Abstract
Abstract 574
Many of the most prevalent inherited disorders currently affecting the human population could potentially be treated with IUHSCT. IUHSCT does not require myeloablation, eliminating the risks inherent to this procedure. Moreover, performing IUHSCT during the period of preimmunity removes the recipient's immune response as a barrier to successful engraftment. Unfortunately, despite numerous clinical trials over the last 20 years for a variety of disorders, IUHSCT has only been successful in treating SCID, highlighting the need for a better understanding of the factors governing engraftment following IUHSCT. We hypothesized that the success of IUHSCT likely depends upon: 1) the maturity/receptivity of the bone marrow (BM) niches at the time of transplant; 2) the degree of donor/recipient MHC compatibility; and 3) the source of donor HSC. To test our hypothesis, we began by performing confocal analyses of fetal sheep bones at gestational days 30–145, using sheep as a model based on its immunologic, physiologic and developmental similarities with the human fetus. These analyses showed that the BM microenvironment commences development with the formation of the vascular niche, beginning by day 40 of gestation. In contrast, the osteoblastic niche, which is known to be critical for the engraftment and maintenance of primitive HCS, only starts developing at roughly day 51 of gestation, and does not reach maturity until day 65. These findings thus indicate that to achieve maximal levels of engraftment following IUHSCT, the transplant should likely be performed at gestational day 65 or later, to ensure that the crucial osteoblastic niche has fully developed. Unfortunately, partial immune-competence is also achieved by roughly day 65, suggesting that immune compatibility between the donor and the recipient will also need to be addressed to maximize engraftment. This issue was addressed by transplanting 65 day-old sheep fetuses (n=21) with eGFP-transduced CD34+ cells isolated from related and unrelated BM and cord blood (CB) donors and comparing the levels of engraftment. Cell counting, PCR, and flow cytometry at 63 days post-IUHSCT revealed that the engraftment levels of the related CB HSC were nearly double those of the unrelated CB HSC in BM (1.7% vs. 0.86%) and more than 4-fold higher in PB (2.77% vs. 0.66%). Interestingly, however, there was no significant difference between the engraftment levels of related and unrelated BM-derived HSC (1.77% vs. 1.88%). We presumed that a greater degree of MHC matching could explain the higher engraftment seen with related versus unrelated CB-derived HSC. This was experimentally confirmed by performing PCR-based MHC microsatellite typing at 12 selected MHC loci on the donor CB cells and on amniotic fluid-derived recipient cells. All related donor/recipient animals exhibited 5–7 perfect matches and 1–6 half matches at the 12 MHC loci. In contrast, non-related donor/recipient animals exhibited only 1–2 perfect matches and 4–6 half matches at these loci. To evaluate the impact of the source of HSC on IUHSCT outcome, we compared these same animals without consideration to donor/recipient relatedness. These analyses demonstrated that the CB-derived HSC engrafted in PB at nearly four-fold higher levels than their BM-derived counterparts (2.75% vs. 0.93%). Conversely, the BM-derived HSC engrafted within the recipient BM at three-fold higher levels than the CB-derived HSC (0.66% vs. 2.24%). Collectively, these results suggest that transplanted HSC show a predilection for engraftment in their site of origin.
In conclusion, our data collectively indicate that: 1) delaying IUHSCT until a point in gestation when the osteoblastic and vascular niches of the BM are mature is likely to improve engraftment; 2) transplanted HSC exhibit a predilection for engraftment in their site of origin; and 3) higher degrees of CB donor/recipient MHC matching doubles the engraftment levels obtained following IUHSCT, but does not seem to impact upon engraftment levels of BM-derived HSC. By simultaneously addressing these issues, it is expected that curative levels of HSC engraftment can be obtained prior to birth, making it possible to establish clinically successful and reliable IUHSCT-based treatments for a wide range of inherited disorders.
Disclosures:
No relevant conflicts of interest to declare.
Population Size and Human-Grivet Monkeys (Chlorocebus aethiops) Conflict in Zegie Peninsula, Bahir Dar, Ethiopia
