Routing Pathway of Syngeneic Donor Hematopoietic Stem Cells After Simple Intra-amniotic Delivery

Background: In utero hematopoietic stem cell (HSC) administration constitutes an unparalleled alternative for the early treatment of genetic hemoglobinopathies, immunodeficiencies, inherited metabolic syndromes, as well the induction of postnatal allogeneic transplant tolerance. Clinical application of this strategy has been significantly limited by current invasive methods of prenatal HSC delivery. Recently, simple transamniotic HSC administration has been shown experimentally to allow for these cells to reach the fetal circulation and bone marrow robustly, at least in a syngeneic healthy model. We sought to determine the pathway of this unique cell kinetic route in a comparable model.

Methods: After IACUC approval, Lewis rat fetuses (n=181) were divided into two groups based on the content of volume-matched (50μL) intra-amniotic injections performed on gestational day 17 (E17; term=E21-22): either a concentrated suspension (2x10^6 cells/mL) of HSCs labeled with a luciferase reporter gene (n=137), or an acellular suspension of recombinant luciferase (n=44). Donor HSCs consisted of syngeneic cells isolated from the amniotic fluid being at least 80% positive for CD34, CD117, SSEA3, SSEA4, Sox2, Oct4, and alkaline phosphatase on flow cytometry. Samples from placenta, chorion, amnion, amniotic fluid, and umbilical cord, as well as from 8 fetal sites, were procured at 5 daily time points thereafter, until term (E18-22) (figure 1). Specimens were screened for luciferase activity via microplate luminometry. Positive luminescence was defined as relative light unit (RLU) values exceeding the mean plus 2 standard deviations of blank wells. Statistical analysis was by the Mann-Whitney U-test and Fisher’s exact test, with significance set at p<0.05.

Results: Overall survival was 57% (78/137), with n=12-21 fetuses analyzed per each of the five time points. When controlled for the acellular luciferase group, donor HSCs were identified in the amnion, chorion, placenta, and amniotic fluid of fetuses receiving cells at all time points (p=0.033 to <0.001), peaking first at the amnion and subsequently at the chorion and placenta. Importantly, the time-dependent luminescence pattern observed at these sites was not compatible with passive cell clearance, but rather suggestive of active cell transport (figure 2). Also when controlled for the acellular luciferase group, donor HSCs could be detected in the fetal liver as early as E18, with their presence progressively expanding to all the other fetal sites (peripheral blood, bone marrow, spleen, thymus, lungs, brain, bowel) over time, in parallel to their increased presence in the chorion and placenta.

Conclusion: The chronology of donor hematopoietic stem cell trafficking after concentrated intra-amniotic injection in a syngeneic healthy model is suggestive of controlled routing through the gestational membranes and placenta, with the latter apparently serving as gateway to the fetal circulation. Hematogenous donor cell routing is a constituent of transamniotic hematopoietic stem cell therapy, significantly expanding its practicality and potential applications.