Blood cells最新文献

Cord blood is a recently recognized source of hematopoietic stem cells. It can be employed successfully to reconstitute hematopoiesis following allogeneic transplantation. One current drawback of cord blood as a treatment has been a risk of transfusion reactions attributable to ABO blood group mismatch. Removal of red cells from the cord blood has led to reduction of the stem cells by 30-50%. In this paper we report red cell depletion by a method that employs 3% gelatin to effectively sediment the erythrocytes and selectively deplete red cells but permits 94% recovery of nucleated cells and enrichment of colony-forming cells by granulocyte-macrophage colony-forming units, erythrocyte burst-forming units, and granulocyte-macrophage-megakaryocyte colony-forming units in the cord blood preparation. This technique has been employed in our study to remove red cells from the cord blood of a male infant delivered by cesarean section, which has permitted treatment of a female sibling suffering from leukemia. The recipient was 8 years old and weighted 36.7/kg. Complete HLA identity between the two siblings was established. A cord blood cell transplant of cryopreserved and later thawed cells (4 x 10(7) nucleated cells per kilogram) was administered to the patient after intensive myeloablative chemotherapy. The patient exhibited a prompt hematologic recovery (absolute neutrophil count > 500 by day 31, 100% male cells in bone marrow and peripheral blood by day 25) and has experienced a 13-month disease-free survival to date.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of maternal blood cell contamination in cord blood as hematopoietic stem cell transplant could be an important factor driving the graft vs. host reactivity. Therefore, the efficiacy of fluorescence-labeled chromosome probes for the X and Y chromosomes to study contaminating maternal cells (XX+, Y-) in nucleated cells of male cord blood specimen (X+, Y+) by fluorescence in situ hybridization (FISH) was evaluated here. Unfortunately, the FISH technique evaluated by standard indirect immunofluorescence was not sufficiently sensitive to safely detect maternal contamination below 2-3%. Thus, the analysis of FISH had to be adapted with confocal laser scanning microscopy, which then gave no false-negative reading values. A Bio-Rad MRC-600 laser scanning microscope with a very high sensitivity for low-intensity and hidden signals was used for the observation of cord blood cells. Sixteen samples from the whole cord blood (CB), 10 analyses of mononuclear cells, 6 of the granulocytic fraction, 6 of picked erythroid burst-forming unit (BFU-E) and granulocyte-macrophage colony-forming unit (CFU-GM) colonies and 5 of enriched CD34+ cells were performed. Only in 1 of 16 cases was a contamination by maternal cells detected: 10% of all nucleated cells from the whole CB, 5% of the CD3+ T-cell fraction, 15% of the myelomonocytic cells from mononuclear cells (MNCs), and 15% of picked BFU-E and CFU-GM colonies were of maternal origin.(ABSTRACT TRUNCATED AT 250 WORDS)

The reasons for prenatal HLA typing for potential cord blood stem cell donor evaluation are reviewed and the strategies for doing so are summarized. Cultured fetal cells are class I HLA typed by modified serological techniques. When indicated, the fetal cells are also class II HLA typed by one or another DNA typing method. The same procedures can be used for the prenatal identification of a potential donor for an affected fetus.

Human umbilical cord blood is an abundant source of long term repopulating stem cells and therefore we investigated the utilization of these cells as targets for genetic manipulation directed towards human gene therapy. Using two different retroviral vectors, one which transfers the neomycin resistance gene and the other which transfers therapeutically relevant adenosine deaminase gene, we have demonstrated increased gene transfer efficiency into committed progenitor cells (CPCs) and long term culture-initiating cells (LTC-IC) derived from cord blood versus adult bone marrow. We further identified a chymotryptic fragment of the extracellular matrix molecule fibronectin (FN 30/35), to which primitive hematopoietic cells adhere. Gene transfer efficiency into hematopoietic cells adherent to FN 30/35 is significantly increased when compared to infection on bovine serum albumin-coated control plates. Utilization of this fragment allowed retroviral mediated gene transfer into cord blood derived CPCs and LTC-ICs with high efficiencies, similar to that observed after coculture of hematopoietic cells on virus producer cells. These data imply cord blood may be a promising source for efficient gene delivery to the human hematopoietic system, and the utilization of the FN 30/35 fibronectin molecule may provide a clinically applicable protocol to achieve this aim.

Human umbilical cord blood (UCB) may be used as an alternative source of bone marrow repopulating cells in allogeneic bone marrow transplantation. The quality and quantity of UCB harvests for transplantation is affected by several factors. In this study we analyzed the influence of delivery, in particular stress during delivery, on the numbers of leukocytes and leukocyte subsets in UCB. Four groups of women with different types of deliveries were included in the study, and from each group samples of UCB were analyzed. Blood samples from healthy adults were used as control. In UCB there was a higher absolute number of leukocytes than in peripheral blood (PB). UCB leukocytes were highest after deliveries with a prolonged second stage of labor, which was mainly due to granulocytosis. The percentage of T cells in UCB was lower than in PB, in particular when stress during delivery was higher. In all groups, however, the absolute concentration of T cells per milliliter of UCB was higher than in adult PB. The differences in T cells in stressful deliveries were mainly due to a relative decrease in CD3+/CD4+ cells in UCB. The relative frequency and absolute concentration of the CD56+ cell population in UCB was higher than in PB, which was mostly due to an increase of CD2-/CD56+ cells, in particular in stressful deliveries. The absolute number of CD34+ cells as well as hematopoietic progenitor cells as determined in semisolid medium cultures was high in UCB and was increased in cases of prolonged secondary stage of labor. This study demonstrates that the quality of UCB transplants is influenced by the course of delivery, in particular by stress during delivery.

A 2 3/4 year old male with thrombocytopenia secondary to Wiskott-Aldrich Syndrome (WAS) and a history of two intracranial hemorrhages as well as hemolytic anemia and neutropenia received a placental blood infusion from an HLA-identical female sibling born by caesarian section at 35 weeks gestation. The patient was prepared with Thiotepa and Cytoxan and received a nucleated cell dose of 3.0 x 10(7)/kg. Cyclosporin A and Methylprednisolone was given for graft versus host disease (GVHD) prophylaxis. An ANC of 0.5 x 10(9)/L and 1.0 x 10(9)/L were achieved on post-transplant days 18 and 28, respectively. Platelet recovery was rapid with a platelet count > or = 100 x 10(9)/L on day +39. On posttransplant day +11, the patient developed an erythematous rash consistent with grade I acute GVHD that resolved without therapy. He was discharged day on +60 and has remained free of infections with a normal platelet count off all immunosuppression therapy 30+ months post-transplantation. Chimerism studies performed on peripheral blood mononuclear cells by fluorescent in situ hybridization indicated that the percentage of donor cells ranged between 55 and 80%. The phenotype and function of peripheral blood lymphocytes are completely normal and the patient has responded in vivo with production of antibodies to both diphtheria and tetanus immunizations. This study demonstrates the feasibility of collecting placental blood after a multiple birth delivery and the ability of umbilical cord blood to provide complete hematopoietic and immunologic reconstitution in a patient with WAS.

The long-term marrow culture (LTC) system allows the sustained production of primitive normal and neoplastic (chronic myeloid leukemia [CML]) hematopoietic cells in vitro for many weeks. This is achieved in the absence of exogenously added hematopoietic growth factors because of the presence in the cultures of supportive "stromal" cells of the fibroblast-endothelial-adipocyte lineages. These latter cells form a confluent adherent layer with which the most primitive hematopoietic cells become associated and which locally regulates their behavior. The LTC system has thus been considered as a model of the microenvironment of the bone marrow and used to delineate potentially physiologically relevant mechanisms that regulate the proliferation, self-renewal and differentiation of primitive normal hematopoietic cells. It has also been used to analyze the molecular basis of the altered proliferative behavior that characterizes primitive neoplastic cells from patients with CML. Most of the information obtained to date has emerged from experiments designed to shift the balance of stimulatory and inhibitory factors present in order to favor either the cycling or quiescence of primitive normal or CML cells in LTC. This has been achieved either by addition of soluble factors (or antagonists) to the LTC medium or by the use of genetically engineered factor-producing stromal cells. Such experiments have allowed the identification of a number of cytokines that promote one or the other of these responses (i.e., primitive progenitor cycling or quiescence), including some that are involved in control mechanisms endogenous to the LTC system. Recent studies suggest that the retention of primitive normal cells in a reversible G(o) state in this system is mediated by the cooperating action of limiting concentrations of at least two endogenously produced inhibitory factors (transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-1 alpha (MIP-1 alpha)), either of which, however, if added exogenously at a sufficient concentration, can exert this action on its own. Interestingly, the heightened turnover characteristic of primitive CML cells appears to be due to a selective unresponsiveness to only one of these two inhibitors (MIP-1 alpha). These findings are consistent with a complex model of the extrinsic regulation of primitive hematopoietic cells in which a multiplicity of intracellular signaling intermediates within the target cells converge at different points ultimately to control their entry into S phase. Our findings further suggest that only some of these pathways may be affected by intracellular expression of the BCR-ABL fusion gene.

The usefulness of placental/umbilical cord blood as a source of stem cells for marrow reconstitution of HLA-matched siblings has now been extended to the unrelated-donor setting. The need for HLA-matched donor tissue makes it essential to have available a frozen inventory of ready-to-use placental blood units. The New York Blood Center's Placental Blood Project, designed to evaluate the practical feasibility of unrelated placental blood transplantation, consists of four basic modules: collection of placental blood, maternal samples and donor data, accession and testing for genetic and infectious disease markers, freezing placental blood units, and data organization and retrieval. Additional modules include a computerized HLA matching algorithm and organization of data about patients requiring transplantation, which may be best taken up by organ-sharing organizations in the future. In this report, we describe the organization and discuss the methods and overall experience after collecting the first 1,000 units and supplying the tissue for the first two unrelated-donor placental blood transplants.

Steel factor (SF) increases the frequency of colony formation by CD34+ CD38- cycling cells, but it does not reverse the effect of an autocrine production of transforming growth factor (TGF)-beta 1 by early progenitors of the stem cell compartment. We have used optimal culture conditions supplemented with SF and anti-TGF-beta serum to estimate the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU), twice as many granulocyte-macrophage (GM) CFU, and three times as many erythroid burst-forming units as the same population from an average bone marrow sample used in adult transplantation. These results suggest that umbilical cord blood is a suitable source of cells for adult transplantation.

Lymphohemopoietic progenitors that are capable of expressing B-cell and myeloid lineages may be cultured from bone marrow cells of adult mice by using a two-step methylcellulose culture system. In this system, the primary colonies expressing myeloid lineages are plated in secondary culture for B-lymphoid colony formation. We have observed that combinations of two factors based on steel factor (SLF), such as SLF plus interleukin (IL)-6, SLF plus granulocyte colony-stimulating factor (G-CSF), and SLF plus IL-11 support the differentiation and proliferation of B-cell progenitors from the lymphohemopoietic progenitors. Surprisingly, IL-3 failed to support B lymphopoiesis either alone or in combination with other factors. In addition, when added to permissive culture conditions, IL-3 and IL-1 independently inhibited the B-cell potential of the primary colonies. The inhibitory effects of IL-3 and IL-1 observed in this in vitro system may be significant in the selection of cytokine combinations for in vitro expansion of hemopoietic stem cells.