Lola Markling

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.

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.

Mixed lymphocyte culture of human fetal liver cells.

Objective: In order to study the immunological function of the human fetus in the first and second trimesters, mixed lymphocyte culture (MLC) of fetal liver and thymic cells was performed. MLC is a functional test to determine human lymphocyte antigen-D incompatibilities. Methods: Human fetal liver and thymic tissue was obtained from abortions in gestational weeks 7–17.5. Forty-seven fetuses were studied with one-way MLC. The cells were stimulated by adding irradiated fetal liver cells, adult bone marrow and peripheral blood lymphocytes. The activity was measured as DNA incorporation of radiolabeled thymidine. Results: The results indicate that the human fetus is competent to react as early as 11–12 weeks of gestation and in some cases even earlier. In very immature fetal livers (< 8 weeks), the MLC seems to be inhibited. Conclusions: Our data suggest that the human fetus can react against foreign transplantation antigens earlier than previous papers have claimed. The onset of reactivity seems to differ considerably among fetuses. The present findings may explain some of the limited success of in utero transplantations of hematopoietic stem cells in human fetuses of normal immunological status.

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.

Recombinant Parvovirus B19 Empty Capsids Inhibit Fetal Hematopoietic Colony Formation in vitro

Erythroid lineage cells are target cells for human parvovirus B19, and a natural infection often results in transient anemia. To determine whether recombinant B19 capsid proteins (VP1/VP2) also inhibit human hematopoietic progenitor growth, a model system was set up. The B19 capsids were inoculated into primary cultures of hematopoietic stem cells derived from human fetal liver, resulting in a 70–95% reduction of BFU-E (burst-forming unit erythroid cells) as compared with the medium control. A similar effect was seen in human hematopoietic stem cell cultures derived from cord blood and adult bone marrow. Preincubation of the B19 capsids with either a monoclonal antibody to the virus or with B19 IgG positive human sera reduced the inhibitory effect. Furthermore, the inhibitory effect could be reduced by preincubating the target cells with a monoclonal antibody to the cellular receptor for the virus, the P antigen. These findings thus show that the inhibition of colony formation of human hematopoietic stem cells can occur in the absence of parvovirus B19 nonstructural proteins. We speculate that B19 capsid could provide a possible strategy to downregulate indigenous hematopoiesis in fetal stem cell transplantations.

MHC antigen expression in human first trimester spinal cord with implications for clinical transplantation procedures

We report human leukocyte antigen (HLA) class I expression in 5-17% and class II in 0-9% of first trimester human spinal cord cells. After 8 days in culture with gamma-interferon, >87% of the spinal cord cells expressed HLA class II. However, mixed cultures of adult human peripheral lymphocytes and immature human spinal cord cells, showed no induction of lymphocyte proliferation prior to or after gamma-interferon exposure in culture. In conclusion, we report non-immunogenic expression of HLA antigens in the human first trimester spinal cord.

In vitro studies of the role of CD3+ and CD56+ cells in fetal liver cell alloreactivity.

Background. Previous functional studies have suggested that fetal liver cells (FLC) are capable of responding to allogeneic stimulation and have allostimulatory capacity by early in the second trimester. The present study was designed to analyze whether the allogenic response in human FLC is restricted to HLA class II expression and to evaluate the role of CD3+ cells (T cells) and CD56+ cells (natural killer cells) in the allogenic response. Methods. Mixed lymphocyte culture (MLC) experiments were performed on human FLC, at 14 to 18 gestational weeks, before and after depletion of HLA class II+ cells from the stimulator FLC pool (6‐11 gestational weeks) and before and after depletion of CD3+ and CD56+ cells from the responder population. Results. Depletion of HLA class II+ cells from the stimulator FLC pool resulted in a decreased response in 9 of 12 initially positive MLCs. CD3+ cell depletion from responder FLC resulted in a decreased response in four of seven experiments with peripheral blood lymphocytes as stimulators and in two of five experiments with FLC as stimulators. Depletion of CD56+ cells from responder FLC resulted in a decreased response in five of seven initially positive MLCs with peripheral blood lymphocytes as stimulators and two of five with FLC as stimulators. Conclusions. The results indicate that FLC are capable of alloresponsiveness when studied in MLC and that the response seems to be HLA class II dependent. Depletion of CD3+ or CD56+ cells led to a reduction in the MLC in a majority of the experiments. The question remains how these cells interact; it is likely that both cell types exhibit complementary effects in the early allogenic response.

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.

In vitro Studies of Haematopoietic Colony-Forming Capacity of Human Fetal Liver Cells at Exposure to Cytotoxic and Immunomodulatory Drugs

Objective: Intrauterine transplantation with haematopoietic stem cells (SC) as a treatment for immunological, haematological and metabolic inherited disorders has not been effective in fetuses with a normal immunological status. Inhibition of the fetal haematopoiesis or immunosuppression might therefore be a therapeutic alternative in fetal SC transplantation. The aim of the present study was to evaluate the effects of drugs that might be considered as therapeutic options in fetal transplantations on the colony-forming capacity of fetal haematopoietic SC. Methods: Fetal liver cells were incubated with doxorubicin, daunorubicin, antithymocyte-globulin (ATG), OKT-3 (Orthoclone) and betamethasone. The effects of these drugs on colony formation by fetal hematopoietic SC were evaluated. Colony-forming capacity assays were cultured during 10 days after drug incubation on day 1 and evaluated for differences in number of colonies compared to unexposed cells. For betamethasone, similar studies were performed with adult bone marrow. Results: A significant reduction in fetal liver haematopoietic colony formation of BFU-E, CFU-GM and CFU- GEMM was detected when 0.1 M doxorubicin (p < 0.05) and 0.5 µM daunorubicin (p < 0.05) were added. OKT-3 (5 µg/ml) and ATG (4 µg/ml) significantly reduced BFU-E, CFU-GM and CFU-GEMM (p < 0.05). A concentration of 0.2 µg/ml betamethasone caused a significant reduction of BFU-E, CFU-GM and CFU-GEMM (p < 0.05). Conclusions: The drugs investigated in this in vitro study were capable to different degrees to decrease the colony-forming capacity of fetal haematopoietic progenitor cells. Whether this strategy will become an alternative in fetal SC transplantations is an open question and further studies are required to elucidate the potential use of these drugs in fetal transplantation.