CFU-GM Production Research Articles

An oral HemokineTM, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo

An oral therapeutic which reduces duration of cytopenias and is active following accidental radiation exposures is an unmet need in radiation countermeasures. Alpha methylhydrocinnamate (ST7) prolongs STAT-5 phosphorylation, reduces growth-factor dependency of multi-lineage cell lines, and stimulates erythropoiesis. Here, ST7 and its isomers were studied for their effects on myeloid progenitors and hematopoietic stem cells (HSCs) following radiation, in nonhuman primates, and murine irradiation models. Addition of ST7 or ST7-S increased CFU-GM production by 1.7-fold (p<0.001), reduced neutrophil apoptosis comparable to G-CSF, and enhanced HSC survival post-radiation by 2-fold, (p=0.028). ST7 and ST7-S administered in normal baboons increased ANC and platelet counts by 50–400%. In sub-lethally-irradiated mice, ANC nadir remained >200/mm3 and neutropenia recovered in 6days with ST7 treatment and 18days in controls (p<0.05). In lethally-irradiated mice, marrow pathology at 15days was hypocellular (10% cellularity) in controls, but normal (55–75% cellularity) with complete neutrophil maturation with ST7-S treatment. Following lethal irradiation, ST7, given orally for 4days, reduced mortality, with 30% survival in ST7-animals vs 8% in controls, (p<0.05). Collectively, the studies indicate that ST7 and ST7-S enhance myeloid recovery post-radiation and merit further evaluation to accelerate hematologic recovery in conditions of radiation-related and other marrow hypoplasias.

MTOR Signaling Is Required for the Survival and Proliferation of Hematopoietic Stem/Progenitor Cells

Abstract 4787The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates protein synthesis, gene transcription, cell growth and cell proliferation. Previous studies have demonstrated that abnormalities in the Pten-mTOR pathway may contribute to the development of leukemia and lead to premature exhaustion of hematopoietic stem cells (HSCs). These findings suggest a role for mTOR in the regulation of HSC self-renewal and cell transformation. The aim of this study was to investigate the involvement of mTOR signaling in the survival and proliferation of HSCs and hematopoietic progenitor cells (HPCs). Immunofluorescent staining with antibodies against phosphorylated mTOR and S6 kinase revealed that Thrombopoietin (Tpo) activates mTOR in cultured mouse bone marrow (BM) lineage negative cells, suggesting that Tpo may promote the survival and proliferation of HSCs/HPCs via activation of the mTOR signaling pathway. Further studies revealed that treatment with an mTOR specific small molecule inhibitor (Ku-69734) significantly suppressed the colony-forming ability of HPCs as evidenced by a dose-dependent decrease in the production of CFU-GM, BFU-E and CFU-GEMM. We also examined the clonogenic function of HSCs using cobblestone-area forming cell (CAFC) assays and found that Ku-69734 treatment markedly reduced the number of CAFCs in long-term BM culture. Moreover, immunophenotyping and flow cytometric analyses showed that inhibition of mTOR induced apoptosis primarily in HSCs and to a lesser degree in HPCs, indicating that mTOR inhibition may suppress the clonogenic function of HSCs and HPCs via the induction of apoptosis. Together, these data demonstrate that mTOR signaling is required for the survival and proliferation of HSCs and HPCs. Given that many mTOR inhibitors are currently in clinical trials for the treatment of cancers, our findings provide the rationale to further evaluate the potential toxicity of mTOR inhibitors in HSCs/HPCs in vivo. Disclosures:No relevant conflicts of interest to declare.

Effects of Gamma Radiation on Bone-Marrow Stromal Cells

The effects of in vitro irradiation on proliferation and hematopoietic supportive functions of stromal cells were studied. To assess the effects of radiation on stromal cells proliferation, marrow cells were exposed to a single dose of gamma radiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that stromal cell proliferation was significantly suppressed after radiation in a dose-dependent manner. Stromal layers obtained from irradiated marrow cells failed to establish adherent layers after 6, 8, or 10 Gy of radiation. To assess the functions of stromal cells that survived radiation, stromal layers derived from irradiated marrow cells were cocultured with freshly isolated autologous hematopoietic cells and assayed for their capacity to support prolonged granulocyte–macrophage progenitors (CFU-GM) production. Stromal layers derived from 2-Gy-irradiated marrow cells resulted in similar CFU-GM production as control cells, while stromal layers derived from 4- to 10-Gy-irradiated marrow cells significantly decreased CFU-GM production. To study the influence of radiation on hematopoietic supportive capacity in established stromal layers, stromal layers generated from non-irradiated marrow cells were irradiated and cocultured with freshly isolated autologous hematopoietic cells. Established stromal layers irradiated up to 10 Gy sustained prolonged CFU-GM production, suggesting that hematopoietic stromal supportive functions remained intact at this dose of radiation. In conclusion, our results indicated that proliferation of stromal cells and bone-marrow stromal layer formation from stromal cells are sensitive to radiation in vitro, while established bone-marrow stromal layer originating from stromal cells is relatively resistant to radiation. Data generated may have implications in future bone-marrow transplantation research.

Telintra™ (TLK199) Enhances Granulocyte Colony-Stimulating Factor Production.

TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. GST P1-1 has been shown to be a negative regulator of c-Jun N-terminal kinase, which has been implicated in the control of cellular growth and differentiation. Studies in rodents have shown that TLK199 treatment increases the levels of circulating white blood cells and stimulates the production of CFU-GM in bone marrow. In a rodent model of 5-FU-induced neutropenia, treatment with TLK199 accelerated the recovery of neutrophil levels at a rate similar to that observed with G-CSF. In a recently reported Phase 2 clinical trial in patients with refractory MDS involving multilineage blood cell dysfunction, treatment with TLK199 resulted in a 65% Hematological Improvement of neutrophils, erythrocytes and/or platelets, confirming the myelorestorative effect of TLK199 in humans. The present study was designed to determine if the myelostimulative effects of TLK199 on neutrophils and their precursors are due in part to increased levels of endogenous G-CSF. TLK199 was tested in normal and neutropenic rodents for its effects on G-CSF production. A single administration of TLK199 (p.o. or i.p.) to normal mice resulted in up to 500% increase in G-CSF serum levels. In a rat model of 5-FU-induced neutropenia, TLK199 administration accelerated the recovery of circulating neutrophils, which corresponded to maximal G-CSF serum levels being observed earlier than vehicle control. The ability of TLK199 to enhance G-CSF production in primary human bone marrow stromal cells was also examined. Treatment of these cells with TLK199 potentiated IL-1b-induced production of G-CSF, GM-CSF and IL-6 from 200 to 400%. These data suggest that the enhancement of G-CSF production may contribute to the accelerated recovery of neutrophils observed with TLK199 treatment under neutropenic conditions. Together with the results from the Phase 2 clinical trial in MDS, these data support further clinical assessment of TLK199 treatment for chemotherapy-induced cytopenia.

Properties of MSC Derived Stromal Cell Lines Developed from Bone Marrow of TNF Knockout Mice.

Mesenchymal stem cells (MSC) forming adherent stromal cell lines have been revealed in suspension fraction (SF) of 70+ weeks old long-term bone marrow culture (LTBMC) of TNF knockout mice.The proportion of CD45+ cells in such old cultures decreased significantly while cellularity of SF increased, accompanied by the first successful generation of cell lines. More than 100 cell lines were established.Almost all cell lines were Sca1 positive, expressed SDF1, had fibroblast-like morphology and expressed collagens type II and IV. Most of cell lines could maintain hematopoiesis (CFU-S, CFU-GM and CAFC production) for 10 weeks. Some of the cell lines were able to maintain growth without differentiation of murine ES cells and some expressed LIF or LIF-receptor, VEGF and FGF1. All cell lines produced hematopoietic growth factors - their supernatants stimulated growth of CFU-GM in semisolid media. The expression of SCF was revealed in all cell lines tested for growth factors production.Quantitative RT-PCR analysis revealed that the expression of hematopoietic growth factors - M-CSF, G-CSF and TPO increased sometimes up to 6 times in comparison with the expression of these factors in adherent cell layer (ACL) of LTBMC. The expression of N-cadherin - one of the main regulators of hematopoietic stem cells (HSC) in niche - increased 7–32 fold in 7 tested cell lines regarding the expression of this RNA in ACL of LTBMC. Whereas the expression level of N-cadherin was lower in wild type bone marrow, NIH3T3 fibroblasts and MS5 cells, maintaining the hematopoiesis and CAFC growth, showed the expression on the higher level than in ACL. The expression of ICAM 1, the molecule also regulating HSC, was reduced in all cell lines. The expression level of genes participating in activating of hematopoietic stem cells in niche such as Jagged, Dll 1 and Ang1 more than halved in 5 out of 7 tested lines. The production of Wnt 5a RNA fluctuated from 0,5 to 12 fold and did not correlate with any other changes in gene expression and the ability of cell lines to maintain hematopoiesis. The level of Ihh, Shh was downregulated in cell lines.The cell line 26dD3 developed in the media containing dexamethasone demonstrated 70 fold increased expression of Kirre - gene supporting HSC but there was no difference in maintaining hematopoiesis and ES cells. High expression of human homolog of this gene (NEPH2) was known to be detected in brain only. At the same time this cell line expressed tubulin b3. These data support a model of lineage specification in which unilineage commitment is prefaced by a “promiscuous” phase of multilineage locus activation. Thus the developed stromal cell lines are not terminally differentiated and use different pathways to maintain hematopoiesis. The TNF deficiency leads to dramatic alterations in regulation of MSC. Mechanisms of this regulation should be scrutinized.

Ex vivo expansion of stem and progenitor cells in co-culture of mobilized peripheral blood CD34+ cells on human endothelium transfected with adenovectors expressing thrombopoietin, c-kit ligand, and Flt-3 ligand.

To optimize conditions for ex vivo expansion of adult hematopoietic stem cells, we evaluated the co-culture of G-CSF mobilized human peripheral blood (PB) CD34(+) cells with endothelial cells engineered to overexpress various hematopoietic growth factors. Immortalized human bone marrow endothelial cells (BMEC) transfected with an expression vector carrying cDNA encoding the human telomerase reverse transcriptase (hTERT) and human umbilical vein endothelial cells (HUVEC) were transfected with combinations of adenovectors expressing murine c-kit ligand (KL), human thrombopoietin (TPO), human Flt3 ligand (FL), and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Ex vivo expansion of PB CD34(+) cells from normal donors and non-Hodgkin lymphoma (NHL) patients in endothelial co-culture was evaluated weekly for total cell production, progenitor (CFU-GM, BFU-E) cell production, and stem cell production as measured by Week-5 Cobblestone Area Forming Cell assay (Wk-5 CAFC). HUVEC transfected with adenovectors expressing TPO, KL, and FL provided the best co-culture system for expanding CD34(+) cells. Maximal total nuclear cell, CFU-GM, and Wk-5 CAFC production occurred between weeks 2 and 3 with 113-fold, 25-fold, and 2.2-5.5-fold expansions, respectively. We did not detect significant differences when GM-CSF was added to the co-culture system. Expansion was also obtained using recombinant human cytokines, but was not maintained beyond 3 weeks. We demonstrated that continuous generation of high levels of TPO, FL, and KL as well as other factors secreted by endothelium provided a clinically relevant co-culture method for ex vivo expansion of stem and progenitor cells from cryopreserved CD34(+) populations.

Combination of interferon alpha with either Ara-C or ATRA in vitro reduces the selective action of interferon against CML CFU-GM.

Although interferon (IFN)-alpha has no specific inhibitory effect on the plating efficiency of granulocyte-macrophage colony-forming cells (CFU-GM) from patients with chronic myeloid leukaemia (CML), it does selectively inhibit the replating ability (secondary colony formation) of CML CFU-GM. Thus, amplification of CFU-GM may be a target for IFN-alpha and other agents used in the treatment of CML. Here we examined whether cytarabine (Ara-C) or all-trans retinoic acid (ATRA) exert similar effects and whether they might in combination with IFN-alpha enhance its efficacy. We found that Ara-C preferentially inhibits the formation of CML CFU-GM compared to normal CFU-GM, but this inhibition was not increased by addition of IFN-alpha. When Ara-C was added to cultures containing IFN-alpha, the inhibition of replating by CML progenitors was abrogated. ATRA increased significantly the plating efficiency of normal CFU-GM. The addition of IFN-alpha to ATRA had no effect on CML or normal colony numbers. However, addition of ATRA to cultures containing IFN-alpha reversed the selective inhibition of CML CFU-GM replating seen in cultures containing IFN-alpha alone. In four IFN-alpha/Ara-C experiments, secondary CML patient-derived colonies were examined by fluorescence in situ hybridisation (FISH). All of them were Ph chromosome positive. No significant effects on CFU-GM production were observed when CML primitive haemopoietic progenitor cells were investigated in a delta (delta) assay. Thus we conclude that combining IFN-alpha with Ara-C or ATRA neutralises the effect of IFN-alpha on CML CFU-GM. This observation provides a rationale for treating patients with alternating courses of IFN-alpha and Ara-C or ATRA, rather than giving either of these two agents in combination with IFN-alpha.

Evaluation of mmp and timp expression in the hematopoietic microenvironment

It is now recognized that the extracellular matrix (ECM) of bone marrow (BM) plays a role not only in supporting BM structure but also in regulating cellular functions. Matrix metalloproteinases (MMPs) and their nature tissue inhibitors (TIMPs) regulate ECM turnover and TIMPs also regulate cell growth. As human long-term bone marrow culture (LTMC) mimics the hematopoietic microenvironment, we used this model to examine the secretion of MMPs and TIMPs. To evaluate the contribution of individual lineages we examined the expression of MMP-2, MMP-9, MT-1 MMP, MT-4 MMP and TIMPs-1, -2 and -3 by hematopoietic cells of erythroid, myeloid and megakaryocytic lineages (expanded in vitro from human normal BM CD34+ cells) and by BM stromal cells (CFU-F, endothelial cells). We found that MMP-9 is expressed and secreted by precursors of all hematopoietic lineages while MMP-2 and MT-1 MMP are expressed mainly by stromal cells and cells of myeloid and megakaryocytic but not erythroid lineages. BM stromal cells expressed and secreted high levels of all the TIMPs tested. This pattern of secretion of MMPs and TIMPs was reflected in the LTMC system: during the first week of LTMC MMP-9 levels were high and then decreased with reduced number of hematopoietic cells while levels of MMP-2, TIMP-1 and TIMP-2 increased with establishment of an adherent layer and expansion of stromal cells. TIMP-3 was released from the ECM matrix by the end of week 8–9 when the production of CFU-GM by adherent and nonadherent layers decreased significantly. Thus we present clear evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment and we are now investigating their role in hematopoiesis.

Effect of Antisense Oligonucleotides on CD34+ Cells from Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by a specific hybrid gene BCR-ABL (formed as a result of t(9;22)). This leads to two possible mRNA usually present in leukemic cells, either B2A2 or B3A2. Targeting these mRNA by antisense oligonucleotides (AS) might offer the opportunity to decrease leukemic growth. We have tested the ability of AS to inhibit the in vitro proliferation of CD34 positive (CD34+) blood cells from 16 patients with newly diagnosed CML. CD34+ cells were isolated by an immunomagnetic technique and incubated for 16 to 18 hours with an 18 mer AS (0.25mM). Sense oligonucleotides served as controls. The effects of AS were evaluated by clonogenic test (production of CFU-GM). Moreover, colonies were picked out and studied by RT-PCR to analyse the presence of BCR-ABL transcript. For nine patients with B3A2 transcript, the median inhibition of CFU-GM formation at day 14 was 64.0 ± 11.2% (68.0 ± 11.4% at day 21) and for the seven patients with a B2A2 transcript: 59.0 ± 11.4% (72.5 ± 12.0% at day 21). AS showed no effect on CD34+ cells from three normal volunteer donor cells. However, for every patient studied, colonies picked out remained BCR-ABL positive with the RT-PCR technique.

Pharmacologic Effects of the Red Blood Cell Substitutes Cross-Linked and Non-Cross-Linked Hemoglobins on Hematopoiesis in Rabbits

The red blood cell substitutes β-β cross-linked (DECA-Hb, XLBV-Hb) and non-cross-linked (HbA) hemoglobins (Hbs), were transfused into rabbits and their effects on hematopoiesis examined. All rabbits receiving DECA-Hb or XLBV-Hb tolerated the Hbs well, whereas 50% of the animals transfused with similar doses of non-cross-linked HbA died. Analysis of peripheral blood and bone marrow BFU-E and CFU-GM production revealed that there was no significant variation in the generation of BFU-E and CFU-GM numbers for each cross-linked Hb transfusion group, but there were significant reductions in the HbA group. In animals transfused with cross-linked Hbs, splenic heme oxygenase (HO) activity was similar to that of controls; liver HO activity was slightly elevated, whereas HO activity was significantly increased in kidneys. Transfusion with non-cross-linked HbA produced greater inductions of HO activity in the liver and kidneys. Hepatic δ-aminolevulinic acid synthase (ALAS) activity was significantly reduced in HbA-transfused animals, whereas transfusion with cross-linked Hbs produced only minor, statistically nonsignificant, reductions in ALAS activity.

Haematopoietic damage persists 1 year after autologous peripheral blood stem cell transplantation.

In the present study we have used cell culture assays in order to assess the damage in the haematopoietic system 1 year after peripheral blood stem cell transplantation (PBSCT), and to establish at what level, haematopoietic progenitor cells (HPC) or stroma, this damage occurs. Thirty-one patients, nine breast cancer (BC), 17 non-Hodgkin lymphoma (NHL) and five Hodgkin disease (HD), who had received autologous PBSCT were included. Forty-eight normal subjects who had given informed consent were used as controls. Results were also compared with a matched group of patients (25 cases) prior to PBSCT. Progenitor cells were analysed using CFU-GM and plastic adherent delta (Pdelta) assays. Long-term bone marrow cultures (LTBMC) in one and two stages were established. One year after transplant both the number of committed progenitor cells and the CFU-GM production in LTBMC were significantly reduced in the three groups of patients when compared with controls (P < 0.05 or P < 0.01). Two-stage LTBMC experiments showed that the impairment in CFU-GM production was due to damage in both patients' stroma and haematopoietic progenitor cells (HPC). All patients, except those with HD, showed a decreased stromal layer confluence (P < 0.05), with significant differences in cell composition as compared to normal bone marrow (P = 0.001). When all these variables were compared with pretransplant results, we observed that stroma formation was significantly lower after PBSCT (P < 0.05), while the number of progenitor cells analysed by the Pdelta assay was significantly increased (P < 0.05). We can conclude that even 1 year after PBSCT, both the committed HPC and BM stroma remain damaged.

Changes in the Functional Capacity of Marrow Stromal Cells after Autologous Bone Marrow Transplantation

Marrow stromal cells were evaluated several months after autologous BMT for their capacity to support both normal hemopoiesis and secrete the main growth factors involved in its control, G-CSF, GM-CSF, IL-3 and SCF. Stromal layers (SL) were obtained by long-term marrow cultures (LTMC) established from 15 patients (9 with hematologic malignancies and 6 with solid tumors) 3 months after autologous BMT and were compared to pre-graft patients. After irradiation, both post-graft and pre-graft SL were recharged with the same inoculum of normal marrow cells. As compared to pre-graft values, CFU-GM production on post-graft SL was significantly increased during the first 2 weeks of culture whereas it was decreased from week 3 to week 8. These findings were only observed in patients with hematologic malignancies and not in patients with solid tumors. Growth factor secretion was evaluated by ELISA in the supernatants of unstimulated and IL-1 -stimulated SL from 10 post-graft patients, 13 pre-graft patients and 5 normal controls. In any group of patients, IL-3 was undetectable either spontaneously or after IL-1-stimulation. As compared to controls, secretion by IL-1 -stimulated SL was not different for GM-CSF in pre-and post-graft patients but tended to be decreased for G-CSF in post-graft patients. SCF secretion, which was not induced by IL-1, appeared dramatically decreased in both pre-and post-graft patients. The capacity of post-graft SL to support CFU-GM growth in LTMC was correlated at week 1 with G-CSF secretion and from week 3 to week 8 with SCF secretion. These results suggest that microenvironment remains qualitatively damaged several months after BMT involving a decreased capacity both to support early hemopoiesis and to secrete SCF, particularly in patients grafted for hemopoietic malignancies.

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors.

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.

Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor.

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte-macrophage colony-forming cells (CFU-GM). Here we have sought a biological explanation for this phenomenon. Most CD34-positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU-GM production by primitive plastic-adherent (Pdelta) cells. Also, Pdelta cells in PBPC harvests, unlike normal marrow Pdelta cells, were insensitive to cell-cycle restraint imposed by contact with marrow-derived stromal cells. We found that Pdelta cells in PBPC collections produce relatively more CFU-GM and relatively fewer BFU-E than Pdelta cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently. neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.

Stromal cells negatively regulate primitive haemopoietic progenitor cell activation via a phosphatidylinositol‐anchored cell adhesion/signalling mechanism

We have tested the effect of stromal cells on the proliferation in long- and short-term cultures of primitive (Thy-1+, CD34+, CD33-, CD38- , HLA-DR , adherent in vitro and quiescent in vivo) progenitors in normal human bone marrow. These primitive cells produce granulocyte-macrophage colony-forming cells (CFU-GM) that are measured in secondary clonogenic assays. Addition of stromal cells to normal adherent haemopoietic progenitor cells reduced CFU-GM production by 80% (P =0.0002) after 1 week of incubation. In long-term culture (LTC), in the presence of stroma. the normal adherent cells did not produce significant numbers of CFU-GM until 3-4 weeks later which suggests that stromal cells reduce the probability of quiescent cell activation. This effect could not be attributed to soluble inhibitory factors and was specific to stroma grown with, rather than without, methylprednisolone. It was blocked by heparanase (H'ase) II treatment of stromal cells, by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of progenitor cells, by antibody blocking of beta1 integrin molecules or by exposure to glucose/N-acetyl-D-glucosamine/alpha-methyl-D-mannoside, but not by exposure to galactose or fructose. Moreover, these interventions enabled the progenitor cells to respond to stimulatory factors in the culture supernatant. We interpret these results as support for a model involving primitive progenitor cell binding to stroma by PI-CAM/HS, beta1 integrin activation via lectin-like interactions and the transduction of signals which reduce the ability of primitive cells to respond to ambient stimulators. This model provides a mechanism for the maintenance of the quiescent state of stem cells by adhesion to stromal cells.

Evaluation of "discordant maturation' in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (CFU-GM).

The 'discordant maturation hypothesis' proposes that the most mature proliferating cells in chronic-phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph-positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (P delta assay for P delta cells) which allows investigation of the kinetics of granulocyte-macrophage progenitor (CFU-GM) production. The frequencies of these primitive (P delta) cells were similar in CML blood (14.5/10(5) mononuclear cells), CML marrow (17.3/10(5)) and normal marrow (11.6/10(5)) The average frequency in normal blood is only 0.58/10(6). The absolute numbers of P delta cells in CML patients are therefore greatly increased. The average numbers of CFU-GM produced by individual P delta cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU-GM produced by individual CML P delta cells was subnormal there was a relative increase in the number of day 7 CFU-GM compared with the number of day 14 and 21 CFU-GM, which agrees with the 'discordant maturation hypothesis'. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU-GM produced by P delta cells or, alternatively, the production of CFU-GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.

Interferon-γ Constitutively Expressed in the Stromal Microenvironment of Human Marrow Cultures Mediates Potent Hematopoietic Inhibition

Clinical and laboratory studies have suggested involvement of interferon-gamma (IFN-gamma) in the pathophysiology of aplastic anemia. T cells from aplastic anemia (AA) patients secrete IFN-gamma in vitro, activated cytotoxic lymphocytes infiltrate aplastic bone marrow (BM), and IFN-gamma mRNA, not detected in normal BM, is present in BM from most AA patients. Many patients respond to immunosuppressive therapy with antithymocyte globulin and cyclosporine. Using long-term BM cultures (LTBMC) as a tissue culture model of hematopoiesis, we show that IFN-gamma is a potent inhibitor in the long-term culture-initiating cell (LTC-IC) assay, the best in vitro surrogate test for human hematopoietic stem cells, as well as of the output of committed progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]). In LTBMC, continuous addition of relatively high IFN-gamma concentrations (1,000 U/mL weekly or 200 U/mL every 2 days) was required for inhibition of secondary colony formation, a measure of LTC-IC number and clonogenicity. To mimick local production of IFN-gamma, human stromal cells were engineered by retroviral-mediated gene transfer to express a transduced IFN-gamma gene. IFN-gamma secreted by stromal cells was far more potent than exogenous IFN-gamma in its effects in the LTC-IC assay. For purified CD34+ cells culture in the presence of IFN-gamma stroma dramatically reduced secondary colony numbers as well as production of CFU-GM and BFU-E. Supernatants from these cultures contained only about 20 U/mL of IFN-gamma; this quantity of cytokine, when added to LTBMC, had little effect on hematopoiesis. The mechanism of hematopoietic suppression was related to the inhibition of cell cycle progression and induction of apoptosis of CD34+ cells. There was no apparent effect of local low-level IFN-gamma production on stromal cell function, as reflected in cell morphology, cell surface phenotype, or expression of hematopoietic growth factor genes. LTBMC with genetically altered stromal cells offers an in vitro model of immune suppression of hematopoiesis in AA and may be helpful in testing certain therapeutic modalities. We infer from our data that local production of low levels of inhibitory cytokine is sufficient to markedly inhibit hematopoiesis and to destroy stem cells and more mature progenitor cells.

Neutralization of stem cell factor in vitro and in vivo: dose-dependent inhibition of stromal cells and induction of granulocyte/monocyte differentiation.

Stem cell factor (SCF), also termed mast cell growth factor or c-kit ligand, plays a central role in the regulation of hematopoiesis and maintenance of viability of hematopoietic cells. We used a new murine monoclonal antibody (MAb) specific for canine SCF to further dissect the role of SCF in vitro and in vivo. This neutralizing MAb, RG7.6 (IgG1), recognizes the soluble form as well as the membrane-bound form of SCF on marrow-derived stromal cells. Treatment of long-term bone marrow cultures (LTMC) with RG7.6 suppressed or stimulated the production of CFU-GM, depending on the MAb concentration and the time of addition to cultures. At concentrations of 0.1-10 micrograms/ml given on the day of recharge of the LTMC, RG7.6 resulted in sustained suppression of CFU-GM grown from nonadherent cells. In contrast, higher doses of RG7.6 (20-100 micrograms/ml) led to a two- to threefold increase in CFU-GM formation from nonadherent cells after 3 days of RG7.6 exposure; after longer RG7.6 exposure there was a rapid decline in the number of CFU-GM. The early increase of CFU-GM was even more distinct when RG7.6 addition to LTMCs was delayed until 1 day before cells were plated for the CFU-GM assay. The early increase of CFU-GMs in the presence of high-dose RG7.6 was mimicked by the addition of granulocyte colony-stimulating factor (G-CSF) to cultures containing suboptimal concentrations of RG7.6, suggesting the possibility that the "positive" response to high-dose RG7.6 was due to an overriding effect of other growth factors, e.g., G-CSF. In stromal cells expressing the membrane-bound form of SCF, the presence of MAb RG7.6, even at low concentrations, interfered with thymidine uptake and proliferation. RG7.6 was also tested in vivo. RG7.6 was given intravenously immediately (days 0-4) after total body irradiation and autologous bone marrow transplantation, and granulocyte counts were followed. The post-irradiation nadir of peripheral blood granulocytes was indistinguishable from controls at low doses of RG7.6 but became more shallow as higher doses of RG7.6 were infused, again suggesting a positive effect on granulocyte differentiation. Thus, the SCF-specific MAb appears to interfere with both stromal and hematopoietic cell function. While only inhibition was observed at lower concentrations, a transient increase in granulocyte production was seen at higher MAb concentrations.

Probability of progenitor renewal (PPR) and production of clonogenic progeny (CFU-GM) by primitive adherent progenitor cells in adult human bone marrow and umbilical cord blood.

Human haemopoietic tissues contain primitive plastic-adherent progenitor cells (P delta cells) that can be detected by measurement of their granulocyte-macrophage colony-forming cell (CFU-GM) progeny. Limiting dilution analysis and Poisson statistics are necessary for determining the frequency of P delta cells because each of them produces several CFU-GM. Limiting dilution also permits measurement of the abilities of individual P delta progenitors to produce CFU-GM. Here we report that the frequencies of P delta progenitors in cord blood and adult marrow are similar (5.6 and 7.8/10(5) mononuclear cells respectively) and individual cord blood P delta progenitors produce fewer CFU-GM than adult P delta progenitors. To test the possibility that the lower production of differentiated progeny by cord blood cells was the result of a higher rate of self-renewal, we devised a two-stage limiting dilution assay relying on the relative production of CFU-GM after two consecutive weeks of incubation. The probability of progenitor renewal (PPR) was derived from the number of wells (progenitors) that produced CFU-GM on both occasions compared with the number that produced CFU-GM on the first occasion only. The total number of CFU-GM produced on the second occasion compared with the number produced on the first occasion provided an index of the overall change in the size of the P delta cell population. The data indicate that P delta cells in cord blood have a higher PPR (0.59) than those in adult marrow (0.36). Also, the relative numbers of CFU-GM produced in the second and first weeks were greater for cord blood (1.2) than for adult marrow (0.36). Therefore P delta cells in cord blood have a greater capacity for self-maintenance and possibly for expansion than P delta cells in adult marrow.

Impaired cytokine production by bone marrow stromal cells of immunodeficient mice.

The aetiology of the bone marrow suppression in HIV-infected patients is unknown. We have demonstrated previously that the ability of bone marrow cells, derived from mice made immunodeficient by infection with the retrovirus LP-BM5, to establish long-term stromal cultures is impaired. In this study we determined the ability of bone marrow stromal cells from these immunodeficient mice to produce cytokines important in haemopoiesis. Neither SCF, IL-3, GM-CSF nor TNF alpha were found in conditioned media of long-term bone marrow cultures (LTBMC) of normal or MAIDS mice. We failed to detect mRNA for TNF or IL-1 alpha, by reverse transcription polymerase chain reaction (RT-PCR), in cultures derived from either normal or immunodeficient mice. Steady-state levels of transcripts for IL-6 were equal in cells from normal and MAIDS mice. Steady-state levels of mRNA for TGF beta 1, a known inhibitor of haemopoiesis, were decreased in cultures derived from MAIDS mice at late stages of infection. The mRNA level of the multipotent haemopoietic regulator stem cell factor was also decreased in MAIDS cultures as compared with normals. Transcripts encoding the transmembrane form of the growth factor were almost absent. Addition of soluble GM-CSF and SCF only transiently increased the production of CFUs (BFUE and CFU-G/M) in MAIDS LTBMC. These findings suggest that derangements in cytokine production in stromal cells of immunodeficient mice may contribute to the suppression of haemopoiesis observed in this disease. One mechanism of HIV-induced depression of haemopoiesis may be via alterations of the haemopoietic microenvironment.