Thrombopoietin enhances the production of myeloid cells, but not megakaryocytes, in juvenile chronic myelogenous leukemia

We previously reported the aberrant growth of granulocyte-macrophage (GM) progenitors induced by a combination of stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile chronic myelogenous leukemia (JCML). We examined here the effects of thrombopoietin (TPO) on the proliferation and differentiation of hematopoietic progenitors in JCML. In serum-deprived single-cell cultures of normal bone marrow (BM) CD34+CD38high cells, the addition of TPO to the culture containing SCF + GM-CSF resulted in an increase in the number and size of GM colonies. In the JCML cultures, in contrast, the number of SCF + GM-CSF-dependent GM colonies was not increased by the addition of TPO. However, the TPO addition caused an enlargement of GM colonies in cultures from the JCML patients to a significantly greater extent compared with the normal controls. There was no difference in the type of the constituent cells of GM colonies with or without TPO grown by JCML BM cells. A flow cytometric analysis showed that the c-Mpl expression was found on CD13+ myeloid cells generated by CD34+CD38high BM cells from JCML patients, but was at an undetectable level in normal controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM colony-forming cells by JCML CD34+CD38neg/low population, indicating the stimulatory effects of TPO on JCML primitive hematopoietic progenitors. Normal BM cells yielded a significant number of megakaryocytes as well as myeloid cells in response to a combination of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells were barely produced by the JCML progenitors. Our results may provide a fundamental insight that the administration of TPO enhances the aberrant growth of GM progenitors rather than the recovery of megakaryocytopoiesis.     

Retroviral-mediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.     

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.     

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured smooth muscle cells of human pulmonary artery

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.     

Human granulocyte colony-stimulating factor (G-CSF) stimulates the in vitro and in vivo development but not commitment of primitive multipotential progenitors from transgenic mice expressing the human G-CSF receptor

Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and restricted differentiation of hematopoietic progenitors into neutrophils. To clarify the effects of G-CSF on hematopoietic progenitors, we generated transgenic (Tg) mice that had ubiquitous expression of the human G-CSF receptor (hG-CSFR). In clonal cultures of bone marrow and spleen cells obtained from these mice, hG-CSF supported the growth of myelocytic as well as megakaryocytic, mast cell, mixed, and blast cell colonies. Single-cell cultures of lineage-negative (Lin-)c-Kit+Sca-1(+) or Sca-1(-) cells obtained from the Tg mice confirmed the direct effects of hG-CSF on the proliferation and differentiation of various progenitors. hG-CSF also had stimulatory effects on the formation of blast cell colonies in cultures using 5-fluorouracil-resistant hematopoietic progenitors and clone-sorted Lin-c-Kit+Sca-1(+) primitive hematopoietic cells. These colonies contained different progenitors in proportions similar to those obtained when mouse interleukin-3 was used in place of hG-CSF. Administration of hG-CSF to Tg mice led to significant increases in spleen colony-forming and mixed/blast cell colony-forming cells in bone marrow and spleen, but did not alter the proportion of myeloid progenitors in total clonogenic cells. These results show that, when functional G-CSFR is present on the cell surface, hG-CSF stimulates the development of primitive multipotential progenitors both in vitro and in vivo, but does not induce exclusive commitment to the myeloid lineage.     

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU-MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti-IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human megakaryocytopoiesis.     

Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo-expanded CB cells, purified CD34(+) cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo-expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34(+) cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45(+) repopulation in NOD/SCIDs BM: 3. 7% +/- 1.2% v 26.2% +/- 5.9%, respectively, at 20 days posttransplantation; P <.005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% +/- 1.7% of CD45(+) cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo-expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45(+) and CD45(+)/CD34(+) cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo-expanded samples were found. The analysis of the engrafted CD45(+) cells showed that both the fresh and the in vitro-incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.     

Fluorescence in situ hybridization of progenitor cells obtained by fluorescence-activated cell sorting for the detection of cells affected by chromosome abnormality trisomy 8 in patients with myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is believed to be a stem-cell disorder involving cytopenia and dysplastic changes in three hematopoietic lineages. However, the involvement of pluripotent stem cells and progenitor cells has not been clarified conclusively. To address this issue, we used fluorescence in situ hybridization (FISH) of blood and bone marrow (BM) smears for mature cells and FISH of cells sorted by fluorescence-activated cell sorting for progenitor cells. Seven patients with MDS associated with trisomy 8 were studied. FISH showed +8 in granulocytes, monocytes, and erythroblasts, but not in lymphocytes. Sorted cells of T (CD3(+)), B (CD19(+)), and NK cells (CD3(-)CD56(+)) from peripheral blood did not contain +8, nor did CD34(+) subpopulations from BM including B (CD34(+)CD19(+)), T/NK (CD34(+)CD7(+)) progenitors, and pluripotent stem cells (CD34(+)Thy1(+)). The +8 chromosome abnormality was identified in stem cells only at the level of colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM; CD34(+)CD33(+)). It may thus be concluded that cells affected by trisomy 8 in the context of MDS are at the CFU-GEMM level and that cells of lymphoid lineage are not involved. These results provide new insights into the biology of MDS and suggest that intensive chemotherapy and autologous BM transplantation may become important therapeutic strategies.     

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.     

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background

Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic-severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar "humanization" rationale may be required.     

CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells

Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi-potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and committing to the T-cell lineage.     

Human natural killer cells

Human natural killer (NK) cells comprise 10 to 15% of peripheral blood lymphocytes, characterized by their morphologic appearance of large granular lymphocytes (LGLs) and phenotype CD3- CD56+ CD16+ or CD16-. Functionally, these cells are defined by their ability to lyse target cells without prior sensitization and without restriction by major histocompatibility (MHC) antigens. These cells play an important role in immune defenses, especially after hematopoietic transplantation. They contribute to the defenses against virus-infected cells, graft rejection, and neoplasias; they also participate in the regulation of hematopoiesis through cytokine production and cell to cell interaction. In this mini-review we attempt to summarize the most relevant findings about these cells in terms of their origin and differentiation, their cell surface characteristics including activation and their cytolytic pathways. We have also provided a brief approach of their potential clinical use. Increased knowledge of NK cell differentiation, ontogeny and regulatory mechanisms may be of use for the planning of immunotherapeutic strategies.     

Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.     

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.