In Utero Hematopoietic Stem Cell Transplantation Research Articles

O27: MICROGLIAL CORRECTION AFTER FETAL THERAPY WITHOUT CONDITIONING IN MICE WITH MUCOPOLYSACCHARIDOSIS TYPE VII

Abstract Introduction Mucopolysaccharidosis type 7 (MPS7) is a lysosomal storage disorder typically fatal in utero. Postnatal enzyme replacement therapy (ERT) to replace missing glucuronidase (GUS) does not penetrate the blood-brain barrier (BBB). We investigated whether in utero ERT (IUERT) specifically targets brain microglia (natural GUS storehouses and key brain inflammation mediators) and whether in utero hematopoietic stem cell transplantation (IUHSCT) results in microglial engraftment as a strategy for permanent correction. Methods We performed IUERT by injecting GUS into MPS7 fetuses mid-gestation, and analyzed tissue homogenates (via colorimetric substrate) and brain microglia (via flow cytometry) for enzyme activity after 4-7 days. We performed IUHSCT by transplanting HSCs mid-gestation from CX3CR1-GFP donors. We examined blood, bone marrow, and brain for engraftment. We assessed brain inflammation by staining for CD68. We performed RNA sequencing to characterize engrafted microglia. Results IUERT resulted in detectable brain GUS activity. Flow cytometry showed that GUS activity after IUERT was near wild-type levels, and brains harvested in adulthood had decreased inflammation via CD68 immunohistochemistry. IUHSCT resulted in multilineage engraftment of hematopoietic cells in blood and bone. Confocal microscopy revealed multifocal engraftment of donor-derived microglia. RNA sequencing indicated that engrafted microglia were nearly identical to endogenous microglia. MPS7 chimeras had evidence of reduced brain inflammation near donor microglia. Conclusion Both IUERT and IUHSCT are complementary treatment modalities that can penetrate the BBB and ameliorate neurologic manifestations of diseases such as MPS7. These results lay the foundation for future studies using in utero molecular therapies for MPS7 as well as other storage disorders.

Genetic Engineering to Induce Fetal-Like Hematopoietic Stem Cells.

Bone marrow transplantation (BMT) or hematopoietic stem cell transplantation (HSCT) is an archetype of cellular therapy. However, to date BMT still suffers from several complications. Recent technological advances have encouraged us to think about an alternative to traditional BMT. Specifically, we propose in utero HSCT (IUHSCT). For this purpose, we suggest that induced fetal-like hematopoietic stem cells (ifHSCs) might be suitable for IUHSCT, and should be seriously evaluated.

Defining Engraftment Patterns and Interactions Between Hematopoietic Stem Cells and Stromal Cells after in Utero Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) has only been fully successful in recipients with severe combined immunodeficiency disorders. Because IUHSCT must be performed without myeloablation or immunosuppression, immunologic barriers and absence of stress-induced signaling have been considered significant contributors to the limited success of this procedure. Other potential barriers to successful engraftment of allogeneic adult donor hematopoietic stem/progenitor cells (HSC) in a fetal recipient result from the unique characteristics of fetal HSC biology and the fetal microenvironment. Transplanted adult cells could be out-competed by fetal HSC, and the fetal microenvironment might not support engraftment and/or expansion of HSC derived from ontogenically disparate sources, as differences in membrane composition and response to cytokines exist between fetal and adult cells. We have previously shown that increased levels of HSC engraftment can be achieved in a fetal setting, if bone marrow (BM)-derived mesenchymal stromal cells (MSC) or (BM)-derived endothelial progenitor cells (EPC) are administered prior to, or at the time of, HSC transplantation. Using a fetal sheep model of IUHSCT, we interrogated mechanisms by which transplanting sheep EPC or MSC prior to, or concurrently with, sheep HSC leads to statistically significant higher levels of HSC engraftment when compared to HSC alone. Using confocal microscopy and fluorescently-labeled cells, we first determined the sites of engraftment/localization of donor EPC, MSC, and HSC at 2-2.5 months after IUHSCT when transplanted at the does of 7.1 x 106 /Kg, 2.5 x 106 /kg, and 2.1 x 106 /kg, respectively, at 60-65 days of gestation (human equivalent of 16 gestational weeks). We demonstrated that EPC and MSC engraft at different sites after IUHSCT than the transplanted HSC. MSC and EPC engrafted in the diaphysis, where they integrated into the vascular and perivascular niches and produced SCF and CXCL12. Confocal analysis demonstrated that transplanted EPC produced higher levels of CXCL12 than endogenous cells, explaining, at least in part, the higher levels of hematopoietic engraftment seen in the BM and PB of animals transplanted with EPC 3 days prior to HSC. HSC engrafted mainly in the metaphysis, a metabolically active tissue in the fetus, which is comprised of large sinusoidal veins expressing high levels of hyaluronan (HA) synthase. In addition, donor HSC in the metaphysis were actively cycling (>90%) as determined by Ki67 staining. Fewer donor HSC were found in the diaphysis, and only a small percentage of these cells were dividing, but they localized in clusters in close proximity to donor MSC or EPC. Moreover, a direct correlation was found between the levels of donor-derived perivascular cells engrafted within the vasculature, and the numbers of donor-derived HSC present in the perivascular areas (defined as being within a distance of 5 cell nuclei from vessel), suggesting that, in the diaphysis, adult MSC and EPC provided support/attracted adult HSC. In summary, we have shown that, in a non-myeloablative fetal setting without stress-induced signaling, allogeneic adult donor HSC engraft and proliferate efficiently along with endogenous hematopoietic cells within the metaphysis, and that the high levels of CXCL12 produced by adult EPC were potentially responsible for their ability to significantly enhance HSC engraftment after IUHSCT. DisclosuresNo relevant conflicts of interest to declare.

Correction of murine hemoglobinopathies by prenatal tolerance induction and postnatal nonmyeloablative allogeneic BM transplants

AbstractSickle cell disease (SCD) and thalassemias (Thal) are common congenital disorders, which can be diagnosed early in gestation and result in significant morbidity and mortality. Hematopoietic stem cell transplantation, the only curative therapy for SCD and Thal, is limited by the absence of matched donors and treatment-related toxicities. In utero hematopoietic stem cell transplantation (IUHCT) is a novel nonmyeloablative transplant approach that takes advantage of the immunologic immaturity and normal developmental properties of the fetus to achieve mixed allogeneic chimerism and donor-specific tolerance (DST). We hypothesized that a combined strategy of IUHCT to induce DST, followed by postnatal nonmyeloablative same donor “booster” bone marrow (BM) transplants in murine models of SCD and Thal would result in high levels of allogeneic engraftment and donor hemoglobin (Hb) expression with subsequent phenotypic correction of SCD and Thal. Our results show that: (1) IUHCT is associated with DST and low levels of allogeneic engraftment in the murine SCD and Thal models; (2) low-level chimerism following IUHCT can be enhanced to high-level chimerism and near complete Hb replacement with normal donor Hb with this postnatal “boosting” strategy; and (3) high-level chimerism following IUHCT and postnatal “boosting” results in phenotypic correction in the murine Thal and SCD models. This study supports the potential of IUHCT, combined with a postnatal nonmyelablative “boosting” strategy, to cure Thal and SCD without the toxic conditioning currently required for postnatal transplant regimens while expanding the eligible transplant patient population due to the lack of a restricted donor pool.

A Novel Hematopoietic Stem Cell (HSC) Mobilizing Agent Enhances Long-term Allogeneic Engraftment After In Utero Hematopoietic Stem Cell Transplantation (IUHCT)

A Novel Hematopoietic Stem Cell (HSC) Mobilizing Agent Enhances Long-term Allogeneic Engraftment After In Utero Hematopoietic Stem Cell Transplantation (IUHCT)

Optimization Of Vascular Niches To Increase Hematopoietic Engraftment

In utero hematopoietic stem cell transplantation (IUHSCT) is a promising approach for correcting selected congenital hematologic and immunologic disorders. However, if higher levels of donor hematopoietic stem/progenitor (HSC) cell engraftment could be achieved, a wider range of inherited disorders could be targeted. We have previously shown that adult bone marrow (BM) derived-CD34+ cells adhere less efficiently to fetal stromal cells than to their adult counterpart. Furthermore, it has been shown that perivascular cells are able to support, through cellular interactions, the long-term engrafting HSC. Here, we hypothesized that by transplanting bone marrow (BM)-derived endothelial progenitor cells (EPC) prior to HSC transplantation, it would be possible to establish HSC donor-optimized vascular niches within the recipient’s BM, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Adult sheep BM HSC were immunoselected with an antibody against sheep CD34, while EPC were isolated by adherence to collagen type I. Characterization of these cells demonstrated that they were spindle-shaped, and they expressed fetal liver kinase (flk-1/KDR), vonWillebrand factor (vWF), and melanoma cell adhesion molecule (MCAM/CD146). In addition, these cells formed capillary-like structures in Matrigel-based media. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered, intraperitoneally, 1.4X105 CD34+ cells transduced with an eGFP-encoding lentiviral vector (HSCeGFP) in combination with 7.1X105 EPC transduced with an mKate-encoding lentiviral vector (EPCmKate) (n=4), from the same donor, either concurrently, or 3 days prior to HSCeGFP transplantation. At 60 days post-transplant, we performed flow cytometry on peripheral blood (PB) and BM to assess the levels of donor cell engraftment. We also performed confocal microscopic analysis of bone sections to identify the localization and interaction between transplanted cells. Our results demonstrate that animals receiving EPCmKate 3 days prior to HSC transplantation displayed 13-fold higher levels of eGFP(+) hematopoietic cells in their BM (6.5±0.5%), when compared with animals receiving EPC and HSC simultaneously (0.39±0.29%). Confocal microscopy analysis showed that, regardless of the time-point of transplant, donor cells that engrafted in the diaphysis localized to the perivascular area, and a correlation was found between the levels of CD146(+)mKate cells and HSCeGFP engraftment. By contrast, in the metaphysis, only eGFP(+) cells were detected, and these cells co-expressed osteopontin, a marker of osteoblasts. These results show that in IUHSCT, delivery of EPC,CD146(+), cells prior to CD34+HSC results in modification of the vascular niches by donor-derived cells, leading to significantly higher levels of HSC engraftment. Furthermore, a considerable percentage of CD34+eGFP(+) cells did not contribute to the hematopoietic pool, but rather, contributed to the developing bone, suggesting that a more effective selective process for HSC might be necessary for improving engraftment in IUHSCT. Disclosures: No relevant conflicts of interest to declare.

In utero hematopoietic cell transplantation—recent progress and the potential for clinical application

In utero hematopoietic stem cell transplantation (IUHCT) is a potential therapeutic alternative to postnatal hematopoietic stem cell transplantation (HSCT) for congenital hematologic disorders that can be diagnosed early in gestation and can be cured by HSCT. The rationale is to take advantage of normal events during hematopoietic and immunologic ontogeny to facilitate allogeneic hematopoietic engraftment. Although the rationale remains compelling, IUHCT has not yet achieved its clinical potential. This review will discuss recent experimental progress toward overcoming the barriers to allogeneic engraftment and new therapeutic strategies that may hasten clinical application.

In utero Hematopoietic Stem Cell Transplantation in Canines: Exploring the Gestational Age Window of Opportunity to Maximize Engraftment

Objective: In utero hematopoietic stem cell transplantation (IUHSCT) is a promising therapy for a variety of congenital disorders. Our objective was to determine the optimal time in gestation for IUHSCT in a canine model. Methods: IUHSCT was performed in day 31-50 (term 63) fetal canines with CD34+ cells isolated from paternal bone marrow at doses of 0.09-3.4 × 10⁹ CD34+ cells/kg and T cells (CD3+/CD5+) from paternal blood at 0.11-1.1 × 10⁹ cells/kg. Engraftment was assayed using PCR-based chimerism analysis (SRY gene detection for female recipients, and unique microsatellite loci for both sexes). Results: Microchimerism and chimerism were present in multiple recipients across most gestational ages at transplant. Maximal engraftment was obtained in hematopoietic tissues in transplants performed at 42 days. At extremes of recipient gestational age, minimal to no engraftment was seen. Conclusion: Fetal age at the time of IUHSCT plays an important role in achieving engraftment in our canine model.

Clonal Deletion of Donor-Reactive T Cell Subsets in Prenatal Allogeneic Chimeras is Preserved During Potent Viral Infection

Long-term engraftment following in utero hematopoietic stem cell transplantation (IUHCT) requires the development of T cell tolerance through the deletion of alloreactive T cell clones and anergy in the remaining non-deleted clones. In response to viral illness, however, a dramatic T cell expansion occurs which may include the expansion of anergic alloreactive T cells. Expansion of these subsets could result in an abrupt rejection of previously engrafted donor cells, permitting devastating relapse of disease in the patient.

Successful In Utero Hematopoietic Stem Cell Transplantation (IUHSCT) Likely Depends on Niche Maturity, Recipient/Donor MHC Compatibility and Donor Cell Origin

AbstractAbstract 574Many 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.

Intravascular Route Is Not Superior to an Intraperitoneal Route for In Utero Transplantation of Human Hematopoietic Stem Cells and Engraftment in Sheep

Previously, we have reported in utero hematopoietic stem-cell transplantation (IUT) in sheep (1) and nonhuman primates (2) using an intraperitoneal (IP) route, as performed in clinical trials in humans (3,4) to treat congenital disorders, including X-linked severe combined immunodeficiency. Recent studies (5,6) have demonstrated that, for IUT in mice, the intravascular (IV) route facilitates the injection of cell doses higher than those that can be injected IP, thereby resulting in increased levels of donor-derived chimerism after birth. The studies are tremendous in that they have demonstrated the feasibility of such narrow route injection in mouse fetuses and yielded superior outcomes. However, no study has compared the IV and IP approaches with respect to the efficiency of engraftment in large animals. In this study, we compared the efficacy of these approaches for IUT in sheep. Human cord blood CD34+ cells were transplanted into the right ventricular cavity (IV group, n=4) or abdominal cavity (IP group, n=3) of the preimmune fetal sheep (at gestation days 59–61; full term, 147 days). The cells were injected through the uterine wall using a 23-gauge needle under ultrasound guidance. For the IV approach, we transplanted cells into the right ventricular cavity instead of the vitelline vein (5,6), because the vitelline vein was too small to be detected under ultrasound guidance at the gestation days. The number of cells transplanted per fetus ranged from 1.4×105 to 6.3×105; there were no intergroup differences. After birth (4 months after transplant), few transplant-derived cells were detected in the peripheral blood, but the engraftment could be properly examined by clonogenic hematopoietic progenitor assay to detect human hematopoietic colony-forming units in the bone marrow (BM). The fractions of human colony-forming units were 1.3%±0.1% and 1.3%±0.9% in the IP and IV groups, respectively (Table 1), indicating that there was no difference in the engraftment between the two groups.TABLE 1: In utero transplantation and engraftment of human hematopoietic stem cells in sheepInitially, we had hypothesized that the engraftment of the transplanted cells that were injected by the IV route in the BM at approximately day 60 of the gestation period in sheep would be better than that of the cells injected by the IP route because during this period, hematopoietic stem cells IV move from the liver into the BM (7). However, we found no difference in the efficacies of both approaches. Furthermore, bradycardia was often detected in the fetuses that were injected by the IV route, thereby implying that the IV approach might have some risk associated with it. Considering that both approaches are equally efficacious for the engraftment of the transplanted cells and because IP injection is evidently easier and safer than IV injection, we hypothesize that the IP approach is more meritorious than the IV approach for IUT experiments in large animals and clinical studies in humans. Therefore, we believe that further clinical trials of IUT can be conducted using the IP approach. Yujiro Tanaka1 Shigeo Masuda1 Tomoyuki Abe1,2,3 Satoshi Hayashi4 Yoshihiro Kitano4 Yoshikazu Nagao2,3 Yutaka Hanazono1 1 Division of Regenerative Medicine Center for Molecular Medicine Jichi Medical University Tochigi, Japan 2 Department of Agriculture Utsunomiya University Tochigi, Japan 3 Division of Biological Production Science United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology Tokyo, Japan 4 Department of Obstetrics and Gynecology National Center for Child Health and Development Tokyo, Japan

Breast Milk Transmission of Maternal Alloantibodies Activates a Postnatal Immune Response That Limits Allogeneic Engraftment after in Utero Hematopoietic Stem Cell Transplantation in the Murine Model

In utero hematopoietic stem cell transplantation (IUHCT) is a potential alternative to postnatal HSCT for treatment of congenital hematologic disorders. We have recently documented in the murine model that, in contrast to the results for congenic IUHCT where 100% of recipients maintain permanent mixed hematopoietic chimerism, 70% of recipients of allogeneic IUHCT lose their chimerism by four weeks of age. We hypothesized that there was an adaptive immune barrier that was differentially activated in non-chimeric versus chimeric mice. To directly test this hypothesis, we compared the frequency of allospecific T-cells by the quantitative in vivo MLR assay in chimeric versus non-chimeric mice, as well as immunized and non-injected control mice. After IUHCT of GFP+ B6 BM cells into E14 Balb/c fetuses, lymphocytes were harvested from 4 week old Balb/c recipients, CFSE stained, and injected into F1 recipients, and F1 spleens were then FACS analyzed for frequency of alloreactive T cells at 96hrs. The frequency of alloreactive T cells in chimeric mice was 2.034 ± 0.562 percent of CD4+, CFSE+ input lymphocytes as compared to 6.323 ± 1.185 percent in non-chimeric mice (p=0.010). We also compared allospecific humoral response in the same groups by flow cytometric assay for anti-donor IgG. Serum was isolated from 4 week old Balb/c IUHCT recipients and incubated with B6 target cells and anti-IgG secondary antibody for detection of alloantibodies. Anti-IgG immunofluorescence was increased 9.512 ± 3.183 fold as compared to 1.097 ± 0.113 fold over controls in non-chimeric and chimeric mice respectively (p=0.013), and showed dose dependence with regard to serum concentration. These results confirm that loss of chimerism after allogeneic IUHCT in the murine model is associated with activation of an allospecific adaptive immune response. We further hypothesized that the source of immune activation was the mature maternal immune system, rather than the immature fetal immune system. To test this hypothesis, we performed IUHCT and used non-injected foster moms as the source of breast milk. Remarkably, we found that 17/17 pups raised by foster moms maintained long-term (> 4 months) donor cell chimerism, as compared to 15/52 pups raised by injected moms (p<0.0001). We then analyzed the injected moms for the presence of alloantibodies at P1 (one week after IUHCT), P8 and P28. At P1, 10/10 were negative for the presence of alloantibodies, while 8/10 were positive for the presence of alloantibodies at P8 as well as P28 (p=0.0007). We have subsequently identified a positive correlation between the number of aborted fetuses and the magnitude of the maternal humoral response (correlation coefficient=0.6, n=15, p=0.018). In fact, the absence of a maternal anti-donor humoral response is only observed in pregnancies with no aborted fetuses and the injected pups from these pregnancies are all chimeric. These results indicate that IUHCT at E14 in the murine model results in maternal immunization through fetal loss and production of alloantibodies which are then transferred in breast milk to pups, triggering an adaptive immune response and resulting in loss of chimerism in 70% of pups by four weeks of age. These findings explain the apparent contradiction of activation of an alloimmune response by IUHCT in the pre-immune fetal recipient and once again validate the concept of fetal immune tolerance as permissive for allogeneic engraftment after IUHCT. The importance of this mechanism of loss of chimerism after IUHCT in large animal or clinical settings is unknown and will be the focus of future investigations. In addition, our observations may have broad implications for autoimmune disease and the immune consequences of maternal-fetal cellular trafficking.

In Utero Hematopoietic Cell Transplantation Using Haploidentical Parental Donors Reverses the Lethal Phenotype in Dogs with Canine Leukocyte Adhesion Deficiency.

Juvenile dogs with the genetic disease leukocyte adhesion deficiency or CLAD, like children with leukocyte adhesion deficiency or LAD, experience recurrent life threatening bacterial infections due to the inability of leukocytes to migrate to sites of infection. Both CLAD and LAD result from defects in the leukocyte integrin CD18 molecule. We have used the CLAD model to develop new therapeutic approaches to children with LAD. Previous studies demonstrate that low levels of donor chimerism following matched littermate transplant reverses the disease phenotype in CLAD. However, most children with LAD lack a matched sibling donor. In utero hematopoietic stem cell transplantation (IUHCT) has been shown to result in low levels of allogeneic chimerism in the normal mouse model. In the current study we evaluate IUHCT in the CLAD model using a haploidentical paternal donor. A previously transplanted CLAD female was mated with a paternal CLAD carrier. IUHCT was performed at gestational day 50 by ultrasound guided intraperitoneal injection of 1.7E+08 paternal CD34+ enriched BM cells/kg estimated fetal weight reconstituted with nonenriched paternal BM to provide 2.4% CD3+ cells. Seven fetuses were injected. One pup was still born and one died on day 2 from maternal neglect. Flow cytometry for CD18 expression in PB, spleen, liver, thymus and BM from the two deceased pups confirmed the diagnosis of CLAD with donor cell engraftment (PB:1.3–3%, spleen:3.5–4%, liver:3.4–4.2%, thymus:1.7–4.4%, BM:3.3–21.3%). Histology demonstrated no evidence of GVHD. Of the 5 surviving pups, 3 are CLAD carriers (Louie, Miles, Ella) and 2 are CLAD offspring (Billie, Duke). Currently all 5 pups are alive at 5 months of age. Engraftment analysis in those in which it is possible by CD18 expression (Billie and Duke) or the presence of the Y chromosome (Billie and Ella) demonstrates donor cells in all analyzable pups at 5 months. The PB levels of CD18+ donor cells in CLAD offspring are low but stable and contribute to multiple lineages (Fig 1A). Clinically, Billie and Duke are alive and active at 5 months compared to historical controls with 4 and 6 month mortalities of 75% and 100%. Both have had mild leukocytosis (Billie:17.6–23.8K/uL, 21.4K/uL @ 5 months; Duke:23.4–39.5K/uL, 23.8K/uL @ 5 months) compared to historical CLAD controls of 50–100K/uL. Billie has had no clinical episodes consistent with the CLAD phenotype. Duke has experienced five CLAD phenotypic episodes which have resolved without the need for intensive care (Fig 1B). There was no evidence of GVHD in any injected animals. This study highlights the ability to safely achieve levels of haploidentical donor CD18+ leukocytes following IUHCT which markedly improve the lethal disease phenotype in a disease-specific large-animal model of a human genetic disease. It supports the potential therapeutic value of IUHCT for diseases, such as LAD, which can be successfully treated with low levels of hematopoietic chimerism. [Display omitted]

In utero hematopoietic stem cell transplantation in canines: The gestational window of opportunity to maximize engraftment

In utero hematopoietic stem cell transplantation (IUHSCT) is a promising therapy for a variety of congenital disorders. Establishing the optimal time in gestation for IUHSCT is one of the critical factors to be determined for successful engraftment of donor cells. Our objective was to determine this gestational age window in a canine model.

High-level allogeneic chimerism achieved by prenatal tolerance induction and postnatal nonmyeloablative bone marrow transplantation

AbstractClinical application of allogeneic bone marrow transplantation (BMT) has been limited by toxicity related to cytoreductive conditioning and immune response. In utero hematopoietic stem cell transplantation (IUHSCT) is a nonablative approach that achieves mixed chimerism and donor-specific tolerance but has been limited by minimal engraftment. We hypothesized that mixed chimerism achieved by IUHSCT could be enhanced after birth by nonmyeloablative total body irradiation (TBI) followed by same-donor BMT. To test this hypothesis, mixed chimerism was created by IUHSCT in a major histocompatibility complex-mismatched strain combination. After birth, chimeric animals received nonmyeloablative TBI followed by transplantation of donor congenic bone marrow cells. Our results show that: (1) low-level chimerism after IUHSCT can be enhanced to high-level chimerism by this strategy; (2) enhancement of chimerism is dependent on dose of TBI; (3) the mechanism of TBI enhancement is via a transient competitive advantage for nonirradiated hematopoietic stem cells; (4) engraftment observed in the tolerant, fully allogeneic IUHSC transplant recipient is equivalent to a congenic recipient; and (5) host-reactive donor lymphocytes are deleted with no evidence of graft-versus-host disease. This study supports the concept of prenatal tolerance induction to facilitate nonmyeloablative postnatal strategies for cellular therapy. If clinically applicable, such an approach could dramatically expand the application of IUHSCT.