CK-18) markers in only 1/17 treated mice (chimerism 95.6%). No epithelial co-staining was noted in the bowel. (Figure 2).

Conclusion: As previously demonstrated, the IV route of IUHCT efficiently allows the creation of chimeric mice able to maintain consistent engraftment in the hematopoietic niche without myeloablation or immunosuppression. Though donor-derived epithelial cells and hepatocytes were identified on histologic analysis in 1 of 17 treatment mice, it is clear that the intact epithelium remains a significant barrier to engraftment. Transplantation earlier in development might allow better epithelial engraftment but is less clinically relevant. Alternatively, induced injury to the epithelium might create space and promote epithelial-directed donor cell differentiation, as has been seen in previous studies of postnatal BMT following conditioning. Though donor-derived epithelial cells were observed following IUHCT, the intact epithelial barrier limits engraftment in sites likely to drive differentiation down an epithelial pathway, which limits the potential of IV IUHCT for use in epithelial cell disorders.