Blood cells最新文献

Over the past 6 years, umbilical cord blood has emerged as an efficacious alternative source of hematopoietic stem cells in related bone marrow transplantation. These encouraging results led us to extend this technology to the mismatched related and unrelated settings in three high-risk leukemic children lacking a matched-related donor for transplantation. Two of the three children also lacked identifiable donors through the National Marrow Donor Program, while the third was in relapse and did not have time to wait for completion of a search. The first child was transplanted with haploidentical umbilical cord blood-derived mononuclear cells from his sister, while the remaining two children were transplanted with partially mismatched, unseparated, unrelated umbilical cord blood banked through the Placental Blood Project at the New York Blood Center. All three children demonstrated trilineage engraftment with donor cells within 6 weeks of transplantation. The patient transplanted with haploidentical marrow developed grade 2 graft vs. host disease (GVHD), which was controlled with steroid and anti-thymocyte globulin (ATG) therapy. One of the two patients grafted with unrelated umbilical cord blood developed mild grade 1 GVHD of the skin, which rapidly cleared with steroid therapy. One patient remains alive, in good health and disease-free 12 months from transplantation.

Cytokines regulate the growth and differentiation of hematopoietic cells through their interaction with receptors of the cytokine receptor superfamily. This family of receptors has conserved motifs in the extracellular domain but share only limited similarity in the cytoplasmic domains. Although lacking catalytic domains, a variety of studies demonstrate that the cytokine receptors function by coupling ligand binding to induction of tyrosine phosphorylation. Recent studies have shown that the JAK family of kinases associate with cytokine receptors and are activated by ligand binding. Interaction occurs with the membrane proximal region of the cytoplasmic domain, a region that has been found to be essential for mitogenesis. One of the substrates of tyrosine phosphorylation is the receptor and, in the case of the receptor for Epo, the membrane distal region of the cytoplasmic domain is phosphorylated. Once phosphorylated, this site becomes a binding site for the amino-terminal SH2 domain of hematopoietic cell phosphatase (HCP). HCP is an important negative regulator of hematopoietic cell growth and its recruitment to the receptor complex is speculated to be important for this effect. The role of HCP is best indicated by the observation that the murine mutation, motheaten, is due to a mutation that results in the inability to make HCP. Motheaten mice die soon after birth due to the overproliferation of a variety of hematopoietic lineages. Together the results demonstrate an essential role in both protein tyrosine phosphorylation and de-phosphorylation in the growth regulation of hematopoiesis.

Lymphohematopoiesis occurs in the densely packed environment of the intramedullary spaces. Primitive lymphohematopoietic stem cells exist in close apposition to a variety of supportive cells including both hemopoietic and nonhemopoietic lineages. Using an in vitro long-term Dexter liquid culture system, we have established that a variety of cytokines are produced constitutively by such stromal cells in culture. These cytokines include Steel factor, interleukin-6 (IL-6), and colony-stimulating factor (CSF-1). Granulocyte-CSF and granulocyte-macrophage-CSF mRNA can be detected after refeeding of cultures, although in quiescent cultures message for these factors is difficult to detect. Interleukin-3, IL-4, and IL-5 are not detectable by standard Northern blot analysis or bioassay of condition media. However, IL-3--detectable by reverse-transcriptase PCR and biologic activity--was confirmed by growth of factor-dependent cells on stromal cells with IL-3 antibody blocking of such growth. Stem cells resident on such stromal cells are mirrored by the high proliferative potential colony-forming cell assay and are responsive to a relatively large number of cytokines, with Steel factor being of central importance, appearing to be a critical component of various synergistic combinations. Steel factor allows reduced levels of other factors in such combinations and works early in a temporal sequence. Hematopoietic stem cells can engraft in normal nonmyeloablated hosts. Using a male/female BALB/c transplantation model, we have shown high rates of engraftment into normal animals, out after marrow infusion to 25 months, after marrow infusion and that post-5-fluorouracil bone marrow is quite deficient in such engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cases of HLA partially matched related cord blood transplantation were reported. The first patient remains healthy more than 4 years after graft. The second patient died from acute GVHD. In the first case, the immune reconstitution was studied over a 2 year period after graft: the alloreactive cytotoxic T-cell precursor (CTLp) frequencies were comparable between the donor and the recipient. Indirect evidence suggested that the resolution of GVHD was not due to a specific deletion of CTLp against the HLA antigens unshared between donor and recipient.

Stem and progenitor cells from a variety of sources including bone marrow, cord blood, and peripheral blood have been used for transplantation. This study compares CD34 cells from all three sources. Flow cytometry analysis of CD34 cells in multiple samples of normal peripheral blood and patient peripheral blood mobilized with chemotherapy (cyclophosphamide/VP16), chemotherapy plus granulocyte colony stimulating factor (G-CSF), and G-CSF alone were compared to bone marrow and cord blood. Although the relative distribution of CD34 percentages in each preparation of cells varied widely, on average the percentage of CD34 cells in these different preparations was 0.15%, 0.6%, 2%, 0.45%, 1.68%, and 0.83% respectively. CD34 subset analysis was performed on these cell preparations using multicolor flow cytometry and antibodies to CD33, CD13, CD45RA, CD19, CD71, and CD38. The major differences observed were that bone marrow CD34 cells contain high percentages of CD19+ cells not found in significant quantity in the other cell preparations and cord blood CD34 cells contained a higher percentage of CD38-cells than the other cell preparations. A magnetic bead system was used with anti-CD34 antibody to purify CD34 cells from mobilized peripheral blood apheresis products, cord blood, and bone marrow. Efficient selection with high purities of CD34 cells was achieved with each of the cell preparations. Comparison of colony-forming activity of each of the cell preparations showed cord blood and mobilized peripheral blood to have slightly higher cloning efficiencies than bone marrow with higher numbers of erythroid blast-forming units (BFU-E) also observed in cord blood CD34 cells. Culture of isolated CD34 cells in liquid culture with interleukin-3, stem cell factor, G-CSF, and granulocyte-macrophage GM-CSF showed over a 100-fold expansion in cell numbers after 25 days, with the peak expansion of colony-forming cells occurring between days 11 and 16. Analysis of day-10 cells from these cultures showed them to be predominantly promyelocytes, myelocytes, and metamyelocytes, with cord blood CD34 cultures showing more promyelocytes than peripheral blood or bone marrow and bone marrow showing more metamyelocytes. Comparison of the proliferation of CD34 cells from these different cell preparations showed that cord blood CD34 cells cultured for 10 days averaged an 85-fold increase in cell numbers followed by mobilized peripheral blood CD34 cells, with an average 56-fold increase, and bone marrow CD34 cells, with an average 49-fold increase.

Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma. Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor. Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum. In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14. As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification of human hematopoietic stem cells (HSC) may be useful clinically for preparation of tumor-free grafts to be used for autologous transplantation and as targets for gene therapy. To analyze the phenotype of the human HSC, assays were used that measure the unique properties of stem cells, i.e., their long-term repopulating ability and their multilineage potential. These assays include: (1) an in vitro long-term hematopoietic culture system, using the murine bone marrow stromal cell line SyS1, which supports both B lymphopoiesis and myelopoiesis; (2) fetal human bone grafts implanted in SCID-hu mice, in which maintenance of CD34+ cells and B and myeloid differentiative capacity of candidate stem cell populations may be measured; (3) fetal human thymus grafts in SCID-hu mice, which allow the analysis of in vivo T-cell potential of a candidate stem cell population. Stem cells in adult bone marrow (ABM) or cytokine-mobilized peripheral blood (MPB) are thought to express CD34 but lack expression of markers indicating lineage commitment. This CD34+ Lineage- (Lin-) subpopulation has been isolated by fluorescence-activated cell sorting and tested for activity in the assays described here. CD34+ Lin- cells from both ABM and MPB demonstrated long-term engraftment in the SCID-hu bone model. This CD34+ Lin- population can be subfractionated further using an antibody to Thy-1. The Thy-1+ subset of CD34+Lin- cells is enriched for both long-term culture-initiating cells (LTC-1C) and has the ability to engraft in vivo.

We have studied the frequency of colony-forming cells (CFC) in fetal and neonatal blood in comparison with adult blood and marrow. Fetal or neonatal blood contains at least as many CFC as adult marrow and higher numbers of the more primitive CFC--those CFC (mixed-cell CFC) giving rise to colonies composed of erythroid and myeloid cells. CD34+ cord blood cells (selected by one of several means) proliferate in culture over time and generate more CFC (from pre-CFC) and differentiated cells in response to stem cell factor (SCF) plus different hematopoietic growth factors. For its effect, SCF requires the synergistic action of erythropoietin (Epo), granulocyte colony-stimulating factor (G-CSF), or interleukin-3 (IL-3). In the presence of Epo or G-CSF, CFC and differentiated cells are generated for 15 days and are mainly erythroid or granulocytic, respectively. In contrast, SCF plus IL-3 generate multilineage CFC and differentiated cells for more than 1 month. When the conditions for these long-term suspension cultures were optimized, CFC and differentiated cells were generated for more than 2 months. At this time, CFC were no longer detectable, but cells continued to be generated, and the cells had a mast cell phenotype. These cells have been maintained and propagated for more than 8 months in the presence of IL-3 and SCF and may represent a useful tool to study human mast cell differentiation.

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.

In the present study, the biological properties of cord blood cells were investigated. Cord blood mononuclear cells and T cells responded normally to activation by alloantigens in primary mixed leukocyte reactions (MLRs), indicating that cord blood T cells can be normally activated via their TcR and have normal proliferative capacities. In addition, they expressed normal levels of accessory molecules such as CD28 and LFA-1, which contribute to amplify their responses. In contrast, cord blood mononuclear cells, but not cord blood monocytes, had a reduced capacity to stimulate allogeneic cells in primary MLRs. In addition, cord blood monocytes express lower levels of HLA-DR and ICAM-1 compared to adult peripheral blood monocytes. Cord blood mononuclear cells were also impaired in their capacity to generate allogeneic cytotoxic activity in primary mixed leukocyte cultures (MLCs). In contrast, cord blood B cells were similar to adult B cells in their capacity to switch to immunoglobulin E producing cells when incubated with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody. We also demonstrated that IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) production by activated cord blood mononuclear cells was comparable to that observed with peripheral blood mononuclear cells isolated from normal adult donors. In contrast, interferon-gamma (IFN-gamma) was significantly decreased, whereas IL-4 and IL-5 were absent. Granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were in general higher in the supernatants of cord blood cells. Thus, cord blood immune responses differ from those of peripheral blood at several levels. Whether these differences account for a reduced capacity of transplanted cord blood cells to modulate graft vs. host disease remains to be determined.