O1095: Despite Homing to Epithelial Structures, Donor Cells Fail to Engraft Following Intra-amniotic in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) exploits fetal immunologic immaturity to facilitate 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 engraftment in previous studies. Recent studies suggest that donor cells home to multiple fetal sites following intra-amniotic (IA) delivery, but evidence of long-term engraftment has not been established. We sought to examine homing and engraftment after IA injection in hopes of improving delivery of transplanted cells to the fetal airway and GI tract through normal fetal swallowing and breathing events.

Methods: Following 24 hour pairings, time-dated BALB/cJ dams underwent laparotomy and E15 intra-amniotic injection of donor bone marrow (BM) harvested from adult B6-GFP mice. Following ficoll separation, 10 x 106 GFP+ donor BM-mononuclear cells were injected into each amniotic sac. Amniotic fluid was collected at 24 hours post-injection to evaluate presence and viability of donor cells by flow cytometry. Fetuses were collected at 24 and 72 hours post-injection to evaluate homing and distribution of cells by stereomicroscopy and immunohistochemistry. An additional group was allowed to deliver and placed with a foster within 24 hours of birth. Chimerism was measured monthly in peripheral blood using flow cytometry with terminal histological analysis conducted between three and six months.

Results: Cells aspirated from the amniotic fluid 24 hours after IA injection had a population of GFP+/CD45+ cells present with mean viability of 89.1% by flow cytometry using DAPI as a viability marker. At 24 and 72 hours, donor cells were present in multiple GI and pulmonary tissues including stomach, liver, and lung as well as other fetal sites (Figure 1), suggesting that donor cells home effectively to epithelial surfaces following IA injection. In a separate cohort, 12 fetuses were observed long-term for donor engraftment beginning at one month of age. Flow cytometry revealed no detectable GFP+/CD45+ cell population in any treated mouse through three months. At terminal analysis, there was no evidence of GFP+ cells in any target tissue including bowel, liver, pancreas, or lung despite the presence of donor cells at homing timepoints (Figure 2).

Conclusion: Consistent with published data, donor cells home to hematopoietic and epithelial sites after IA injection of BM-MNCs including luminal structures of the GI tract and airway. However, monthly peripheral blood chimerism and terminal histologic analysis do not show evidence of long-term donor cell engraftment in epithelial target tissues or within the hematopoietic niche. The lack of engraftment limits the therapeutic potential for IA injection as monotherapy or as an adjunct to IV IUHCT.