Fetoscopic versus Ultrasound-Guided Intravascular Delivery of Maternal Bone Marrow Cells in Fetal Macaques: A Technical Model for Intrauterine Haemopoietic Cell Transplantation

Abstract

<b><i>Introduction:</i></b> Significant limitations with existing treatments for major haemoglobinopathies motivate the development of effective intrauterine therapy. We assessed the feasibility of fetoscopic and ultrasound-guided intrauterine haemopoietic cell transplantation (IUHCT) in macaque fetuses in early gestation when haemopoietic and immunological ontogeny is anticipated to enable long-term donor cell engraftment. <b><i>Material and Methods:</i></b> Fluorescent-labelled bone marrow-derived mononuclear cells from 10 pregnant <i>Macaca fascicularis</i> were injected into their fetuses at E71–114 (18.9–170.0E+6 cells/fetus) by fetoscopic intravenous (<i>n</i> = 7) or ultrasound (US)-guided intracardiac injections, with sacrifice at 24 h to examine donor-cell distribution. <b><i>Results:</i></b> Operating times ranged from 35 to 118 min. Chorionic membrane tenting and intrachorionic haemorrhage were observed only with fetoscopy (<i>n</i> = 2). Labelled cells were stereoscopically visualised in lung, spleen, liver, and placenta. Donor-cell chimerism was highest in liver, spleen, and heart by flow cytometry, placenta by unique polymorphism qPCR, and was undetected in blood. Chimerism was 2–3 log-fold lower in individual organs by qPCR than by flow cytometry. <b><i>Discussion:</i></b> Both fetoscopic and US-guided IUHCT were technically feasible, but fetoscopy caused more intraoperative complications in our pilot series. The discrepancy in chimerism detection predicts the challenges in long-term surveillance of donor-cell chimerism. Further studies of long-term outcomes in the non-human primate are valuable for the development of clinical protocols for IUHCT.

Maternal microchimerism and cell‐mediated immune‐modulation enhance engraftment following semi‐allogenic intrauterine transplantation

“…Our experimental strategy of adult donor cells was chosen to reflect the clinical situation in which adult (maternal) BM‐HSC are used for IUT, 6 with non‐IUT pups treated only postnatally as the clinically relevant controls. Though large animals are necessary to determine feasibility in humans, there are challenging technical limitations in quantifying the chimerism of unmanipulated donor cells (unless there is sex mismatch) in wild‐type animals which reflect the anticipated translational difficulties in human IUT 6,16 . This mixed‐breed technical model allows parallel study of trafficked maternal immune cells and transplanted unmarked maternal donor cells in the recipient due to the use of coisogenic breeds of B6 females, as we begin to delineate the complex interactions between the parental donor cell exposure, maternal cell influx, and the fetal immune response.…”

“…6,33 This level of engraftment was not repeatable in the NHP model of mIUT, where boluses of 0.2-1.7E+8 maternal BM cells/fetus resulted only in microchimerism in most organs. 16 Although the mechanisms of graft rejection were not studied in the NHP, a brisk immunological rejection was suspected due to minimal donor cells recovered 24h post-IUT. Mouse IUT studies demonstrate stable DCC following congenic and fully allogenic transplantation, determined the threshold chimerism of 1.8% required to induce and sustain DST, and confirmed the association between initial antigen exposure and host T or NK cell interaction and subsequent induction of either allospecific tolerance or rejection.…”

“…Though large animals are necessary to determine feasibility in humans, there are challenging technical limitations in quantifying the chimerism of unmanipulated donor cells (unless there is sex mismatch) in wild-type animals which reflect the anticipated translational difficulties in human IUT. 6,16 This mixed-breed technical model allows parallel study of trafficked maternal immune cells and transplanted unmarked maternal donor cells in the recipient due to the use of coisogenic breeds of B6 females, as we begin to delineate the complex interactions between the parental donor cell exposure, maternal cell influx, and the fetal immune response. We used uniform intrauterine doses of 10E+9 cells/kg per pup (~0.5 g) and 4.2E+8 cells/kg per pup postnatally (4w, ~12 g).…”

“…This may be circumvented by transplanting semi‐allogenic maternal bone marrow (BM)‐derived HSC which demonstrate better tolerance and more efficient engraftment, 4,6 a strategy used in a current clinical trial for alpha thalassemia major (NCT02986698) which has successfully reported the delivery of the first two treated infants. However, we were unable to replicate the same engraftment in the non‐human primate (NHP) model and thus questioned the influence of this maternal microchimerism (MMc) on donor cell survival and engraftment, as well as the influence of semi‐allogenic donor cells from either the paternal or maternal donor on the responsiveness of trafficked maternal cells 16 . We investigated if maternal donor cells were better tolerated and engrafted more efficiently than paternal cells due to the anticipated lower immune‐responsiveness of trafficked maternal immune cells resulting in a more favorable influence on engrafted donor cells 12,14 .…”

“…However, we were unable to replicate the same engraftment in the non-human primate (NHP) model and thus questioned the influence of this maternal microchimerism (MMc) on donor cell survival and engraftment, as well as the influence of semi-allogenic donor cells from either the paternal or maternal donor on the responsiveness of trafficked maternal cells. 16 We investigated if maternal donor cells were better tolerated and engrafted more efficiently than paternal cells due to the anticipated lower immune-responsiveness of trafficked maternal immune cells resulting in a more favorable influence on engrafted donor cells. 12,14 We used a cross-bred murine model (CD57BL/6 x BALB/c) for a direct comparison of maternal (mIUT) and paternal donor cell IUT (pIUT), interrogating engraftment, maternal immune microchimerism, and fetal immune response (Figure 1A).…”