Blood cells最新文献

Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogeneic bone marrow transplantation, for the treatment of a number of genetic diseases. To determine conditions that yield maximal gene transfer into UCB progenitor cells, we examined a number of variables. We used cell-free retroviral vector supernatants that convey neomycin (G418) resistance and measured the percentage of G418-resistant progenitor-derived colonies. Adding retroviral supernatant to the UCB cells in basal medium once a day for 3 days produced a threefold increase of G418-resistant colonies (9.8%) compared to a single exposure to supernatant (3.1%). To establish whether recombinant human growth factors are beneficial during transduction, the presence of interleukin-3 (IL-3), IL-6, and mast cell growth factor (MGF, a c-kit ligand) were compared in different combinations. Inclusion of the three factors together caused a threefold increase of gene transfer (30.4%) compared to transduction in basal medium. When the UCB cells were precultured in medium containing IL-3, IL-6, and MGF for 3 days before addition of the retroviral supernatant on days 4, 5, and 6, the average extent of gene transfer was 21.8%, compared with an average of 34.4% when UCB cells were transduced on days 1, 2, and 3. The presence of marrow stroma during the transduction of the UCB cells did not further increase gene transfer. We conclude that UCB progenitor cells can be efficiently transduced with the use of recombinant human growth factors IL-3, IL-6, and MGF and may be a suitable source for gene therapy.

Human umbilical cord bloods were fractionated by unit gravity sedimentation in 1% (v/v) dextran, followed by immunoaffinity selection for CD34+ stem and progenitor cells. Dextran sedimentation alone enabled recovery of more than 80% of the nucleated cells present and 90% of the CD34+ cells, as determined by flow cytometry. The addition of an immunoaffinity selection step for CD34+ cells resulted in a 134-fold enrichment for CD34+ cells, with a mean yield of 64 +/- 15%. The resultant CD34+ population contained almost half the CFU-GM activity initially present in the cord bloods and could be expanded ex vivo in liquid culture.

To carry out cord blood transplants from allogeneic unrelated donors, cord blood stem cell banks must be established. This report proposed the policies and procedures that can be used to establish cord blood banks. The areas covered include donor consent and suitability criteria; infectious and genetic disease testing; collection, processing, and preservation of the cord blood; retention of specimens for special testing; confidentiality; documentation and record keeping; establishment of a quality control program; and related regulatory issues. There are no technologic impediments to establishing cord blood banks. Agreement on some standard policies and procedures would facilitate exchange of cord blood stem cells among transplant centers and should increase the number of transplants that can be done.

When 200 x 10(6) male BALB/c cells are given by tail vein injection to female nonmyeloablated hosts in one injection, a relatively low engraftment percentage is seen, but when the same total number of cells is given over five injections (separated by 24 hours), the observed engraftment is much higher. A further increase in engraftment appears to occur when the same number of cells is given in 10 injections separated by at least 24 hours. These data suggest that somewhere between 5 and 10% of marrow niches are available at intervals of 24 or more hours and that the keys to high levels of engraftment are the cell cycle status of the engrafting stem cell and the schedule of engraftment.

Human cord blood cells have been shown to highly engraft the marrows of sublethally irradiated SCID mice. Herein we report our experience with this system and the use of immunohistochemistry to identify human cell engraftment. Immunohistochemistry results correlated well with those of flow cytometry, human progenitor-cell cultures, and molecular analysis of human specific markers. Immunohistochemistry should play a useful role in the in vivo analysis of human stem/progenitor cell engraftment in xenogeneic transplantation models.

Cord blood (CB) was analyzed for its alloreactive immune potential to evaluate its capacity to mediate graft vs. host disease (GVHD) and graft vs. leukemia (GVL) effects. Cord blood was observed to display minimal innate cytotoxic capacity but was capable of rapidly developing significant nonspecific natural killer cell-like effector mechanisms. Cord blood was unable to generate effective alloantigen-specific cytotoxic T lymphocytes (CTL), which was at least partially due to an altered lymphokine profile. The frequency of alloreactive T cells present in CB (whether assessed as total responding T-cell or CTL precursors) was greatly reduced as compared to adult peripheral blood lymphocytes (PBLs). However, the frequency of nonspecific effector cells was equivalent to PBLs. Significantly, CB T cells seemed to have undergone some type of developmental tolerance to maternal HLA antigens in utero, which could greatly increase the utility of CB in familial transplants. That is, CB T cells were unresponsive to noninherited maternal HLA antigens. Finally, CB demonstrated significant GVL capacity whether measured in vitro or in an animal model in vivo. Thus, the use of CB in most transplant settings should be free of the immunological problems associated with GVHD yet still be an effective mediator of GVL.

Human umbilical cord blood (CB) is increasingly used as an alternative to bone marrow as a source of stem and progenitor cells for pediatric patients. We have evaluated the alloreactive potential of cord blood T cells in vitro. Our findings demonstrate that in a primary mixed leukocyte culture CB T cells demonstrate strong proliferative responses to allogeneic stimulation but little to no generation of cytotoxic effector function. Furthermore, restimulation of primary cultures results in a state of proliferative unresponsiveness. Such diminished cytotoxic and proliferative responses may, in part, be related to the low incidence of graft vs. host disease thus far noted in human CB transplants.

Cord blood contains stem cells in amounts similar to or slightly less than those present in a bone marrow collection to be used for bone marrow transplantation (BMT). Too few cord blood transplants (CBT) have yet been performed to define the ability to achieve engraftment and the rate of engraftment. Two cord blood transplants have been performed using granulocyte-macrophage colony stimulating factor (GM-CSF) to hasten engraftment. Two children, aged 5 and 6 years received a CBT using HLA-identical stem cells collected at the birth of a sibling. One child had X-linked lymphoproliferative disease (XLP), and the other, acute lymphoblastic leukemia in second complete remission. One had an ABO and one an Rh blood group mismatch. Conditioning therapy consisted of cyclophosphamide, melphalan, and antithymocyte globulin or busulphan and cyclophosphamide. Graft-versus-host disease prophylaxis was methotrexate and cyclosporine or cyclosporine. Both children were given GM-CSF at 5 micrograms/kg/day from day 1 until the absolute neutrophil count (ANC) reached 1.0 x 10(9)/L for 3 consecutive days. If this level was not reached by day 14, the dose of GM-CSF was doubled. Both children engrafted rapidly, with ANCs reaching 0.5 x 10(9)/L in 12 and 16 days. Engraftment was confirmed by blood group in both and sex chromosome typing in one. Both children developed mild GVHD localized to skin, which resolved with steroid therapy. The child with XLP was cured and has survived for 34 months; the second child has survived 27 months with normal marrow function but has had a relapse of leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)