Cells In Long-term Bone Marrow Cultures Research Articles

Chlorella vulgaris treatment ameliorates the suppressive effects of single and repeated stressors on hematopoiesis

The reports regarding the mutual influence between the central nervous system and the immune system constitute a vast and somewhat controversial body of literature. Stress is known to disturb homeostasis, impairing immunological functions. In this study, we investigated the hematopoietic response of Chlorella vulgaris (CV)-treated mice exposed to single (SST) and repeated stress (RST). We observed a reduction in the numbers of hematopoietic progenitors (HP) in the bone marrow and long-term bone marrow cultures (LTBMC) using flow cytometry and a coinciding decrease in the number of granulocyte-macrophage colonies (CFU-GM) after treatment with both stressors, but SST caused a more profound suppression. We observed a proportional increase in the colony-stimulating activity (CSA) of the serum of animals subjected to SST or RST. In the bone marrow, SST and RST induced a decrease in both mature myeloid and lymphoid populations but did not affect pluripotent hematopoietic progenitors (Lin(-)Sca-1(+)c-kit(+), LSK), and again, a more profound suppression was observed after SST. We further quantified the levels of interleukin-1α (IL-1α) and interleukin-6 (IL-6) and the number of myeloid cells in LTBMC. Both SST and RST reduced the levels of these cytokines to similar degrees. The myeloid population was also reduced in LTBMC, and SST induced a more intense suppression. Importantly, CV treatment prevented the changes produced by SST and RST in all of the parameters evaluated. Together, our results suggest that CV treatment is an effective tool for the prophylaxis of myelosuppression caused by single or repeated stressors.

Increased Levels of the High Mobility Group Box 1 Protein Sustain the Inflammatory Bone Marrow Microenvironment in Patients with Chronic Idiopathic Neutropenia via Activation of Toll-Like Receptor 4

Chronic idiopathic neutropenia (CIN) is a granulocytic disorder characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells and an inflammatory BM microenvironment. The aim of this study was to investigate the possible involvement of Toll-like receptors (TLRs) in the production of pro-inflammatory mediators in CIN BM. We evaluated the expression of TLRs in patient BM cell subsets and adherent cells of long-term BM cultures (LTBMCs) using flow cytometry. We also examined the activation of TLR-mediated signaling using real-time PCR arrays and explored for potential endogenous TLR-specific ligands in CIN BM. CIN patients (n = 30) displayed significantly increased expression of surface TLR4 in monocytes of BM and LTBMC adherent cells compared to controls (n = 27). The TLR signaling gene array study in purified BM CD14(+) cells showed that numerous TLR-related genes displayed at least two-fold increase in patients compared to controls. Among the over-expressed genes were genes related to the MyD88-dependent and MyD88-independent pathway suggesting a TLR4-mediated signaling. BM plasma from CIN patients induced the production of pro-inflammatory mediators including interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and IL-8 by autologous BM monocytes, and this effect was abrogated by a specific TLR4 inhibitor. The levels of the high mobility group box 1 protein (HMGB1), representing a TLR4 ligand, were significantly increased in patient LTBMC supernatants compared to controls. These data demonstrate a significant role of BM monocytes in the pathophysiology of CIN through the production of pro-inflammatory cytokines in a TLR4-mediated mechanism under the influence of endogenous ligands such as HMGB1.

Inhibition of p38 MAPK Attenuates Ionizing Radiation-Induced Hematopoietic Cell Senescence and Residual Bone Marrow Injury

Exposure to a moderate or high total-body dose of radiation induces not only acute bone marrow suppression but also residual (or long-term) bone marrow injury. The induction of residual bone marrow injury is primarily attributed to the induction of hematopoietic cell senescence by ionizing radiation. However, the mechanisms underlying radiation-induced hematopoietic cell senescence are not known and thus were investigated in the present study. Using a well-established long-term bone marrow cell culture system, we found that radiation induced hematopoietic cell senescence at least in part via activation of p38 mitogen-activated protein kinase (p38). This suggestion is supported by the finding that exposure to radiation selectively activated p38 in bone marrow hematopoietic cells. The activation was associated with a significant reduction in hematopoietic cell clonogenic function, an increased expression of p16(INK4a) (p16), and an elevated senescence-associated β-galactosidase (SA-β-gal) activity. All these changes were attenuated by p38 inhibition with a specific p38 inhibitor, SB203580 (SB). Selective activation of p38 was also observed in bone marrow hematopoietic stem cells (HSCs) after mice were exposed to a sublethal total-body dose (6.5 Gy) of radiation. Treatment of the irradiated mice with SB after total-body irradiation (TBI) increased the frequencies of HSCs and hematopoietic progenitor cells (HPCs) in their bone marrow and the clonogenic functions of the irradiated HSCs and HPCs. These findings suggest that activation of p38 plays a role in mediating radiation-induced hematopoietic cell senescence and residual bone marrow suppression.

Chlorella vulgaris Modulates Immunomyelopoietic Activity and Enhances the Resistance of Tumor-Bearing Mice

We studied the effects of Chlorella vulgaris (CV) on the interaction between stromal and hematopoietic stem cells in normal and Ehrlich ascites tumor (EAT)-bearing mice. Long-term bone marrow culture (LTBMC), cytokine production, spleen mononuclear cells (SMC) proliferation (SCP), colony stimulating activity (CSA), and NK cells activity were evaluated. In tumor bearers, reduced capacity of stromal cell layer to support the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM), concomitantly to decreased numbers of total nonadherent cells in LTBMC and reduced local production of IL-6 and IL-1α, were observed. Presence of the tumor has not altered the number of stromal adherent cells. CV treatment restored the ability of stromal cells from EAT-bearing mice to produce IL-6 and IL-1α, which was consistent with increased number of nonadherent cells and higher ability to display CFU-GM in vitro. EAT growth increased SCP, serum CSA, and IL-10 production and concurrently depressed NK cell activity and the secretion of IL-2, IFN-γ, and TNF-α. Treatment of tumor-bearing mice with CV augmented CSA, SMC proliferation, NK cell activity, and the production of IL-2, IFN-γ, and TNF-α, whereas IL-10 levels where reduced. Our results suggest that CV modulates immunehematopoietic cell activity and disengages tumor-induced suppression of these responses.

Inhibition of p38 MAPK Reduces Ionizing Radiation (IR)-Induced Hematopoietic Suppression by Preventing Hematopoietic Stem/Progenitor Cell Senescence.

Bone marrow (BM) suppression is the most common dose-limiting side effect of conventional cancer therapy, particularly with certain alkylating agents and/or ionizing radiation (IR). It has been shown that p38 mitogen-activated protein kinase (p38 MAPK) plays a critical role in regulation of hematopoiesis and its activation mediates IR- and chemotherapy-induced cell injury and oxidative stress-induced hematopoietic stem cell (HSC) exhaustion. Therefore, we examined the role of p38 MAPK in radiation-induced BM injury. First, we monitored the activation of p38 MAPK in BM hematopoietic cells at different time after exposure to IR using our well-established long-term BM cell culture (LTBMC) model system. The activation of p38 MAPK was detected within 24 hours after BM hematopoietic cells were exposed to 4 Gy IR and this activation sustained up to 5 weeks after radiation. Inhibition of p38 MAPK activity with a p38 MAPK specific inhibitor (SB202190) attenuated IR-induced suppression of the hematopoietic function of BM hematopoietic cells in an in vitro colony forming cell (CFC) assay. Moreover, the number of hematopoietic progenitor cells produced by SB202190-treated BM cells was significantly greater than that by the cells without SB202190 treatment after exposure to IR and followed by a five-week LTBMC. Interestingly, p38 MAPK inhibition showed no effect on IR-induced apoptosis in both HSCs and hematopoietic progenitor cells (HPCs), whereas the radioprotection effect of SB202190 was associated with a significantly reduction of p16INK4a expression and senescence-associated β-galactosidase (SA-β-Gal) activity in irradiated BM cells after five weeks of LTBMC. These findings suggest that activation of p38 MAPK may mediate IR-induced hematopoietic suppression by induction of hematopoietic stem/progenitor cell senescence and pharmacological inhibition of the p38 MAPK pathway may have the potential to be developed as an innovatively therapeutic strategy to ameliorate IR- and chemotherapy-induced BM toxicity.

Leukemia-Derived Fibroblast Cells Produce Vascular Endothelial Growth Factor Significantly in Chronic Myelogenous Leukemia Cases.

Objective: We reported recently that myofibroblast cells in bone marrow cells of chronic myelogenous leukemia (CML)-patients were partly derived from CML clone, which was identified after long-term bone marrow cell-cultures. The generated BCR-ABL-positive fibroblast cells produced various kinds of cytokines more than that observed in the normal clone-derived fibroblast cells. Among these cytokines, we focus on vascular endothelial growth factor (VEGF)-system in this report.Materials and Methods: Bone marrow cells were collected from chronic myelogenous leukemia cases (20 of chronic phase (CP), 3 of accelerated phase (AP), 8 of blast crisis phase (BC)), which were separated with gravity sedimentation. Obtained non-adherent mononuclear cells were cultured in DMEM with 10% FCS in the humidified 5% CO2 incubator. When cells showed morphological changes into fibroblastoid cells, cells were treated with trypsin and further cultured. The obtained stromal fibroblast cells were sub-cloned into culturing in 96 well plates, and from which RNAs were extracted. cDNAs were synthesized, and RT-PCR was performed to identify BCR-ABL fusion product. The selected clones were further analyzed on DNA levels with FISH to identify BCR-ABL. The concentration of VEGF in the patients' sera and in the conditioned media of non-adherent mononuclear cells, of fibroblast cells derived from normal clone and of fibroblast cells with BCR-ABL was measured with ELISA kit (R&D Systems). The expression of VEGF, VEGF receptor type-1 and VEGF receptor type-2 was also compared at RNA levels. The effect of anti-VEGF antibody to the culturing CML cells was determined using 3H-incorporation assay system.Results and Discussion: The levels of VEGF in sera from CML patients were elevated significantly. However, in patients with CP the expression of VEGF was not observed in non-adherent mononuclear cells. In contrast, VEGF production was observed in non-adherent mononuclear cells from the patients with AP and BC. Stromal fibroblasts produced VEGF in CP, AP and BC, in which BCR-ABL-positive fibroblast cells produced at the significant levels. The expression of VEGFR-1 and -2 was detected in non-adherent mononuclear cells from CP, AP and BC. When non-adherent mononuclear cells from CML patients were cultured onto the stromal fibroblast cells, significant additive effects were observed with 3H-thymidine incorporation assays. And when anti VEGF antibody was added to the cultures, the proliferation activity was significantly decreased. These observations indicate that in CML cases, especially in CP, VEGF plays an important role for the cellular proliferation with paracrine system.

Inhibition of p38 MAPK Reduces Ionizing Radiation-Induced Hematopoietic Cell Injury.

Our previous studies demonstrated that exposure to ionizing radiation (IR) causes long-term bone marrow (BM) damage by induction cellular senescence in hematopoietic cells. The senescent hematopoietic cells exhibited an elevated activity of the senescence associated β-galactosidase (SA-β-gal), a biomaker for senescent cells, and expressed increased levels of p16Ink4a (p16), whose expression has been implicated in the establishment and maintenance of cellular senescence. Activation of the p38 MAPK pathway has been implicated in the induction of cell cycle arrest and senescence in response to a variety of stressors in part by up-regulating the expression of p16. Therefore, in the present study we examined the role of the p38 MAPK pathway in IR-induced hematopoietic cell senescence. The results showed that exposure of male C57BL/6 mice to a sublethal dose (6.5 Gy) of total body irradiation (TBI) caused a sustained activation (>4 weeks) of p38 in Lin− c-kit+ Sca-1+ (LKS+) hematopoietic stem cells (HSCs). This activation was associated with a persistent up-regulation of p16 and quantitative and qualitative reduction of LKS+ HSCs. Inhibition of p38 activity with SB202190 (a specific p38 inhibitor) attenuated IR-induced inhibition of the hematopoietic function of BM hematopoietic cells in an in vitro colony forming cell (CFC) assay. Moreover, the production of hematopoietic progenitor cells (HPCs) in SB202190-treated BM cells was 7.5 fold higher than that of irradiated cells without SB202190 treatment after five weeks of long-term BM cell culture (LTBMC); and the cells treated with SB202190 after IR retained their clonogenic function while the control irradiated cells lose their ability to form colonies. These findings suggest that p38 may play an important role in mediating IR-induced suppression of hematopoietic cell function and pharmacological inhibition of the p38 pathway may potentially be developed as novel therapeutic strategy to ameliorate IR- and chemotherapy-induced BM toxicity.

Three‐Dimensional Bioreactor Cultures: A Useful Dynamic Model for the Study of Cellular Interactions

The ex vivo expansion of hematopoietic cells is a developing area with emphasis on bioreactor systems for amelioration of culture conditions. A rational design of bioreactors, especially those allowing microgravity, could permit the production of stem cells and will offer new approaches for studying the mechanisms of proliferation, differentiation, and signal transduction of cultured cells. The efficacy of two commercially available bioreactors (rotating-vessel miniPERM and static INTEGRA CL 350) to support long-term bone marrow cell cultures (LTBMCC) and three-dimensional growth of Hodgkin's lymphoma HD-MY-Z cells was investigated. In the miniPERM system, the growth of LTBMCC spheroids (containing 30-40 cells) was obtained. An essentially higher content of hematopoietic precursor cells (colony-forming units-granulocyte macrophage) was registered in the rotating-vessel system. In this bioreactor, a growth of large HD-MY-Z spheroids (containing 100-200 cells) was achieved. The composed mathematical models of the physicomechanical behavior of spheroids enabled the evaluation of the revolution frequency increase schedule. The differential equations took into account all inertial effects caused by the production module rotation movement as well as those caused by the relative movement of the spheroid in the fluid. The models aimed at the optimization of the rotation frequency increase schedule for different types of cells to reduce shear stress, augment productivity, and tolerate the growth of large spheroids. The models were numerically tested using MATLAB-SIMULINK software, and the trajectories of prestained HD-MY-Z spheroids were filmed. The coincidence of the theoretical and experimental trajectories was sufficient.

Ehrlich Ascites Tumor as a Tool in the Development of Compounds with Immunomodulatory Properties

In previous works, we have demonstrated that the myeloprotective properties of several natural and synthetic compounds are partly responsible for their antitumor activity in the Ehrlich ascites tumor (EAT) model. In this work, we present information that may be useful to the study of pharmacological and toxicological properties of compounds that affect the hematological compartment. Clonogenic studies in EAT‐inoculated mice demonstrated a rapid decrease in bone marrow CFU‐GM, whereas a progressive increase in splenic CFU‐GM and cellularity was observed, followed by splenomegaly. Bone marrow cellularity declined on the third day after tumor challenge, returning to normal values thereafter. Serum from EAT‐bearing mice produced detectable colony‐stimulating activity in vitro. Similar results were observed with the conditioned medium from Ehrlich tumor cell cultures, but not with the cell‐free Ehrlich tumor ascitic fluid. Tumor inoculation also resulted in a more striking depletion in the number of non‐adherent cells in long‐term bone marrow cell cultures (LTBMCs) with no bone marrow stroma formation. We speculate that the physiological alterations induced by the EAT growth can be used to assess the ability of compounds to modulate the hematopoietic response.

Increased apoptosis of bone marrow CD34(+) cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus.

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34(+) cells, compared with the control group, reflecting the decrease of both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells. Patient CD34(+)/Fas(+) but not CD34(-)/Fas(+) cells were significantly increased. Apoptotic (7AAD(dim)) cells were higher among CD34(+)/Fas(+) than among CD34(+)/Fas(-) cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34(+)/Fas(+) cells. These findings are suggestive of a Fas-mediated apoptosis accounting for the low CD34(+) cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E), compared with the control group, and that the generation of colony-forming cells in long-term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34(+) cells were increased, apoptotic CD34(+)/Fas(+) cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML).

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.

Recovery of hematopoietic activity in bone marrow from human immunodeficiency virus type 1-infected patients during highly active antiretroviral therapy.

The mechanisms responsible for the hematopoietic failure in human immunodeficiency virus type 1 (HIV-1)-infected patients are still unknown. Several findings indicate that the in vitro proliferative potential of precursor cells from AIDS patients is reduced. The changes seen in bone marrow (BM) morphology and the defective BM functions associated with cytopenias have both been proposed as potential explanations. In patients treated with highly active antiretroviral therapy (HAART) an immune reconstitution associated with increased whole blood cell counts has been described. We have investigated the effects of HAART on the number of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), using long-term BM cell cultures (LTBMC) in a group of subjects with HIV-1 infection enrolled in an open study to evaluate the mechanisms of immune reconstitution during HAART. In each patient, the increase in colony growth was homogeneous, regardless of the type of hematopoietic progenitor cells assayed; in four subjects an increase in the most primitive progenitor cells (LTC-ICs) was observed. These findings were associated with the in vivo data showing increased numbers of BM mononuclear cells (BMMCs) after HAART and with a rise in peripheral CD4(+) T cell counts and decreased levels of plasma HIV-1 RNA. A decreased number of hematopoietic progenitor cells and/or a defective modulation of progenitor cell growth might be the cause of the hematological abnormalities in AIDS patients. Controlling HIV-1 replication by HAART could determine a restoration of stem cell activity, probably because of the suppression of factors that inhibit normal hematopoiesis.

Functional activity of hemopoietic and stromal cells in various types of myelodysplastic syndrome

Using clonal methods for assessment of hemopoietic and stromal cells and long-term bone marrow cell cultures, we have demonstrated heterogeneity of myelodysplastic syndrome. Low content of stromal precursor cells in native bone marrow, peculiarities in the formation of the stromal layer and its hemopoiesis-stimulating capacity in long-term cultures, and altered properties of stromal precursor cells in long-term cultures indicate defect in the stromal microenvironment in myelodysplastic syndrome.

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells.

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo, which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity.

Alkylphosphocholines: Effects on human leukemic cell lines and normal bone marrow cells

The anti-leukemic activity of a series of alkylphosphocholines (APCs) was studied against a panel of human leukemic cell lines (HL-60, K-562, Reh, MOLT-4, Jurkat, Ramos and Raji). Cytotoxic efficacy was measured by the MTT cell survival assay. All cell lines were found to be sensitive, except the multipotential CML-derived K-562 cell line. Flow cytometry of HL-60 cells showed a significant decrease of cells in S phase and the formation of a sub-G1 fraction. DNA fragmentation typical for programmed cell death was detected by DNA gel electrophoresis in these cells but not in any of the other leukemic lines. At concentrations below the cytotoxic range, mitogenic effects were seen in HL-60 cells after 14-hr exposure. Colony formation by K-562 cells revealed an augmented clonogenicity after exposure to APC with a short alkyl chain. In contrast, cells of lymphoid origin did not undergo DNA fragmentation or show mitogenic stimulation after exposure to APC. Normal bone marrow cells were also investigated for mitogenic and genotoxic effects. No decrease was found in the number of hematopoietic progenitors in long-term bone marrow cell cultures after exposure to APC. On the contrary, a significant increase was found after short exposure. Dodecylphosphocholine, hexadecylphosphocholine (HPC) and (octadecyl-[2-(N-methylpiperidino)-ethyl]-phosphate exhibited a mild clastogenicity at equimolar high doses on murine bone marrow cells in vivo,which is unusual for the majority of classical DNA-interacting anti-cancer drugs. In conclusion, APCs are agents with a broad spectrum of in vitro anti-leukemic effects, which lack hematological toxicity. Int. J. Cancer,77:778–786, 1998. © 1998 Wiley-Liss, Inc.

Analysis of hematopoietic stem cell reprogramming with toxigenicity.

The molecular mechanisms by which a stem cell is committed to individual lineage are largely unknown. Two different models, though not mutually exclusive, are currently debated. The first describes the temporal and hierarchical coordination of lineage-specific transcriptional programs. The second suggests that multilineage genes are expressed in a self-renewing and undifferentiated cell prior to lineage commitment. To challenge these two models in in vivo-appropriate conditions, the expression of an exogenous toxigene was used to create transgenic animals in which an inducible, reversible cell knock-out at a specific stage of differentiation could be achieved. Both additional transgenesis using the megakaryocyte specific alphaIIb promoter and targeted transgenesis were used to express the herpes virus thymidine kinase (tk) gene in the megakaryocytic lineage. When the tk gene was targeted to the locus of the megakaryocyte-specific alphaIIb gene, a typical Glanzman thrombasthenic syndrome was created. Despite this bleeding disorder, the lack of expression of the alphaIIb gene did not affect the development of the mice. In both transgenic and targeted animals, all progenitor cells were sensitive to the effect of the gancyclovir (GCV), both in vivo and ex vivo. Long-term bone marrow cell cultures on stromal layers indicated that most of the very early progenitor cells expressed the enzyme. All the results obtained with this inducible toxic phenotype indicated that genetic programs that are in control of the expression of lineage-specific genes are operative in a totipotent stem cell prior to lineage commitment and strongly support the concept that stem cells express a multilineage transcriptome.

Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III.

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing beta-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures

AbstractAttempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Neither Human Immunodeficiency Virus-1 (HIV-1) nor HIV-2 Infects Most-Primitive Human Hematopoietic Stem Cells as Assessed in Long-Term Bone Marrow Cultures

Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38−) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS.

Murine marrow stromal response to myelotoxic agents in vitro.

Previous studies have shown that the haemopoietically active murine long-term bone marrow culture (LTBMC) is a useful model for studying drug-induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit-fibroblasts (CFU-F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 micrograms/ml CEF-treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat-containing and endothelial cells. In contrast, treatment with 1 microM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU-F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 micrograms/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.