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Aortopexy is the therapeutic modality of choice for severe primary tracheomalacia. The thoracoscopic approach has been used with good results in infants and toddlers, but little information exists on the use of aortopexy in older children. We present the case of a boy with a lifelong history of refractory, a steroid-dependent reactive airway disease, and who was found to have severe primary tracheomalacia. He subsequently underwent thoracoscopic aortopexy with immediate resolution of the tracheomalacia as demonstrated by serial bronchoscopy and long-term resolution of his clinical respiratory symptoms at 1 year.

Donor-specific tolerance has been induced after both fetal and neonatal hematopoietic stem cell (HSC) transplantation in mice. However, the relationship between hematopoietic microchimerism and tolerance in these models has not been defined due to the insensitivity of donor cell detection methodology. To address this problem we developed a semiquantitative polymerase chain reaction (PCR)-based assay for detection of microchimerism after major histocompatibility (MHC) class I disparate HSC transplantation. This assay was used to examine the relationship between microchimerism and tolerance after fetal and neonatal transplantation of fully allogeneic bone marrow cells.C57BL/6 mice (H2-Kb) were used as adult bone marrow donors and Balb/c mice (H2-Kd) were used as fetal or newborn recipients. A dose of 10(10) BM cells/kg was injected intraperitoneally into recipient animals. Peripheral blood of animals which survived beyond 3 weeks of age was analyzed by PCR for the presence of donor MHC class I DNA. Tolerance was tested by placement of donor-specific skin grafts after determination of chimerism status.Our assay was found to be specific for H2-Kb donor cells in an H2-Kd background with a sensitivity of0.0001%. Of 49 animals injected in utero 19 (38%) had donor DNA present in peripheral blood at low levels (0.1%) whereas only 1 of 18 neonatally injected animals had detectable donor cells (P0.01). Tolerance to donor-specific skin grafts was found in 6 of 9 animals which were chimeric after in utero HSC transplantation whereas none of the 18 neonatally injected animals including the chimeric animal were tolerant.Our results indicate the following. (CITEREF RID="JR975255RF1"1) Hematopoietic microchimerism can be detected by PCR in peripheral blood after in utero injection of fully allogeneic HSCs. (CITEREF RID="JR975255RF2"2) Fetal injections yield a higher incidence of microchimerism than newborn injections. (CITEREF RID="JR975255RF3"3) Tolerance can be induced across a fully allogeneic barrier by in utero HSC transplantation and this is associated with the presence of peripheral blood microchimerism.

Purpose The purpose of this study was to characterize the growth rate of sacrococcygeal teratomas (SCTs) and determine its relationship to adverse outcomes. Methods A retrospective review of all pathology-confirmed isolated SCT patients evaluated with at least two documented ultrasounds and followed through hospital discharge between 2005 and 2012 was conducted. SCT growth rate was calculated as the difference between tumor volumes on a late- and early-gestation ultrasound divided by the difference in time. Outcomes were death, high-output cardiac failure (HOCF), hydrops, and preterm delivery. Student's t -test, receiver operator characteristics, Fisher's Exact test, and Pearson's correlation were performed. Results Of the 28 study subjects, there were 3 in utero demises and 2 neonatal deaths. Significantly faster SCT growth rates were seen in all adverse outcomes, including death ( p p =0.005), and preterm delivery ( p =0.009). There was a significant association with adverse outcomes at >61cm 3 /week (AUC=0.87, p =0.001, LR=4.52). Furthermore, there was an even greater association with death at >165cm 3 /week (AUC=0.93, p =0.003, LR=18.42). Growth rate was directly correlated with the percent of solid tumor ( r =0.60, p =0.0008). Conclusion Faster SCT growth is associated with adverse outcomes. SCT growth rate determined by ultrasound is an effective prognostic indicator for adverse outcomes and easily applied to patient management.

In extreme cases of breast hypertrophy, amputation of the nipple-areolar complex and transplantation during reduction mammaplasty has been advocated to avoid nipple necrosis. We report our experience with 172 patients having inferior breast pedicle reduction without amputation of the nipple-areolar complex. Mean total weight of resected tissue was 1,946 g (548 to 5,100 g), with a mean nipple-areolar transposition of 10 cm (0.5 to 23 cm). Dividing patients into four groups by weight of resection, we compared complication rates. In this series, where nipple-areola amputation was avoided, there was a 99.6% survival rate of the nipple-areolar complex with 97.1% retention of nipple sensibility. Patients with extreme breast hypertrophy (3,000 g resected tissue) experienced no increase in complications when compared to smaller reductions. In most cases of gigantomastia, amputation of the nipple can be avoided using the inferior breast pedicle technique. Size of breast resection alone should not determine the fate of the nipple.

Background Cell-specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with adeno-associated viral (AAV) vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts. Materials and methods We packaged AAV2 genome with green fluorescent protein reporter in capsids of other serotypes, AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8, respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 h at multiplicities of infection 102, 103, 104, and 105. We assessed transduction efficiency at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10 ng/mL platelet-derived growth factor-B to culture media. Results Transduction efficiency of the pseudotyped AAV vectors was dose dependent. Multiplicity of infection 105 resulted in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy conferred differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared with AAV2/2. Adeno-associated virus 2/8 was more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5, and 2/8 was significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrated similar transduction efficiency, but by day 7, the three pseudotyped vectors transduced significantly more murine cells compared with AAV2/2. However, in human cells, there was no significant difference in the transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 d. Conclusions The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts in both the normal and wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.

Reproductive success among outbred placental mammalian species requires active tolerance in mothers to foreign paternal antigens expressed by the developing fetus. Although this essential necessity for immunological tolerance has primarily been examined from the perspective of maternal responsiveness to foreign paternal antigens expressed by the developing fetus, exposure to maternal tissue during in utero development also imprints tolerance to genetically foreign non-inherited maternal antigens (NIMA) that persists into adulthood. Nonetheless, until now the biological advantages reinforcing conservation of tolerance to NIMA across eutherian mammals remained unclear. We show vertically transferred maternal cells that establish microchimerism in offspring promote systemic accumulation of regulatory T cells with NIMA specificity. In females, NIMA-specific regulatory T cells expand during pregnancies sired by males expressing alloantigens with overlapping NIMA specificity, thereby averting fetal wastage normally triggered by disruptions of fetal tolerance. Thus, reproductive fitness among females carrying embryos expressing paternally inherited antigens overlapping with NIMA is selectively enhanced. These findings demonstrate that genetic fitness, canonically thought to be restricted to Mendelian inheritance, is enhanced in female placental mammals through vertically transferred maternal cells that promote conservation of NIMA and enforce cross-generational reproductive benefits. Future studies will investigate the cellular and molecular phenotype of the microchimeric maternal cells to elucidate specific mechanisms that drive NIMA-specific regulatory T cell accumulation.

We present the case of an 8-year-old girl with acute onset of intermittent lower abdominal pain. The gray-scale US examination showed an enlarged right ovary with peripheral cysts, reflecting ovarian congestion and strongly suggesting the diagnosis of torsion. Normal arterial and venous flow, however, was found on Doppler US.To demonstrate the importance of gray-scale US findings despite the presence of blood flow found on Doppler US in salvaging a viable, torsed ovary.Despite the Doppler findings, a presumptive diagnosis of ovarian torsion was made.Surgery confirmed the presence of a torsed ovary, which was viable and appeared normal after detorsion.This case illustrates that the gray-scale US appearance of the ovary can be more reliable than Doppler US for the diagnosis of ovarian torsion.

Children represent a unique group of patients who are likely to greatly benefit from minimally invasive surgery (MIS). The promise of less postoperative pain, smaller scars, shorter hospital stays, and a faster return to school continues to drive growth in this area. The development of pediatric-specific techniques and documentation of improved outcomes form a critical gateway to widespread application of pediatric MIS. A brief perspective on current approaches to MIS for pediatric congenital and acquired disease is provided in this report. Technical departures from standardized adult MIS and the rationale for their modification are highlighted.

To the best of the authors' knowledge, this report represents the first description of a ventriculoperitoneal (VP) shunt that migrated into the chest cavity where it caused recurrent pneumonias. This 15-year-old boy with a history of hydrocephalus treated with VP shunt therapy as an infant presented with a 2-year history of chronic coughing and recurrent pneumonia. A high-resolution chest computed tomography scan revealed a right lower lobe infiltration and evidence of migration of the peritoneal shunt tubing through the diaphragm into the lung parenchyma. The catheter was pulled back into the peritoneal cavity via a simple abdominal incision. The patient's long-term outcome was excellent, and there was complete cessation of the pneumonia.

Purpose The failure of in utero transplantation in immune-competent recipients suggests the existence of a fetal immune barrier. The importance of donor major histocompatibility complex (MHC) class I expression in the induction of prenatal tolerance remains undefined. We hypothesized that donor cell MHC class I expression facilitates engraftment in prenatal allogeneic recipients rather than promoting immune rejection. Methods B6.Ly5.2 (class I + ) or B6.TAP −/− (class I − ) murine fetal liver cells were transplanted into age-matched allogeneic fetal recipients. Survival to weaning and subsequent growth was assessed. Engraftment rates and peripheral blood chimerism levels were measured serially. Results The presence or absence of class I expression did not affect survival or growth of recipients and no graft-vs-host disease developed. Allogeneic recipients of B6.Ly5.2 cells exhibited significantly higher levels of donor hematopoietic chimerism when compared to recipients of B6.TAP −/− cells (27% + 10% vs 11% + 8%; P = .004) that deteriorated further over time. Conclusions Donor class I MHC antigen expression is essential for stable long-term engraftment and maintenance of donor-specific tolerance. Further studies are needed to better characterize the role of the fetal innate immune system in prenatal allotransplantation.

Background Intestinal malrotation in adulthood may present with a variety of chronic symptoms. Surgical intervention frequently leads to other complications in these patients. We hypothesized that the chronic nature of malrotation in adults could cause a delay in diagnosis and increased perioperative complications. Study Design All patients diagnosed with intestinal malrotation from July 2002 through July 2006 were included. IRB approval was obtained. Outcomes in patients less than 16 years of age were compared with outcomes from those older than 16. Presenting symptoms, initial diagnosis, results of imaging data, and time to diagnosis were evaluated. Surgical management, resulting complications, and rate of reoperation were analyzed. Results Twenty-four patients with intestinal malrotation were identified (age range, 10 days to 89 years old; 10 adults, 14 children). Seventy percent of adults experienced chronic symptoms for 6 months or more before the diagnosis of malrotation was made (children, 14%, p=0.017). No patients in the adult group were initially diagnosed with malrotation, although 57% of children were correctly diagnosed at the time of presentation of symptoms (p=0.006). Postoperative complications occurred in 60% of adults, but in only 29% of children, though this did not reach significance (p=0.211). Forty percent of adult patients required reoperation (p=0.020). Conclusions Intestinal malrotation in adults is often associated with a delay in diagnosis and increased morbidity. Enhanced awareness of this entity in adults may enhance patient counseling and improve therapeutic outcomes in these patients.

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34+CD45− cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34+ and detectable levels of CD45+ cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.

Invariant NKT (iNKT) cells are glycolipid-reactive alpha/beta T cells which have an important role in the regulation of GVHD after allogeneic bone marrow transplantation. During thymic development, murine iNKT cells divide into three transcriptionally distinct lineages-NKT1, NKT2, and NKT17 that differ in their cytokine expression profile both at rest and upon antigen recognition via their TCR. Given that the lineage profile of iNKT cells varies dramatically between inbred strains of mice, it has been postulated that recognition of allospecific glycolipids determines iNKT cell lineage-fate decisions. Therefore, we challenged this hypothesis in a murine model of prenatal allogeneic transplantation to determine if the lineage commitment of immature iNKT cells was intrinsically programmed or extrinsically regulated by the allospecific environment during development. Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature iNKT cells of both donor and host origin have the capacity to participate in education as CD1d on bone marrow-derived cells regulate the maturation of developing iNKT cells. This permitted an analysis of the impact of either host-to-donor or donor-to-host environmental cues in directing iNKT cell lineage-fate decisions. iNKT cell populations were identified using flow cytometric analysis of the transcription factors PLZF and T-bet. The lineage profile for donor and host thymic iNKT cells from chimeric mice were compared to the thymic iNKT cell population in naïve controls. As shown, B6 iNKT cells in prenatal chimeras exhibited a predominance of the NKT1 lineage in either the donor or the host situation similar to their frequency in naïve B6 controls (figure A). Conversely, Balb/c iNKT cells in both the donor and the host situation exhibited skewing toward an NKT1 lineage profile and away from the NKT2 lineage bias seen in naïve Balb/c controls (figure B). Furthermore, the expression of the H-2d MHC class I-reactive Ly49A receptor by Balb/c iNKT cells strongly correlates with the NKT1 lineage fate in control animals. However, both donor and host Balb/c cells demonstrated reduced correlation between Ly49A expression and NKT1 lineage fate indicating that the presence of H-2b expressing B6 cells diminished the ability of H-2d -reactive Ly49A to dictate lineage fate decisions. This study uniquely demonstrates the potential for cell-extrinsic signals in guiding iNKT cell lineage fate in an asymmetric fashion. Specifically, we find that: 1) the B6 iNKT lineage profile is intrinsically determined and unaffected by exposure to allogeneic Balb/c cells during development; 2) the Balb/c iNKT cell lineage profile is extrinsically determined and dominantly skewed toward an NKT1 lineage by exposure to even small numbers of B6 cells during development; and 3) the exposure to B6 cells overrides the contribution of Ly49A to developmental decisions made by Balb/c iNKT cells. Future studies will explore the regulatory interactions that govern allospecific iNKT cell lineage fate decisions and the resulting impact on the pro-inflammatory or immunoregulatory function of iNKT cells in clinically-relevant models. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Prenatal transplantation capitalizes on the unique fetal environment, allowing for life-long engraftment of allogeneic stem cells without the need for harsh conditioning regimens. A prerequisite for stable engraftment of allogeneic cells likely requires the negative selection of donor-specific host effector T cells (Teff) and the support of donor-specific regulatory T cells (Tregs). However, little is known about the interplay between these cell types during development. The purpose of this study was to characterize the dynamic relationship between donor-specific Teff and Tregs as they emerge during development. Prenatal allogeneic chimeras were established by in utero transplantation of E14 fetal liver light density cells into age-matched allogeneic fetal recipients (Balb/c to B6 or B6 to Balb/c). In this model, immature T cells from B6 mice expressing TCRv-beta-5, 11, and 12 are negatively selected by mtv-8 superantigen complexed with I-E class MHC II on Balb/c cells. As alpha/beta TCR rearrangement does not occur until E16, this established transplantation model allows for alloantigen to be present from the earliest stages of thymic selection. Kinetic analysis of donor-specific T cell populations was performed in peripheral blood paired with in depth analysis in thymus and spleen in control and chimeric mice. Negative selection of donor-specific Teff cells occurs at an unexpectedly slow pace in Balb/c to B6 prenatal chimeras. Donor-specific CD4 and CD8 Teff are significantly decreased in frequency at 4 weeks of age but do not reach maximal deletion until 12 weeks of age (TCRv-beta-5 data shown in Figure A). Further analysis demonstrated that this slow elimination of donor-specific Teff was paired with an early increase in the frequency of donor-specific Tregs at 4 weeks of age (TCRv-beta-5 data shown in Figure B.) This increase in donor-specific Tregs likely occurred as a result of peripheral expansion as there was no change in the frequency of donor-specific Tregs in the thymus (Figure B) and no change in the frequency of these cells that expressed the markers of thymically derived natural Tregs neuropilin-1 or helios (data not shown). In agreement with this hypothesis, donor-specific splenic Tregs incorporated BrdU at a higher rate than other Tregs in young mice indicating a potential expansion of donor-specific Tregs in the periphery (TCRv-beta-5 data shown in Figure C.) Collectively, these data demonstrate that prenatal transplantation is characterized by: 1) a surprisingly slow reduction of donor-specific Teff subsets; 2) an early expansion of donor-specific Tregs. Further studies will explore the role of donor-specific Tregs in controlling early immunity to prenatally encountered antigens. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

RESULTS: There were 76 piglets, of which 35 received supplemental PN with enteral feeding and 39 received supplemental oxygen. Incidence of NEC was higher in piglets receiving supplemental PN (91.4% vs 24.4% ; p

The treatment of congenital hematologic disorders before birth by in utero hematopoietic stem cell transplantation remains a challenging goal. Although success has been achieved in X-linked severe combined immunodeficiency, the approach has failed in all other disorders attempted thus far. In this review, we examine relevant experimental data from the perspective of an analysis of why failure has occurred. We will also attempt to pose the important questions that will need to be answered prior to further clinical attempts to treat most target disorders by this approach.

Clinical 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.

In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism and donor-specific tolerance without cytoreductive conditioning or immunosuppression. The primary limitation to the clinical application of IUHSCTx has been minimal donor cell engraftment, well below therapeutic levels for most target diseases. Donor lymphocyte infusion (DLI) has been used in postnatal circumstances of mixed chimerism as targeted immunotherapy to achieve a graft-versus-hematopoietic effect and to increase levels of donor cell engraftment. In this report we demonstrate in the murine model that a combined approach of IUHSCTx followed by postnatal DLI can convert low-level, mixed hematopoietic chimerism to complete donor chimerism across full major histocompatibility complex barriers with minimal risk for graft-versus-host disease (GVHD). Time-dated embryonic day 14 (E14) to E15 Balb/c (H-2Kd, CD45.2) fetuses underwent intraperitoneal injection of 5 × 106T-cell–depleted B6 (H-2Kb, CD45.2) bone marrow cells. Chimeric recipients then received transplants at either 4 or 8 weeks of age with 1 of 3 doses (5, 15, or 30 × 106cells) of donor congenic splenocytes (B6-Ly5.2/Cr, H-2Kb, CD45.1). The response to DLI was dose dependent, with conversion to complete donor peripheral blood chimerism in 100% of animals that received high-dose (30 × 106 cells) DLI. Only 1 of 56 animals receiving this dose succumbed to GVHD. This study directly supports the potential therapeutic strategy of prenatal tolerance induction to facilitate nontoxic postnatal cellular therapy and organ transplantation, and it has broad implications for the potential treatment of prenatally diagnosed genetic disorders.

Background/Purpose: In utero hematopoietic stem cell transplantation (IUHSCTx) has been experimentally or clinically effective only in circumstances in which there is a survival advantage for donor cells. A survival advantage exists for normal muscle cells in muscular dystrophy. Because hematopoietic and mesenchymal stem cells may have the capacity to differentiate into muscle cells, the authors hypothesized that in utero bone marrow (BM) or fetal liver (FL) stem cell transplantation may be used to treat muscular dystrophy. Methods: Time-dated 14-day-gestation fetal muscular dystrophy mice ( mdx ) were injected intraperitoneally with 1 to 5 × 10 6 BM or FL cells per fetus from Rosa26 donor mice (transgenic for lacZ). Four weeks after birth, peripheral blood from the pups was analyzed for hematopoietic chimerism by using fluorescence-activated cell sorting (FACS) for the Ly-9.1 marker. Chimeric mice (6 BM and 2 FL recipients) were sacrificed at 12 to 14 months of age, muscles were stained with X-gal, and analyzed by 1- to 2-μm plastic sections. Polymerase chain reaction (PCR) for lacZ was performed in other organs to determine systemic engraftment. Results: At the time of death, all animals that were chimeric at 4 weeks continued to show hematopoietic chimerism of 0.2% to 9% by FACS. Engrafted donor cells were found in multiple sections from hindlimb skeletal muscles, diaphragms, and hearts from both BM and FL recipients. These cells had incorporated into the host muscles, and their morphology was consistent with myogenic differentiation. PCR of BM, liver, spleen, thymus, kidney, and lung for lacZ was positive in multiple animals. Conclusions: IUHSCTx leads to widespread engraftment of donor cells in multiple muscle compartments of hematopoietic chimeras. The advantage for normal myocytes offered in the mdx model allows engraftment and myogenic differentiation of transplanted BM or FL cells by morphology at a relatively higher frequency in muscle relative to other tissues, without the need for host conditioning. Because muscular dystrophy now can be detected early in gestation, such a strategy may offer a future alternative in the clinical treatment of this disease. J Pediatr Surg 37:1058-1064. Copyright 2002, Elsevier Science (USA). All rights reserved.

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene. Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells. Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.

In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising approach for the treatment of a potentially large number of fetuses affected by congenital hematologic disorders. With technical and molecular advances in prenatal diagnosis, the majority of these diseases can now be diagnosed early in gestation, allowing consideration of prenatal treatment. In addition, technical advances in fetal imaging and intervention make it possible to perform the transplants with relatively minimal risk. It, therefore, stands to reason that there is increasing interest in performing in utero hematopoietic stem cell transplantation at many fetal treatment centers. Although the approach remains experimentally promising, expansion of clinical application will depend on improved understanding of the biological barriers to engraftment in the fetus as well as the development of effective clinical strategies based on the hematopoietic biology of individual disorders. This article presents the current status of this emerging therapeutic approach.

Background/Purpose: In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. Methods: DBA/2 mice (Mls a+ ) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (Mls a− ) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vβ6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vβ6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vβ6 antibody for stimulation with or without exogenous interleukin-2 (IL2). Results: Vβ6+ splenocytes constituted 6.32% of CD4 + T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vβ6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal ( P Conclusions: In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.

Purpose Posterior sagittal anorectoplasty is a well accepted procedure utilized for repair of congenital imperforate anus. This procedure allows acceptable function and quality of life in these unfortunate children. However, for obvious reasons it is seldom performed in the adult patient.

Recurrent or metastatic pediatric solid malignancies, including osteosarcoma, carry a dismal prognosis despite modern multi-modality treatment approaches. Although the success of KIR (Killer Immunoglobulin-Like Receptor) incompatible, haploidentical stem cell transplantation has been documented in hematological malignancies in adults and children, this approach has not been thoroughly examined in pediatric solid tumors. In this study, we evaluated the potential for KIR-incompatible lysis of osteosarcoma cells, in vitro. We hypothesized that the killing of osteosarcoma cell targets could be predicted by the degree of inhibitory KIR receptor/ligand mismatch with NK cell effectors. To test this hypothesis, healthy donor NK cells were isolated by magnetic bead sorting and their KIR phenotype determined by flow cytometry. Consistent with previous studies, donor NK cells exhibited a high prevalence of all three relevant inhibitory KIRs (KIR2DL1, KIR2DL2/KIR2DL3, KIR3DL1). Conversely, examination of three established osteosarcoma cell lines (HOS, SaOS, and U2OS) demonstrated significant variability in cell surface HLA class I expression, by flow cytometry. QRTPCR analysis of these cell lines revealed variable expression of the three known inhibitory KIR ligands (HLA-C groups 1 and 2, HLA-Bw4). This variable KIR-ligand expression allowed evaluation of NK-mediated lysis of targets with varying KIR receptor-ligand incompatibility. Following a 12-hour incubation of donor NK cells in IL-2, lysis of osteosarcoma targets was measured in an annexin V flow cytometric assay. Osteosarcoma cell lines that expressed fewer KIR ligands consistently showed greater susceptibility to NK-mediated cytotoxicity. These findings were consistent using NK effectors from different donors. Additionally, we observed that at high passage number (>20), SaOS cells demonstrated down-regulation of KIR ligand expression. These changes correlated with increased lysis by the same donor NK cells. Our findings suggest: Variable expression of KIR ligands in osteosarcoma cell lines allows potential susceptibility to KIR-incompatible, NK cell-mediated lysis; The killing of osteosarcoma cells by NK cells can be predicted by the degree of receptor/ligand mismatch; and During expansion, osteosarcoma cells may alter expression of KIR ligands, resulting in increased or decreased susceptibility to lysis by KIR-incompatible NK cells. Further studies are needed to explore the utility of KIR-incompatible, haploidentical stem cell transplantation for patients with high-risk osteosarcoma.

The failure to achieve durable engraftment following prenatal transplantation in immunologically normal human fetal recipients calls for a closer examination of the fetal immune response to allotransplantation. Previous studies in mice suggest that the fetal innate immune system functions as a critical barrier to allogeneic engraftment mediated by recognition of MHC class Ib antigens. We hypothesized that Qa-2 (the putative murine homolog for HLA-G) might play an essential role in the modulation of fetal immune response to prenatally transplanted allogeneic cells. To address this hypothesis, we utilized B6.K1 mice as a donor strain. B6.K1 mice are Qa-2 deficient and are congenic with wild-type B6.Ly5.2 mice. Light density mononuclear cells (LDMCs) were harvested from the livers of 14 dpc fetal B6.K1 or B6.Ly5.2 mice and transplanted into age-matched allogeneic Balb/c fetal recipients at a dose of 105 cells per fetus. Following delivery, peripheral blood chimerism was assessed serially in the recipients. Survival to weaning was similar between the groups without evidence of GVHD. At 3 weeks of age, recipients of B6.K1 cells demonstrated significantly lower peripheral blood chimerism levels than recipients of B6.Ly5.2 control cells. By 6 months of age, nearly all of the recipients of B6.K1 cells had lost their chimerism. Conversely, the chimerism levels in recipients of B6.Ly5.2 control cells remained stable suggesting that donor Qa-2 expression was essential for allograft survival. To assess the competitive capacity of the B6.K1 donor cells in the absence of immunologic disparity, B6.K1 or B6.Ly5.2 fetal liver LDMCs were transplanted into congenic B6.Ly5.1 hosts at the same cell dose per fetus. This resulted in stable long-term engraftment of the B6.K1 cells in all recipients. Chimerism levels were identical to those recipients who received B6.Ly5.2 control cells, confirming that the engraftment disparities observed in the allogeneic recipients resulted from immunologic rejection. To assess the resilience of this apparent Qa-2-dependent innate immune barrier, the allogeneic transplantation experiments were then repeated at a ten-fold higher donor cell dose (106 cells/fetus). Early chimerism levels remained significantly lower in allogeneic recipients of Qa-2 deficient cells compared to controls. However, recipients of B6.K1 cells maintained their engraftment for more than 6 months indicating that the Qa-2-dependent fetal immune barrier may be overcome with higher levels of circulating antigen. From these experiments we conclude: Host allorecognition of the class Ib antigen Qa-2 is crucial for durable engraftment following in utero transplantation; The failed engraftment of Qa-2 deficient hematopoietic cells does not result from a defective competitive engraftment capacity; Qa-2 dependent fetal immune rejection may be diminished by higher levels of early chimerism. These experiments provide direct evidence for the critical role of MHC class Ib antigens in regulation of the fetal immune response to allotransplantation. Additionally, the demonstration of reliable engraftment following transplantation of higher cell doses provides a translationally relevant approach to enhance the clinical success of prenatal transplantation in immunologically normal hosts.

Progress toward clinical application of embryonic stem cells (ESC) derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model such as the rhesus macaque. However, in contrast to human ESC’s (hESC’s), efforts to induce conclusive hematopoietic differentiation from rhesus ESC’s (rESC’s) have been unsuccessful. Despite their close phylogenetic relationship, subtle differences exist between the hematopoietic differentiation of rESC’s and hESC’s. We recently reported that although rESC’s have the potential for hematopoietic differentiation; they exhibit an arrest at the hematoendothelial precursor stage of hematopoietic development in culture conditions developed for hESC’s. One possible difference may be in the requirement for fibroblast growth factor (FGF) signaling. Despite documentation of its contribution to the maintenance ESC’s in an undifferentiated state, the role for FGF-2 in the hematopoietic differentiation of hESC’s and rESC’s has not been similarly examined. Given its critical role for the formation and subsequent hematopoietic differentiation of murine ESC-derived hemangioblasts, we wondered if enhanced hematopoietic differentiation from rESC’s could be achieved by culture supplementation with FGF-2. To answer this question, undifferentiated rESC’s were subjected to embryoid body (EB) differentiation with daily FGF-2 supplementation of the cytokine-rich media. Cultures were analyzed by flow cytometry after 16 days of EB culture. We found that the FGF-2 supplemented cultures appeared more robust with an overall higher numbers of cells. More importantly, a dramatic expansion of hematoendothelial precursors (Flk1hi+ VE-cadherin- CD45−), committed hematopoietic progenitors (CD34+CD45+Lin−), and hematopoietic cells (CD45+) was seen in FGF-2 supplemented cultures when compared to controls. These effects were consistent in two separate lines of rESC’s (R420 and R456). Next we wondered if the observed effect of FGF-2 on hematopoietic development was concentration-dependent. Therefore, we compared serial increases in FGF-2 concentration (0, 10, 50 and 100 ng/ml) of the EB differentiation media and found the effect to be concentration-dependent. From these results, we conclude that FGF-2 appears to play a critical role in the hematopoietic differentiation of rESC’s. Both the development of hematoendothelial precursors and the differentiation of committed hematopoietic cell types are augmented. To study this further, the significance of FGF signaling at various stages of rESC-derived hematopoietic differentiation must be evaluated. A better understanding of the requirements for FGF-2 in EB development will likely lead to improved protocols for the production of human and rhesus ESC-derived hematopoietic progenitors.

Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O 6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

Recent studies suggest that development of human embryoid body-derived hematopoietic progenitor cells (EB-HPC’s) mirrors that of primitive yolk sac-derived hematopoietic progenitors (YS-HPC’s) in their expression of transcription factors known to be critically associated with hematopoietic development. However, the findings of these studies were limited to molecular characterization of heterogeneous EB cultures rather than analysis of hematopoietic lineage-committed cell subsets. We wondered: 1) if the expression patterns were consistent at the protein level in both bulk EB cultures and subsets of hematopoietic-lineage committed cells; and 2) if these expression patterns differed from those found in definitive HPC’s harvested from early fetal hematopoietic tissues. To answer these questions, we compared the intracellular expression of critical transcriptional regulatory proteins from human and rhesus EB-derived HPC’s (hEB-HPC’s and rEB-HPC’s) with hematopoietic progenitors from rhesus fetal liver (rFL-HPC’s) and rhesus placental blood HPC’s (rPB-HPC’s). Rhesus macaque fetal tissue was used in lieu of scarce human fetal tissues. Using multi-color intracellular flow cytometry we correlated the expression of transcriptional proteins (SCL/TAL1, GATA-1, GATA-2, Oct-3/4, HOXB4) with cell surface determinants of hematopoietic commitment (CD34, CD31, CD45) in hEB-HPC’s, rEB-HPC’s, rFL-HPC’s and rPB-HPC’s. Human and rhesus EB16 cells were harvested from identical culture conditions and compared to 0.34 gestation rFL-HPC’s and rPB-HPC’s. The frequency of CD45+ cells was higher in hEB-HPC’s (31%) and rPB-HPC’s (46%) compared to rFL-HPC’s and rEB-HPC’s. A much higher frequency of GATA-1 and HOXB4 expressing cells was seen in both of the fetal cell types when compared to either rhesus or human EB’s. Additionally, the frequency of GATA-2 and SCL expressing cells was comparable in all cell types. Lastly, the expression of Oct3/4 was higher in rPB-HPC’s and rFL-HPC’s when compared to bulk EB cultures. However, analysis of subsets of EB cells expressing hematopoietic antigens (CD45, CD34) revealed co-expression of Oct-3/4 on both CD45+ and CD34+ cell fractions. From these findings, we conclude: critical differences exist in GATA-1 and HOXB4 expression between HPC’s derived from EB cultures and HPC’s harvested during early definitive fetal hematopoietic development; and human EB-derived hematopoietic progenitor cells express persistently high levels of Oct-3/4. The differences in the expression of these critical transcriptional factors at the protein level may explain the observed functional differences between EB-derived HPC’s and definitive HPC’s from fetal and adult sources.

Despite clinical success with in utero hematopoietic stem cell transplantation in immunodeficient recipients, only microchimerism ( Table 1. Expression of Ly49A at 3 months and 1 year Chimerism Level Ly49A expression Animal PB-3 months (%) PB-12 months (%) rMFI-3 months (%) rMFI-12 months (%) 7831 0.2 0.1 76 74 7759* 1.33 0.2 38 92 7830 3.22 1.81 45 29 7860 1.91 2.38 42 33 7838 4.9 5.2 38 32