Neonatal Outcomes of Multiple Congenital Thoracic Lesions.

INTRODUCTION: Congenital pulmonary airway malformations (CPAM), intra- and extralobar bronchopulmonary sequestrations (iBPS/eBPS), CPAM-BPS hybrid lesions (HL), congenital lobar emphysema (CLE), bronchial atresia (BA), and foregut duplication cysts (FDC), collectively referred to as congenital thoracic lesions (CTL), are mostly solitary. Patients with multiple CTL are rare, and reports on such cases are scarce. To address this dearth, we analyzed a large multifocal CTL patient cohort. METHODS: Retrospective chart review of patients born between 1/1/2013 and 3/31/2023 who underwent surgery for a CTL at our tertiary center. Patients with radiological and surgical diagnosis of multifocal CTL, defined as ≥2 CTL present in more than one lobe were included to record pre-, peri-, and postnatal patient characteristics. RESULTS: Among 701 CTL patients, 74(10.5%) had multiple CTL. CTL multifocality was prenatally recognized correctly in 8(12.9%) patients. Most multiple CTL were right-sided, unilateral multilobar lesions (n=33, 44%). Bilateral CTL were found in 9(12.1%) patients. CPAM-CPAM lesions were the most prevalent CTL types (n=36, 49%). Genetic syndromes were confirmed in 3(4%) and additional congenital anomalies in 9(12.9%) patients, 5 of those had multiple congenital anomalies. Of 49(65%) patients with multilobar CTL, 25(51%) underwent bilobectomy and 24(49%) lung-sparing surgery. Length of stay was similar. Mortality was 5.4%. CONCLUSION: We report on the largest patient cohort with multiple CTL to date. Multiple CTL occurred in 1/10 patients with CTL, and only 12.9% were recognized prenatally. Lung-sparing surgery can be considered. Multiple additional congenital anomalies and genetic syndromes may be more common and genetic testing should be considered. Overall, outcomes in this patient population are favorable.

Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice.

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

Immune modulation permits tolerance and engraftment in a murine model of late-gestation transplantation.

ABSTRACT: In utero hematopoietic cell transplantation is an experimental nonmyeloablative therapy with potential applications in hematologic disorders, including sickle cell disease (SCD). Its clinical utility has been limited due to the early acquisition of T-cell immunity beginning at ∼14 weeks gestation, posing significant technical challenges and excluding treatment fetuses evaluated after the first trimester. Using murine neonatal transplantation at 20 days postcoitum (DPC) as a model for late-gestation transplantation (LGT) in humans, we investigated whether immune modulation with anti-CD3 monoclonal antibody (mAb) could achieve donor-specific tolerance and sustained allogeneic engraftment comparable with that of the early-gestation fetal recipient at 14 DPC. In allogeneic wild-type strain combinations, administration of anti-CD3 mAb with transplantation resulted in transient T-cell depletion followed by central tolerance induction confirmed by donor-specific clonal deletion and skin graft tolerance. Normal immune responses to third-party major histocompatibility complex and viral pathogens were preserved, and graft-versus-host disease did not occur. We further demonstrated the successful application of this approach in the Townes mouse model of SCD. These findings confirm the developing fetal T-cell response as a barrier to LGT and support transient T-cell depletion as a safe and effective immunomodulatory strategy to overcome it.

The Delivery Room Resuscitation of Infants with Congenital Diaphragmatic Hernia Treated with Fetoscopic Endoluminal Tracheal Occlusion: Beyond the Balloon.

INTRODUCTION: Randomized controlled trials found that fetoscopic endoluminal tracheal occlusion (FETO) resulted in increased fetal lung volume and improved survival for infants with isolated, severe left-sided congenital diaphragmatic hernia (CDH). The delivery room resuscitation of these infants is particularly unique, and the specific delivery room events are largely unknown. The objective of this study was to compare the delivery room resuscitation of infants treated with FETO to standard of care (SOC) and describe lessons learned. METHODS: Retrospective single-center cohort study of infants treated with FETO compared to infants who met FETO criteria during the same period but who received SOC. RESULTS: FETO infants were more likely to be born prematurely with 8/12 infants born <35 weeks gestational age compared to 3/35 SOC infants. There were 5 infants who required emergent balloon removal (2 ex utero intrapartum treatment and 3 tracheoscopic removal on placental bypass with delayed cord clamping) and 7 with prenatal balloon removal. Surfactant was administered in 6/12 FETO (50%) infants compared to 2/35 (6%) in the SOC group. Extracorporeal membrane oxygenation use was lower at 25% and survival was higher at 92% compared to 60% and 71% in the SOC infants, respectively. CONCLUSION: The delivery room resuscitation of infants treated with FETO requires thoughtful preparation with an experienced multidisciplinary team. Given increased survival, FETO should be offered to infants with severe isolated left-sided CDH, but only in high-volume centers with the experience and capability of removing the balloon, emergently if needed. The neonatal clinical team must be skilled in managing the unique postnatal physiology inherent to FETO where effective interdisciplinary teamwork is essential. Empiric and immediate surfactant administration should be considered in all FETO infants to lavage thick airway secretions, particularly those delivered <48 h after balloon removal.

Fetal gene therapy.

Fetal gene therapy was first proposed toward the end of the 1990s when the field of gene therapy was, to quote the Gartner hype cycle, at its "peak of inflated expectations." Gene therapy was still an immature field but over the ensuing decade, it matured and is now a clinical and market reality. The trajectory of treatment for several genetic diseases is toward earlier intervention. The ability, capacity, and the will to diagnose genetic disease early-in utero-improves day by day. A confluence of clinical trials now signposts a trajectory toward fetal gene therapy. In this review, we recount the history of fetal gene therapy in the context of the broader field, discuss advances in fetal surgery and diagnosis, and explore the full ambit of preclinical gene therapy for inherited metabolic disease.

Prenatally Diagnosed Large Lung Lesions: Timing of Resection and Perinatal Outcomes.

INTRODUCTION: Fetuses with large lung lesions including congenital cystic adenomatoid malformations (CCAMs) are at risk for cardiopulmonary compromise. Prenatal maternal betamethasone and cyst drainage for micro- and macrocystic lesions respectively have improved outcomes yet some lesions remain large and require resection before birth (open fetal surgery, OFS), at delivery via an Ex Utero Intrapartum Treatment (EXIT), or immediately post cesarean section (section-to-resection, STR). We sought to compare prenatal characteristics and outcomes in fetuses undergoing OFS, EXIT, or STR to inform decision-making and prenatal counseling. METHODS: A single institution retrospective review was conducted evaluating patients undergoing OFS, EXIT, or STR for prenatally diagnosed lung lesions from 2000 to 2021. Specimens were reviewed by an anatomic pathologist. Lesions were divided into "CCAMs" (the largest pathology group) and "all lung lesions" since pathologic diagnosis is not possible during prenatal evaluation when care decisions are made. Prenatal variables included initial, greatest, and final CCAM volume-ratio (CVR), betamethasone use/frequency, cyst drainage, and the presence of hydrops. Outcomes included survival, ECMO utilization, NICU length of stay (LOS), postnatal nitric oxide use, and ventilator days. RESULTS: Sixty-nine percent (59 of 85 patients) of lung lesions undergoing resection were CCAMs. Among patients with pathologic diagnosis of CCAM, the initial, largest, and final CVRs were greatest in OFS followed by EXIT and STR patients. Similarly, the incidence of hydrops was significantly greater and the rate of hydrops resolution was lower in the OFS group. Although the rate of cyst drainage did not differ between groups, maternal betamethasone use varied significantly (OFS 60.0%, EXIT 100.0%, STR 74.3%; p = 0.0378). Notably, all OFS took place prior to 2014. There was no difference in survival, ventilator days, nitric oxide, NICU LOS, or ECMO between groups. In multiple variable logistic modeling, determinants of survival to NICU discharge among patients undergoing resection with a pathologic diagnosis of CCAM included initial CVR <3.5 and need for <3 maternal betamethasone doses. CONCLUSION: For CCAMs that remain large despite maternal betamethasone or cyst drainage, surgical resection via OFS, EXIT, or STR are viable options with favorable and comparable survival between groups. In the modern era there has been a shift from OFS and EXIT procedures to STR for fetuses with persistently large lung lesions. This shift has been fueled by the increased use of maternal betamethasone and introduction of a Special Delivery Unit during the study period and the appreciation of similar fetal and neonatal outcomes for STR vs. EXIT and OFS with reduced maternal morbidity associated with a STR. Accordingly, efforts to optimize multidisciplinary perinatal care for fetuses with large lung lesions are important to inform patient selection criteria and promote STR as the preferred surgical approach in the modern era. LEVEL OF EVIDENCE: Level IV.

Morphologic Features in Congenital Pulmonary Airway Malformations and Pulmonary Sequestrations Correlate With Mutation Status: A Mechanistic Approach to Classification.

Congenital pulmonary airway malformations (CPAMs) have a range of morphologies with varying cyst sizes and histologic features (types 1 to 3). Evidence suggested they arise secondary to bronchial atresia, however, we recently showed that cases with type 1 and 3 morphology are driven by mosaic KRAS mutations. We hypothesized that 2 distinct mechanisms account for most CPAMs: one subset is secondary to KRAS mosaicism and another is due to bronchial atresia. Cases with type 2 histology, similar to sequestrations, would be related to obstruction and therefore negative for KRAS mutations regardless of cyst size. We sequenced KRAS exon 2 in type 2 CPAMs, cystic intralobar and extralobar sequestrations, and intrapulmonary bronchogenic cysts. All were negative. Most sequestrations had a large airway in the subpleural parenchyma adjacent to the systemic vessel, anatomically confirming bronchial obstruction. We compared morphology to type 1 and 3 CPAMs. On average, type 1 CPAMs had significantly larger cysts, but there remained substantial size overlap between KRAS mutant and wild-type lesions. Features of mucostasis were frequent in sequestrations and type 2 CPAMs, while their cysts were generally simple and round with flat epithelium. Features of cyst architectural and epithelial complexity were more common in type 1 and 3 CPAMs, which rarely showed mucostasis. Similarity in histologic features among cases that are negative for KRAS mutation support the hypothesis that, like sequestrations, the malformation of type 2 CPAMs is related to obstruction during development. A mechanistic approach to classification may improve existing subjective morphologic methods.

Fetal allotransplant recipients are resistant to graft-versus-host disease.

In utero hematopoietic cell transplantation (IUHCT) is an experimental treatment for congenital hemoglobinopathies, including Sickle cell disease and thalassemias. One of the principal advantages of IUHCT is the predisposition of the developing fetus toward immunologic tolerance. This allows for engraftment across immune barriers without immunosuppression and, potentially, decreased susceptibility to graft-versus-host disease (GVHD). We demonstrate fetal resistance to GVHD following T cell-replete allogeneic hematopoietic cell transplantation compared with the neonate. We show that this resistance is associated with elevated fetal serum interleukin-10 conducive to the induction of regulatory T cells (Tregs). Finally, we demonstrate that the adoptive transfer of Tregs from IUHCT recipients to neonates uniformly prevents GVHD, recapitulating the predisposition to tolerance observed after fetal allotransplantation. These findings demonstrate fetal resistance to GVHD following hematopoietic cell transplantation and elucidate Tregs as important contributors.

Molecular and Cellular In Utero Therapy.

Significant advances in maternal-fetal medicine and gene sequencing technology have fostered a new frontier of in utero molecular and cellular therapeutics, including gene editing, enzyme replacement therapy, and stem cell transplantation to treat single-gene disorders with limited postnatal treatment strategies. In utero therapies take advantage of unique developmental properties of the fetus to allow for the correction of monogenic disorders before irreversible disease pathology develops. While early preclinical studies in animal models are encouraging, more studies are needed to further evaluate their safety and efficacy prior to widespread clinical use.

Defining the spatial landscape of KRAS mutated congenital pulmonary airway malformations: a distinct entity with a spectrum of histopathologic features.

The potential pathogenetic mechanisms underlying the varied morphology of congenital pulmonary airway malformations (CPAMs) have not been molecularly determined, but a subset have been shown to contain clusters of mucinous cells (MCC). These clusters are believed to serve as precursors for potential invasive mucinous adenocarcinoma, and they are associated with KRAS codon 12 mutations. To assess the universality of KRAS mutations in MCCs, we sequenced exon 2 of KRAS in 61 MCCs from 18 patients, and we found a KRAS codon 12 mutation in all 61 MCCs. Furthermore, all MCCs from a single patient always had the same KRAS mutation, and the same KRAS mutation was also found in non-mucinous lesional tissue. Next generation sequencing of seven MCCs showed no other mutations or copy number variations. Sequencing of 46 additional CPAMs with MCCs revealed KRAS mutations in non-mucinous lesional tissue in all cases. RNA in situ hybridization confirmed widespread distribution of cells with mutant KRAS RNA, even extending outside of the bronchiolar type epithelium. We identified 25 additional CPAMs with overall histologic architecture similar to CPAMs with KRAS mutations but without identifiable MCCs, and we found KRAS mutations in 17 (68%). The histologic features of these KRAS mutated CPAMs included type 1 and type 3 morphology, as well as lesions with an intermediate histologic appearance, and analysis revealed a strong correlation between the specific amino acid substitution and histomorphology. These findings, together with previously published model organism data, suggests that the formation of type 1 and 3 CPAMs is driven by mosaic KRAS mutations arising in the lung epithelium early in development and places them within the growing field of mosaic RASopathies. The presence of widespread epithelial mutation explains late metastatic disease in incompletely resected patients and reinforces the recommendation for complete resection of these lesions.

In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease.

In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua G→A (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA G→A (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases.

Delivery technologies for in utero gene therapy.

Advances in prenatal imaging, molecular diagnostic tools, and genetic screening have unlocked the possibility to treat congenital diseases in utero prior to the onset of clinical symptoms. While fetal surgery and in utero stem cell transplantation can be harnessed to treat specific structural birth defects and congenital hematological disorders, respectively, in utero gene therapy allows for phenotype correction of a wide range of genetic disorders within the womb. However, key challenges to realizing the broad potential of in utero gene therapy are biocompatibility and efficiency of intracellular delivery of transgenes. In this review, we outline the unique considerations to delivery of in utero gene therapy components and highlight advances in viral and non-viral delivery platforms that meet these challenges. We also discuss specialized delivery technologies for in utero gene editing and provide future directions to engineer novel delivery modalities for clinical translation of this promising therapeutic approach.

Open Fetal Surgical Outcomes for Myelomeningocele Closure Stratified by Maternal Body Mass Index in a Large Single-Center Cohort.

BACKGROUND: Open maternal-fetal surgery for in utero closure of myelomeningocele (MMC) has become an accepted treatment option for prenatally diagnosed open neural tube defects. Historically, this option has been limited to women with BMI < 35 due to concern for increasing complications in patients with obesity. OBJECTIVE: The aim of this study was to evaluate maternal, obstetric, and fetal/neonatal outcomes stratified by maternal BMI classification in women who undergo open maternal-fetal surgery for fetal myelomeningocele (fMMC) closure. METHODS: A single-center fMMC closure registry was queried for maternal demographics, preoperative factors, fetal surgery outcomes, delivery outcomes, and neonatal outcomes. Data were stratified based on maternal BMI: <30, 30-34.99, and ≥35-40, corresponding to normal weight/overweight, obesity class I, and obesity class II. Statistical analysis was performed using statistical software SAS v.9.4 (SAS Institute Inc., Cary, NC, USA). RESULTS: A total of 264 patients were analyzed, including 196 (74.2%) with BMI <30, 54 (20.5%) with BMI 30-34.99, and 14 (5.3%) with BMI ≥ 35-40. Maternal demographics and preoperative characteristics were similar among the groups. Operative time increased with increasing BMI; otherwise, perioperative outcomes were similar among the groups. Obstetric and neonatal outcomes were similar among the groups. CONCLUSION: Increasing maternal BMI did not result in a negative impact on maternal, obstetric, and fetal/neonatal outcomes in a large cohort of patients undergoing open maternal-fetal surgery for fMMC closure. Further study is warranted to determine the generalizability of these results.

Gene and Stem Cell Therapies for Fetal Care: A Review.

Importance: Gene and stem cell therapies have become viable therapeutic options for many postnatal disorders. For select conditions, prenatal application would provide improved outcomes. The fetal state allows for several theoretical advantages over postnatal therapy, including immune immaturity and cellular niche accessibility. Observations: Advances in prenatal diagnostic accuracy and surgical precision, as well as improvements in stem cell and gene therapy methods, have made prenatal gene and stem cell therapy realistic. Studies in mouse models and early human trials demonstrate the feasibility of these approaches. Additional efforts are under way to streamline fetal applications of stem cell and gene therapy while carefully considering best ethical practice and following established regulatory pathways. Conclusions and Relevance: Fetal stem cell and gene therapy bring important therapeutic opportunities for select disorders that present in the fetal and neonatal periods. While this field is in its infancy, these therapies are starting to be available clinically, and clinicians should be aware of their benefits and challenges.

Two's Company: Multiple Thoracic Lesions on Prenatal US and MRI.

BACKGROUND: Congenital pulmonary airway malformations (CPAM), bronchopulmonary sequestrations (BPS), and CPAM-BPS hybrid lesions are most commonly solitary; however, >1 lung congenital lung lesion may occur. OBJECTIVES: To assess the frequency of multiple congenital thoracic anomalies at a high-volume referral center; determine prenatal ultrasound (US) and magnetic resonance imaging (MRI) features of these multifocal congenital lung lesions that may allow prenatal detection; and determine the most common distribution or site of origin. METHODS: Database searches were performed from August 2008 to May 2019 for prenatally evaluated cases that had a final postnatal surgical diagnosis of >1 congenital lung lesion or a lung lesion associated with foregut duplication cyst (FDC). Lesion location, size, echotexture, and signal characteristics were assessed on prenatal imaging and correlated with postnatal computed tomographic angiography and surgical pathology. -Results: Of 539 neonates that underwent surgery for a thoracic lesion, 35 (6.5%) had >1 thoracic abnormality. Multiple discrete lung lesions were present in 19 cases, and a lung lesion associated with an FDC was present in 16. Multifocal lung lesions were bilateral in 3 cases; unilateral, multilobar in 12; and, unilobar multisegmental in 4. Median total CPAM volume/head circumference ratio for multifocal lung lesions on US was 0.66 (range, 0.16-1.80). Prenatal recognition of multifocal lung lesions occurred in 7/19 cases (36.8%). Lesion combinations were CPAM-CPAM in 10 cases, CPAM-BPS in 5, CPAM-hybrid in 2, hybrid-hybrid in 1, and hybrid-BPS in 1. Of 5 unilateral, multifocal lung lesions, multifocality was prenatally established through identification of a band of normal intervening lung or intrinsic differences in lesion imaging features. CONCLUSIONS: Although less common, multiple thoracic abnormalities can be detected prenatally. Of multifocal lung lesions, the most common combination was CPAM-CPAM, with a unilateral, multilobar distribution. Prenatal recognition is important for pregnancy counseling and postnatal surgical management.

Mucinous Cell Clusters in Infantile Congenital Pulmonary Airway Malformations Mimic Adult Mucinous Adenocarcinoma But Are Not Associated With Poor Outcomes When Appropriately Resected.

Congenital pulmonary airway malformations (CPAMs) are abnormalities of the lung arising during development. At our institution the majority of type I infantile CPAMs contain mucinous cell clusters (MCCs). The overlapping histology of MCCs and adult in situ mucinous adenocarcinomas, as well as reports of metastatic mucinous adenocarcinoma arising in CPAMs resected later in childhood raise concerns about the malignant potential of these cells. However, after adequate surgical resection, malignant recurrence has not been reported in infants with CPAMs. Despite benign behavior, MCCs often have histologic features that, in an adult, would be consistent with a diagnosis of adenocarcinoma. Therefore, to assess the spectrum of features that may be seen in these presumed precursor lesions, we characterized the histology of 671 MCCs spread across 44 infantile CPAMs and compared them to 10 adult mucinous adenocarcinomas. MCCs in CPAMS were often numerous, widespread, and located outside of the large cysts. Mucinous and nonmucinous epithelium within CPAMs showed complex architecture, making application of adult adenocarcinoma architectural patterns difficult. The MCCs within CPAMs displayed nuclear features similar to adult mucinous adenocarcinomas. The proliferative index in infantile MCCs was higher than in adult mucinous adenocarcinomas but was also higher in uninvolved infantile lung tissue. This work illustrates that histologic features typically associated with adenocarcinoma frequently occur within CPAMs; however, this does not alter their benign behavior. Therefore, extreme caution should be used if adult lung cancer terminology is applied to avoid significant potential psychological and physical harms associated with the label of adenocarcinoma.

Regulatory T cells promote alloengraftment in a model of late-gestation in utero hematopoietic cell transplantation.

In utero hematopoietic cell transplantation (IUHCT) has the potential to cure congenital hematologic disorders including sickle cell disease. However, the window of opportunity for IUHCT closes with the acquisition of T-cell immunity, beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Here we report that regulatory T cells can promote alloengraftment and preserve allograft tolerance after the acquisition of T-cell immunity in a mouse model of late-gestation IUHCT. We show that allografts enriched with regulatory T cells harvested from either IUHCT-tolerant or naive mice engraft at 20 days post coitum (DPC) with equal frequency to unenriched allografts transplanted at 14 DPC. Long-term, multilineage donor cell chimerism was achieved in the absence of graft-versus-host disease or mortality. Decreased alloreactivity among recipient T cells was observed consistent with donor-specific tolerance. These findings suggest that donor graft enrichment with regulatory T cells could be used to successfully perform IUHCT later in gestation.