Blood cells最新文献

To elucidate the pathophysiologic significance of red blood cell (RBC) filterability, we measured RBC rheology with our own designed nickel mesh with 3-microns pores, smaller than the previously used 5-microns pores. Vertical and cylindrical pores with no pore coincidence were regularly distributed across the filter, the pore entrances of which showed a round and rather smooth transition to the pore inside. An advantage of the nickel mesh is the repeated use (at least 100 times) of the same filter possible after ultrasonic washing. A very low concentration of RBC, i.e., 3 x 10(4) cells per cubic millimeter (hematocrit value of approximately 0.3%), was sufficient for a typical test to examine RBC filterability. The filtration of the dilute RBC suspension was not influenced by contaminating or added leukocytes up to a leukocyte count of approximately seven cells per cubic millimeter; therefore, measurements can be performed using conventionally washed RBCs. This may be practically relevant to routine use, such as in a clinical laboratory. As compared with filtration through 5-micron pores, filtration through 3-micron pores was found to be very sensitive in detecting major determinants of RBC deformability, particularly, changes in viscoelastic properties of the cell membrane, surface area/volume ratio of the cell, perturbing effects of lysophosphatidylcholine, and osmolality of the medium. The 3-micron filtration method revealed a marked impairment in the filterability of Heinz body-containing RBCs from patients with unstable hemoglobin (Hb) disease (Hb Yokohama). Thus, 3-micron-filtration measurements may contribute to several subfields of hematology.

In an attempt to expand the hematopoietic progenitor cell (HPC) content of a single collection of umbilical cord blood (CB), we investigated the ex vivo proliferative potential of CB CD34+ cells and the rate of exit of these cells from G0/G1 phases of cell cycle in response to different cytokine combinations. Initial experiments in which phenotypically defined populations of CB and adult bone marrow (BM) CD34+ cells were examined for their HPC content revealed that, contrary to BM, CB CD34+ human leukocyte A (HLA)-DR+ cells appeared to contain the majority of primitive HPC. In cultures of BM CD34+ HLA-DR+ cells incubated with stem cell factor (SCF)+interleukin-3 (IL-3), CD34+ cells increased five-fold over 5 days, while CD34+ cells from CB CD34+ HLA-DR+ cultures increased 11-fold under these same conditions, illustrating an enhanced proliferative potential of CB CD34+ HLA-DR+ cells vs. similar cells from adult BM. Furthermore, a 6.2-fold increase in the number of CB CD34+ still residing in G0/G1 was observed on day 5 in cultures supplemented with SCF and IL-3, suggesting the generation of large numbers of primitive HPC in vitro. The effect of SCF on the exit of CB and BM CD34+ HLA-DR+ cells from G0/G1 was then examined. Following 36- to 48-hour exposure to SCF, 45% of quiescent CB cells exited G0/G1 in contrast to only 13% of quiescent BM cells. In serum-free media supplemented with either SCF or IL-3 alone, CB CD34+ HLA-DR+ cells did not exit G0/G1 phases of cell cycle as rapidly as when CB plasma was present, unless SCF and IL-3 were added simultaneously. Collectively, these results suggest that CB CD34+ cells are more responsive to cytokine stimulation, especially SCF, and may represent more suitable candidates for ex vivo expansion of HPC than BM cells. Furthermore, these data illustrate potentially important biologic differences between the HPC content of subpopulations of BM and CB cells, and the response of these subpopulations to cytokine stimulation.

It is becoming increasingly clear that the parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors in human gene therapy. Specifically, the adeno-associated virus 2 (AAV), a human parvovirus, has gained particular attention in view of its nonpathogenic nature as well as its remarkable site-specificity of integration into the human chromosome. Using the recombinant AAV vector system, it is feasible to obtain high-efficiency transduction of slow- or non-cycling primary hematopoietic stem and progenitor cells, without the need for prestimulation with cytokines, which could potentially lead to differentiation of these cells before transplantation.

Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation.

In this report we have used an in vitro assay for long-term culture-initiating cells (LTC-IC) to detect primitive hematopoietic progenitor cells (HPC) in the peripheral blood (PB) of cancer patients who received high-dose cyclophosphamide (HD-CTX) followed by a combination of recombinant hematopoietic growth factors (C-HGF) including either interleukin-3 (IL-3) + granulocyte colony-stimulating factor (G-CSF), IL-3 + granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3/GM-CSF fusion protein (PIXY-321). In addition, we have developed a quantitative assay for cells capable of generating additional colony-forming cells (pre-CFC) as a means of determining primitive HPC present in mobilized PB cells. CD34+ human leukocyte A (HLA)-DR- cells isolated from the mobilized PB were capable of initiating long-term hematopoiesis in vitro that persisted for 10 weeks, while CD34+ HLA-DR- cells obtained from the nonmobilized PB or BM were capable of sustaining long-term hematopoiesis in vitro for only 4 weeks and 8 weeks, respectively. As determined by a limiting dilution analysis of mobilized PB CD34+ HLA-DR- cells, the frequency of pre-CFC was 4.3% (range, 1.0-8.3%). Pre-CFC comprised 0.01% (range, 0.001-0.02%) of mobilized PB mononuclear cells, and 151 pre-CFC were calculated to be present in one milliliter of mobilized PB (range, 20-310/ml). These results suggest that PB mononuclear cells collected by leukapheresis following mobilization with HD-CTX + C-HGFs contain not only differentiated HPCs but also more primitive HPC.

Umbilical cord blood (CB) has been shown to contain sufficient hematopoietic stem cells for allogeneic bone marrow transplant (BMT) and to contain progenitor cells susceptible to retrovirally mediated gene transduction. Enrichment of CD34+ cells from fresh unseparated or thawed unseparated CB could offer several advantages for (1) the storage of CB samples in an unrelated stem cell bank, which includes a decrease in volume and thus less storage space and (2) gene transfer into these cells. Cord blood was collected from the umbilical cord vein immediately after vaginal full-term delivery and samples of 5-12 ml (total leucocytes: 100 +/- 50 x 10(6)) of fresh unseparated (n = 6) and thawed unseparated (n = 8) CB were processed to enrich CD34+ cells using the Ceprate LC Biotin-Avidin affinity system. CD34+ cells, present at a frequency of 1.2 +/- 0.8% among leukocytes from CB (calculated by immunophenotyping and fluorescence-activated cell sorter (FACS) analysis), can be rapidly enriched to 54.4 +/- 12.3% (range, 38.6-87.1%) from fresh unseparated CB and 44.6 +/- 28% (range, 13.6-76%) from thawed unseparated CB. Hematopoietic progenitor assays for unseparated (cell concentration 3 x 10(4), 1 x 10(5), 3 x 10(5)) and CD34-enriched cells (cell concentration 1 x 10(3), 3 x 10(3), 1 x 10(4)) were performed in the presence of 5 U human interleukin-3 (hu IL-3), 30 U hu-IL-6, 10 U human granulocyte colony-stimulating factor, 6 U erythropoietin, and 15 ng human stem cell factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Human CD34+ cells were isolated from bone marrow from normal volunteers and expanded under serum-free culture conditions. CD34+ cells were cultured with interleukin-3 (IL-3), IL-1, and stem cell factor and expanded in granulocyte-macrophage colony-forming units, erythroid blast-forming units, and CD34+ cell number during the first 7-14 days of incubation. By contrast, cultures maintained in fetal calf serum under identical conditions showed much reduced expansion, as measured by all of the above parameters. The level of expansion of the CD34+ cells was dependent on the combination of growth factors used during culture. The data establish the feasibility of serum-free expansion of progenitors and suggest the clinical use of this procedure for the generation of expanded progenitor cell products for transfusion after chemotherapy to minimize treatment-related cytopenias.

Human hematopoietic stem cells are contained within a population of marrow cells that expresses the CD34 antigen but not other antigens associated with commitment to specific lineages. Evidence that stem cells capable of maintaining long-term hematopoiesis are within this CD34+ lineage-negative (Lin-) population is reviewed, including in vivo studies in humans and nonhuman primates. In vitro studies of the CD34+ Lin- population have indicated that the blast-sized cells, which are presumably in cycle, proliferate and give rise to colony-forming cells in the presence of combinations of growth factors, including c-kit ligand and interleukin-3 (IL-3). Recent studies have examined the factors required for the growth of the quiescent subset of the CD34+ Lin- cells, identified as small to medium lymphocyte-sized cells that resist treatment with 4-hydroperoxycyclophosphamide, a known characteristic of the marrow-repopulating cell. These studies have shown that an interaction with marrow stromal cells is required, in addition to c-kit ligand and IL-3, to induce these cells to proliferate and form multiple colony-forming cells. These studies have further indicated that this effect of stroma is mediated by a soluble factor(s). This activity may represent a novel factor(s) and/or a novel combination of growth factors.

We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.