Sverker Ek

Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies

The use of fetal hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy. In one fetus with alpha-thalassemia, one with sickle cell anemia, and one with beta-thalassemia, we have transplanted fetal liver cells obtained from legal abortions in gestational weeks 6-11. The fetus with alpha-thalassemia was transplanted twice during pregnancy, in the 15th (20.4 x 10(8) cells/kg) and in the 31st weeks of gestation (1.2 x 10(8) cells/kg), and is now two years of age. One fetus with sickle cell anemia received its transplant in the 13th week of gestation (16.7 x 10(8) cells/kg), and is now one year old. The fetus with beta-thalassemia was transplanted in 18th week (8.6 x 10(8) cells/kg), and is now three months old. Engraftment was evaluated by chromosomal analysis (sex chromosomes), red cell phenotyping, HLA class I and II typing, and PCR (polymerase chain reaction) for Y chromosome-specific sequences and DNA polymorphisms in cord and peripheral blood. The children with alpha- and beta-thalassemia underwent bone marrow aspirations at 3 and 7 months of age, respectively. In neither of these cases were we able to detect convincing evidence of stem cell engraftment. Thus, the administration of fetal stem cells to fetal recipients after the 12th week of gestation did not result in permanent hematochimerism. It remains to be determined whether the engraftment process can be promoted by earlier transplantations and/or higher cell doses.

Fetal hydronephrosis; prevalence, natural history and postnatal consequences in an unselected population

Background. The aim of this study was to estimate the incidence of fetal hydronephrosis, the antenatal development and the postnatal findings in a low risk population. Methods. A 2‐year retrospective study included almost 9,000 patients, and all cases found to have antenatal hydronephrosis, defined as an AP diameter ≥5 mm, had a structuralised ante‐ and postnatal follow‐up. Results. Some 85 cases were found in the antenatal period, and 32 of these cases had a pathologic follow‐up. Of these, 13 had an increase in severity of dilatation during pregnancy, 10 had stable findings, but no case where the dilatation decreased had any postnatal pathology. Furthermore, half of the cases found in the third trimester (18 of 32) also had an abnormal follow‐up. Two cases of Downs syndrome were diagnosed after birth. Conclusion. Antenatal hydronephrosis was found in almost 1% (n = 85) of the study population. There was an almost 2% risk of Downs syndrome in this group. The majority of cases had an increase in severity and some were stable in the antenatal period, but it was not possible to predict the individual postnatal outcome. In the group where the finding diminished in the antenatal period, no patient had an abnormal postnatal follow‐up. Female fetuses with hydronephrosis seem to have an adverse postnatal outcome compared to male fetuses.

Establishment of a tissue bank for fetal stem cell transplantation

Study Objective. To analyse the yield of fetal liver tissue in first trimester abortions and to evaluate the number of nucleated cells obtained from each fetal liver during the sixth to twelfth week of gestation.

Cytokines in fetal blood and amniotic fluid in Rh-immunized pregnancies

Objective To determine fetal serum and amniotic fluid (AF) levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin, and to explore the relationship between cytokines and hemoglobin concentration, white blood cell count (WBC), and platelet count in fetuses affected by Rh immunization. Methods Thirty-four consecutive Rh-immunized patients in gestational weeks 19-33 were included. All patients were investigated by funipuncture and 13 by amniocentesis. The levels of IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin were estimated using commercially available immunoassays. Results There was a significant correlation between erythropoietin concentrations in fetal serum and AF (r = 0.54, P < .05), whereas none of the other cytokines showed a positive correlation between these two compartments. Fetal serum contained higher concentrations of IL-3, granulocyte-macrophage colony-stimulating factor, stem cell factor, and erythropoietin compared with AF. In contrast, the IL-6 level was significantly higher in AF compared with fetal serum (P = .002). Erythropoietin and IL-3 levels were both negatively correlated with fetal hemoglobin concentrations (r = −0.75, P = .02, and r = −0.67, P = .045). The fetal WBC correlated significantly with the fetal serum concentration of granulocyte-macrophage colony-stimulating factor (r = 0.38, P = .04). Conclusion Human fetuses with anemia due to erythrocyte immunization exhibit an increased production of erythropoietin and IL-3. Other studied cytokines (such as stem cell factor, granulocyte-macrophage colony-stimulating factor, and IL-6) did not correlate with the degree of fetal anemia. Among the studied cytokines, only erythropoietin showed a positive correlation between fetal serum and AF.

Tissue distribution of transplanted fetal liver cells in the human fetal recipient

OBJECTIVE Our purpose was to study the tissue distribution and concentrations of transplanted fetal liver cells in the human fetus. STUDY DESIGN Radiolabeled indium 111 fetal liver cells were injected in vivo under ultrasonographic guidance into 10 normal fetuses (13 to 17 weeks of gestation) before a prostaglandin abortion. Six fetuses were injected intraperitoneally and four intracardially. Another two fetuses serving as controls were injected with indium-labeled maternal plasma. The fetuses were all alive, at least until 6 hours before expulsion. After expulsion the fetuses were dissected, and radioactivity was measured in various fetal tissues. Results for each tissue were expressed as percentages of the total injected dose. RESULTS Significantly greater uptake of fetal liver cells in the liver, spleen, thymus, kidney, lung, and placenta was obtained with intracardiac than with intraperitoneal injection. Skeletal uptake did not differ in relation to mode of administration. With intracardiac injection uptake was greater in such parenchymal organs as the liver, spleen, and thymus (4.9%, 4.0%, and 3.9%, respectively). Uptake in the rib, clavicle, humerus, and sternum was 2.7%, 1.8%, 2.1%, and 1.1%, respectively. Placental uptake was 0.1%. The intracardiac route yielded a higher concentration of cells in different fetal organs than did injection of only radiolabeled maternal plasma, suggesting an active uptake of cells in different fetal hematopoietic organs. CONCLUSION The mode of administration of fetal liver cells seems to be a major determinant of donor cell concentration in the transplanted human fetus and may be a significant determinant of the rate of successful engraftment.

Fetal CD103+ IL-17–Producing Group 3 Innate Lymphoid Cells Represent the Dominant Lymphocyte Subset in Human Amniotic Fluid

Amniotic fluid (AF) surrounds the growing fetus, and cells derived from AF are commonly used for diagnosis of genetic diseases. Intra-amniotic infections are strongly linked to preterm birth, which is the leading cause of perinatal mortality worldwide. Surprisingly little is known, however, about mature hematopoietic cells in AF, which could potentially be involved in immune responses during pregnancy. In this study, we show that the dominating population of viable CD45+ cells in AF is represented by a subset of fetal CD103+ group 3 innate lymphoid cells (ILCs) producing high levels of IL-17 and TNF. Fetal CD103+ ILC3s could also be detected at high frequency in second-trimester mucosal tissues (e.g., the intestine and lung). Taken together, our data indicate that CD103+ ILC3s accumulate with gestation in the fetal intestine and subsequently egress to the AF. The dominance of ILC3s producing IL-17 and TNF in AF suggests that they could be involved in controlling intra-amniotic infections and inflammation and as such could be important players in regulating subsequent premature birth.

Screening of fetal stem cells for infection and cytogenetic abnormalities.

Fetal stem cell transplantation may rely on material from therapeutic abortions. It is essential that the stem cell transplant does not transmit any microorganisms that may affect the fetus and that genetically abnormal cells are avoided. To evaluate such contamination, human fetal stem cells collected February 1992 - December 1993 were analyzed for bacterial and fungal growth, and the placentas were karyotyped. Four samples of 70 were positive for different pathogens. Serological screening of 43 women during this period resulted in five seroconversions and revealed one carrier of anti-HCV. Karyotyping revealed two abnormal findings out of 72 samples. Thus, the concept of using material from therapeutic abortions is safe.

Survival and neonatal outcome after fetoscopic guided laser occlusion (FLOC) of twin-to-twin transfusion syndrome (TTTS) in Sweden.

Abstract Aim: To determine infant survival and neonatal outcome after fetoscopic laser treatment of twin-to-twin transfusion syndrome (TTTS). Results: In 53/71(75%) laser-treated TTTS cases, at least one twin was liveborn and in 42/71(59%) cases at least one twin survived infancy. Fetal survival did not differ between donors [41/71(58%)] and recipients [46/71(65%), P=0.36]. Among liveborns, infant survival was 29/41(71%) in donors and 36/46(78%) in recipients (P=0.12). Infant survival did not correlate to maternal characteristics (age, BMI, smoking or parity), gestational age at treatment or severity of TTTS (Quintero stage). No TTTS infant born before 25 weeks of gestation survived the first week. Among the 87 infant survivors, 26 (30%) had an Apgar score <7 at 5 min, 47 (54%) developed respiratory distress syndrome, 10 (11%) showed signs of severe brain damage, nine (10%) renal failure, eight (9%) bronchopulmonary dysplasia, and five (6%) infants developed retinopathy of prematurity ≥stage 3. There was no significant difference in neonatal morbidity between recipients and donors. Conclusions: Fetal survival after laser treatment was comparable to that reported by other international centers. There was no significant difference in survival or neonatal morbidity between donors and recipients. Major neonatal morbidity was common, and combined with extremely preterm delivery the prognosis of TTTS is poor.

Cytokine Stimulation of Human Fetal Hematopoietic Cells

The effects of interleukins 3 and 6, stem cell factor, and granulocyte-macrophage colony-stimulating factor on human fetal hematopoietic, bone marrow, and cord blood cells were studied on the basis of the colony-forming capacity. Fetal hematopoietic cells from 28 elective abortions, three bone marrow samples, and three cord blond samples were incubated with cytokines and investigated for the presence of BFU-E (burst-forming units--erythroid), CFU-GM (colony-forming units--granulocytes, macrophages), and CFU-GEMM (colony-forming units--granulocytes, erythrocytes, macrophages, megakaryocytes). Single and combined cytokines and preincubation versus adding cytokines in culture were investigated. Interleukin-6 alone had the most pronounced effect on BFU-E formation. All four cytokines in combination yielded the highest scores for CFU-GM (p < 0.05) and CFU-GEMM (p < 0.05), whereas BFU-E was not enhanced. The mode of cytokine exposure was not a determinant of colony formation.

Colony formation of human fetal CD34+ hematopoietic cells.

Manipulations to enhance engraftment of donated cells may be advantageous in transplantation of fetal hematopoietic cells (FHC). By assessing the formation of colonies, CD34+ enrichment was evaluated with and without cytokine stimulation (interleukins 3 and 6, stem cell factor, granulocyte-macrophage colony-stimulating factor). Cord blood cells and bone marrow cells served as controls. In FHC, cytokine stimulation and CD34+ enrichment always enhanced the formation of CFU-GM (colony-forming units--granulocytes, macrophages) and CFU-GEMM (colony-forming units-granulocytes, erythroid cells, macrophages, megakaryocytes). However, BFU-E (burst-forming units--erythroid cells) in FHC remained unchanged after cytokine stimulation and CD34+ enrichment. In FHC, the addition of cytokines and the enrichment of CD34+ cells usually contributed equally to enhance CFU-GM and CFU-GEMM colony formation. CD34-negative FHC produced the same number or more BFU-E and half the number of CFU-GM and CFU-GEMM as compared with crude cells. This CD34-negative cell population also responded to cytokine stimulation. Such findings may indicate that purification of CD34+ cells is not meaningful in fetal transplantation.