Alan W. Flake

HUMAN MESENCHYMAL STEM CELLS ENGRAFT AND DEMONSTRATE SITE-SPECIFIC DIFFERENTIATION AFTER IN UTERO TRANSPLANTATION IN SHEEP

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (F.IX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2×1011 vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of F.IX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Correction of congenital diaphragmatic hernia in utero: VI. hard-earned lessons

Extensive experimental work suggests that repair of congenital diaphragmatic hernia (CDH) in utero may salvage severely affected fetuses who otherwise have a high expected mortality despite optimal postnatal care including extracorporeal membrane oxygenation (ECMO). We have reported that repair of CDH in utero is physiologically sound and safe for the mother, but technically difficult especially when the liver is herniated into the fetal chest. In the 3 years since our last report (1989 to 1991), 61 additional patients were referred for consideration of in utero repair. Fetal repair was attempted in 14 with severe isolated left CDH diagnosed before 24 weeks gestation. Five fetuses died intraoperatively, from technical problems related to reduction of incarcerated liver and uterine contractions--problems which have subsequently been surmounted. Nine patients were successfully repaired. Four babies survived, two delivered prematurely and died, and three died in utero within 48 hours of repair. Intraoperative technical problems have been overcome; the factors limiting successful outcome are postoperative physiologic management of the maternal-fetal unit and effective tocolysis to control preterm labor.

Platelets regulate lymphatic vascular development through CLEC-2–SLP-76 signaling

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Plug the lung until it grows (PLUG): A new method to treat congenital diaphragmatic hernia in utero☆

Fetal lungs normally produce fluid that flows through the upper airway into the amniotic fluid. In fetuses with congenital diaphragmatic hernia (CDH), obstructing the flow of lung fluid may expand the lungs and propel the viscera from the chest, alleviating the pulmonary hypoplasia associated with CDH. To test this hypothesis, left-sided diaphragmatic hernias were created in sixteen 75-day-gestation fetal lambs (full-term, 145 days). At 120 days, the trachea was ligated in eight lambs; it was left unligated in the other eight. At 135 to 140 days, the fetuses were delivered, and a tracheostomy performed. Newborns were ventilated for 1 hour and then killed. Blood gas analysis was performed at 0,5,20,40, and 60 minutes. Lung dry weight, DNA, protein, and lipid analyses, as well as plasma cortisol measurements were performed. At autopsy, in the ligated lambs, the abdominal viscera was reduced from the thorax; however, the unligated lambs had viscera completely occupying the left chest. The lungs of the ligated lambs had a higher dry weight (4.22 +/- 1.37 g/kg v 1.95 +/- 0.59 g/kg; P =.001), DNA (193.8 +/- 90.5 mg/kg v 91.5 +/- 66.4 mg/kg; P = .02), and protein (1798 +/- 691.6 mg/kg v 766.6 +/- 201 mg/kg; P = .004). Lung saturated phosphatidyl choline (SPC) levels, DNA:protein ratio, and plasma cortisol were not different between the groups. Neonatal Po2 at 60 minutes was higher in the ligated group (179.4 +/- 127.0 mm Hg v 60.9 +/- 62.4 mm Hg; P < .05), and Pco2 was lower (44.1 +/- 21.4 v 83.9 +/- 23.5; P < .05) in the ligated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Congenital high airway obstruction syndrome (CHAOS): A potential for perinatal intervention

Congenital high airway obstruction syndrome (CHAOS) results in a predictable constellation of findings: large echogenic lungs, flattened or inverted diaphragms, dilated airways distal to the obstruction, and fetal ascites and/or hydrops. The authors report on four fetuses referred for evaluation. None of them survived. Postmortem evaluation showed that three fetuses had laryngeal atresia, and one had tracheal stenosis. Coexistent fetal anomalies were accurately diagnosed by ultrasound in three of the four patients. The finding of CHAOS on prenatal ultrasound examination is diagnostic of complete or near-complete obstruction of the fetal upper airway, most likely caused by laryngeal atresia. A greater understanding of the natural history of CHAOS may permit improved prenatal and perinatal management.

Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero

Although promising, clinical and experimental efforts at in utero hematopoietic stem cell (HSC) transplantation currently are limited by minimal donor cell engraftment and lack of early donor cell expression after transplantation. We reasoned that cotransplantation of stromal elements (ST) might condition the fetal microenvironment for the engraftment of donor HSC and facilitate precocious bone marrow (BM) hematopoiesis. In this study we cotransplanted sheep ST, derived from adult or fetal BM, with either adult or fetal HSC, into preimmune fetal sheep. We analyzed donor cell chimerism in BM and peripheral blood and compared levels of chimerism achieved with recipients of HSC alone. In all experimental groups, stromal cotransplantation markedly increased the level of peripheral blood donor cell expression at 60 days after transplantation relative to controls. Adult BM-derived stroma cotransplanted with adult HSC provided the highest levels of circulating donor cells, whereas fetal-derived stroma was less effective. In addition, ST cotransplantation resulted in increased donor cell engraftment in the BM and led to significantly increased levels of donor hematopoiesis for over 30 months after transplant. Cotransplantation of stroma may represent a valuable clinical strategy for optimal application of in utero HSC transplantation.

DEATH DUE TO HIGH-OUTPUT CARDIAC FAILURE IN FETAL SACROCOCCYGEAL TERATOMA

Fetal sacrococcygeal teratoma (SCT) is being recognized with increasing frequency. Placentomegaly and hydrops fetalis are preterminal events, and it has been suggested that fetal death may be due to high-output cardiac failure from arteriovenous shunting through the tumor. We had a chance to examine this hypothesis when a 21-week fetus presented with a huge sacrococcygeal teratoma. There were marked placentomegaly, cardiomegaly, hyperdynamic ventricles, and a pericardial effusion. Doppler studies showed tremendous flow through the SCT with extreme enlargement of the inferior vena cava, consistent with congestive heart failure from increased flow through the tumor. Hydrops developed, and the fetus was delivered because of placental abruption. This case provides supportive evidence that the teratoma acts as a large arteriovenous shunt, causing high-output cardiac failure. We have now collected 18 more cases of sacrococcygeal teratoma diagnosed in utero. Of the total 45 cases of fetal SCT, 9 had placentomegaly and/or fetal hydrops and all 9 fetuses died in utero or shortly after birth. We conclude that the only hope for survival in these severely affected fetuses is to reduce blood flow to the tumor before birth.

Fetal Surgery for Cystic Adenomatoid Malformation of the Lung

We reviewed our experience with fetal therapy for congenital cystic adenomatoid malformation of the lung (CCAM) at the University of California, San Francisco Fetal Treatment Center. Fetuses with life-threatening CCAM were selected for prenatal treatment according to predetermined guidelines, including the gestational age of the fetus, the size of the intrathoracic lesion, maternal health, and the development of fetal hydrops. The knowledge that fetuses with hydrops are at high risk for fetal or neonatal death led to fetal surgical resection of the massively enlarged pulmonary lobe (fetal lobectomy) in six cases. In the first case, resection was too late, since preoperative labor and maternal preeclampsia could not be reversed, leading to premature delivery of a nonviable infant. In the next four cases, CCAM resection led to resolution of the hydrops, impressive in utero lung growth, and neonatal survival. Right middle and lower lobe resection in the sixth fetus at 21 weeks was successful, but subsequent inexplicable fetal death highlights the need for better postoperative fetal monitoring and treatment. Three other fetuses with a single predominant cyst underwent thoracoamniotic shunt placement alone; two survived after delivery and prompt neonatal surgery with the assistance of high-frequency ventilation or extracorporeal membrane oxygenation. Fetal therapy can now be considered for otherwise fatal space-occupying intrathoracic lesions in the fetus.