Pluripotent Hemopoietic Progenitors Research Articles

The Mll partial tandem duplication: differential, tissue-specific activity in the presence or absence of the wild-type allele

AbstractThe partial tandem duplication of MLL (MLL-PTD) is found in 5% to 10% of patients with acute myeloid leukemia (AML) and normal cytogenetics. Its expression in leukemic blasts is coincident with a silenced wild-type (WT) MLL allele. We therefore generated mice expressing the Mll-PTD in the absence of Mll-WT. These MllPTD/− mice die at birth unlike the normal life expectancy of MllPTD/WT, MllWT/−, and MllWT/WT mice. Using MllWT/WT fetal liver cells (FLC) as baseline, we compared MllPTD/− with MllPTD/WT FLC and found both had increased HoxA gene expression and granulocyte-macrophage colony-forming progenitor cells (CFU-GM); in contrast, only MllPTD/WT FLC had increased pluripotent hemopoietic progenitors (CFU-GEMM). The similarities between MllPTD/WT and MllPTD/− mice suggest that the Mll-PTD mutation can up-regulate target genes in a dominant, gain-of-function fashion. The differences between these 2 genotypes suggest that in select tissues the Mll-PTD requires cooperation with the Mll-WT in the genesis of the observed abnormality.

Evidence for Distinct Intracellular Signaling Pathways in CD34+ Progenitor to Dendritic Cell Differentiation from a Human Cell Line Model

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

Activation of Human Mast Cells through Stem Cell Factor Receptor (KIT) Is Associated with Expression of bcl-2

Background: Mast cells (MCs) are multifunctional effector cells of the immune system. These cells originate from pluripotent hemopoietic progenitors. In contrast to basophils and other leukocytes, MCs exhibit a remarkably long life span (years) in vivo. Although a role for stem cell factor (SCF) and SCF receptor (KIT) in long-term survival of MCs has been proposed, the underlying biochemical mechanisms remain unknown. Materials and Methods:We have examined expression of ‘survival-related’ molecules of the bcl-2 family including bcl-2 and bcl-x_L, in primary human MCs and the human MC line HMC-1. Primary MCs were isolated from dispersed lung tissue by cell sorting using an antibody against KIT. mRNA expression was analyzed by RT-PCR and Northern blotting. Results: As assessed by RT-PCR, purified unstimulated lung MCs (>98% pure) exhibited KIT- and bcl-x_L mRNA, but did not express bcl-2 mRNA. However, exposure of lung MCS to SCF (100 ng/ml) for 8 h resulted in expression of bcl-2 mRNA. Corresponding results were obtained by immunocytochemistry. In fact, exposure of MC to SCF resulted in expression of the bcl-2 protein whereas unstimulated MCs displayed only the bcl-x_L protein without expressing the bcl-2 protein. The human MC leukemia cell line HMC-1, which contains a mutated and intrinsically activated SCF receptor, showed constitutive expression of both bcl-2 and bcl-x_L at the mRNA and protein level. Conclusion: Our data show that human MCs can express members of the bcl-2 family. It is hypothesized that bcl-x_L plays a role in KIT-independent growth of MCs, whereas bcl-2 may be involved in KIT-dependent functions of MCs.

Evidence for Distinct Intracellular Signaling Pathways in CD34+ Progenitor to Dendritic Cell Differentiation from a Human Cell Line Model

Abstract Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-α) CD34+ HPC→DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC→DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-α, or PMA (with or without the calcium ionophore ionomycin, or TNF-α), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14− surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms α, βI, βII, and μ; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

Phorbol Esters Induce Differentiation of Human CD34+ Hemopoietic Progenitors to Dendritic Cells: Evidence for Protein Kinase C-Mediated Signaling

The intracellular signals that mediate the differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have found that the phorbol ester PMA by itself induced 47% +/- 8.7% of input human CD34+ hemopoietic progenitors to differentiate into cells with morphology and surface Ag phenotype characteristic of DC by day 7 of culture. Functionally, PMA-generated DC processed and presented whole soluble Ag and also induced resting T cell proliferation and Ag-specific CTL effector function. Unlike cytokine-driven DC differentiation, PMA suppressed proliferation and induced cell death (in part via apoptosis) in cells that did not differentiate to DC. The effects of PMA were blocked by inhibitors of protein kinase C activation, suggesting a central role for this signaling molecule. PMA-mediated signaling also induced expression of the RelB transcription factor, an NF-kappaB family member implicated in DC differentiation. These findings suggest that phorbol esters activate protein kinase C, which then initiates the terminal component of an intracellular signaling pathway(s) involved in the DC differentiation of CD34+ hemopoietic progenitors.

Hemopoietic stem cell proliferation in Belgrade rats: to complete the parable.

The Belgrade laboratory (b/b) rat is a mutant animal suffering of a severe anemia, resulting from a severe intracellular iron deficiency. This deficiency is determined by an impaired intracellular transport of iron due to a block located between the endocytic vesicles and the cytosolic iron transport to mitochondria. This results in a complex series of disturbances affecting hemopoietic stem and progenitor cells, as well as growth factor production. The gene affected by the "b" mutation is unknown. This review summarizes the current knowledge on b/b rat hemopoiesis, with the focus on pluripotent hemopoietic progenitors, such as Spleen Colony Forming Units scored at day 8 after transplantation (CFU-Sd8) and more primitive stem cells (pre-CFU-S) responsible for Marrow Repopulating Ability. A special effort was made to describe the effects of b/b mutation on hemopoietic stem cells distinguishing between the alterations connected to the primary defect (intracellular iron deficiency), and those secondary to severe anemia (severe chronic hypoxia). The intracellular iron deficiency is responsible for a reversible cytostatic effect on CFU-Sd8, while the overcoming of this proliferative block reveals that the size of bone marrow CFU-Sd8 population is limited by the level of oxygenation. The size of pre-CFU-S population is also limited by the level of oxygenation, implying that, in contrast to the normal situation, in b/b rats pre-CFU-S are actively proliferating and therefore sensitive to hypoxia. This review also explains how to get more reliable data on the effects of intracellular iron deficiency and hypoxia on the proliferation of hemopoietic stem and progenitor cells and points to the potential importance of b/b rat phenomenon for fundamental research in cell biology and in the search of new ways to restrain the proliferation of malignant cells.

Assessment and characterization of hemopoietic stem cells.

The adequate production of blood cells is sustained by pluripotent hemopoietic progenitors whose behavior is influenced by a permissive hemopoietic microenvironment. Hemopoietic progenitors have in common the expression of CD34 surface molecules, but are heterogeneous with respect to other properties. It is commonly accepted that primitive progenitors, considered to be candidates for marrow repopulating cells, are HLA-DR-, without expressing lineage-specific determinants. They are usually quiescent with respect to their cell cycle status. In addition to CD34, they may display adhesion molecules on their surface and express receptors for ligands such as c-kit, FLT2/FLK3 and various other cytokines. Some of these are expressed constitutively, while others emerge as the cells progress through their regular maturation program. This process appears to include a gradual reduction of their proliferative capabilities as demonstrated by a progressive loss of the length of their telomeric structures.

Effects of Prolonged Nitrous Oxide Exposure on Hemopoietic Stem Cells in Splenectomized Mice

We have demonstrated in previous papers that prolonged nitrous oxide exposure suppresses murine hemopoiesis more in the spleen than in the bone marrow and that this is caused by the suppression of the hemopoietic supportive activity of the microenvironment. In the present study, we used splenectomized mice as an experimental model to investigate the direct effect on bone marrow function of prolonged nitrous oxide inhalation. All of the experimental mice were splenectomized at the age of 4 wk. Half of the experimental mice were continuously exposed to 50% nitrous oxide, and the remainder were continuously exposed to air as controls, starting 3 wk after splenectomy and lasting 14 days, and the numbers of pluripotent hemopoietic stem cells (CFU-S) and granulocyte-macrophage progenitor cells (GM-CFC) in bone marrow were counted. The numbers of pluripotent hemopoietic stem cells and granulocyte-macrophage progenitor cells in the bone marrow of mice exposed to air showed no significant change. The numbers of these two types of cells found in nitrous oxide-exposed mice were approximately 60% of control levels. These data are almost the same as our previously reported bone marrow data obtained in nonsplenectomized mice exposed to nitrous oxide for 14 days. The present results suggest that the marked decrease in the number of splenic hemopoietic stem cells in our previous data is not a result of migration of the cells to bone marrow, and that nitrous oxide directly affects murine bone marrow hemopoiesis.

Scanning electron microscopic observation of CFU-Mix of normal adults and leukemic patients including two cases of myeloid-lymphoid bipotent leukemia

We observed CFU-Mix of 4 normal adults and 11 cases of leukemia. The culture system used by us in this study represented a suitable model for studying differentiation of hemopoietic stem cells in vitro. The pathogenesis of myeloid-lymphoid bipotent leukemia were malignant changes in pluripotent hemopoietic progenitor cells. The leukemic clones showed proliferation, but did not mature in culture. It is suggested that differentiation of leukemic stem cell did not pass through CFU-Mix stage.

Studies on human CFU-Mix microvolume culture by use of limiting dilution assay

By use of limiting dilution assay we investigated the bipotent and pluripotent hemopoietic progenitor cells (CFU-Mix) from 21 normal adults, cultured for 32 times. The CFU-Mix counts were 35.7 colonies/10(6) bone marrow cells. According to different cellular elements they could be divided into 5 groups, namely GEMM, GE, GL, GMeg and GM phi. It was suggested that differentiation of the stem cell is of stochastic process, influenced by different hemopoietic growth factors and cellular microenvironment. Our model seems to be suitable for studying differentiation of the stem cells. The rates of 3H-TdR and 55 + 59Fe incorporation were determined, and the 2nd day of culture was found to be the delayed stage of cell growth.

Deficiency of pluripotent hemopoietic progenitor cells in myelodysplastic syndromes.

Pluripotent (CFU-MIX), erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) progenitor cells were examined in bone marrow (BM) from 23 patients with myelodysplastic syndromes (MDS). Patients were grouped according to the FAB classification: Refractory anemia (RA), n = 3; RA with ring sideroblasts (RARS), n = 3; RA with excess of blasts (RAEB), n = 8; RA with excess of blasts in transformation (RAEBt), n = 7; chronic myelomonocytic leukemia (CMML), n = 2. In FAB groups RA, RARS, RAEB and RAEBt CFU-GM concentrations were normal or decreased but both CMML-patients had increased CFU-GM values. Abnormal cluster growth was observed in 9 of 23 MDS-patients. BFU-E colony formation was subnormal in all cases. Mixed-colony assay values were at the lower limit of controls in one patient and decreased in the remaining 22 MDS-patients. A similar growth pattern of hemopoietic progenitor cells was observed in 19 patients with acute nonlymphocytic leukemia (ANLL), who were studied for comparison. These data suggest a quantitative or qualitative/functional defect of the pluripotent progenitor cell compartment as the major cause for the cytopenia in MDS-patients.

Increased lethality and delay in the recovery of hemopoietic stem cells after irradiation in mice exposed to nitrous oxide

Effects of N2O-exposure on the survival rate and growth kinetics of hemopoietic stem cells after irradiation were investigated. LD 50, 30 days after irradiation, was 300 rad for N2O exposed mice and over 550 rad for control mice. Recovery of the pluripotent hemopoietic stem cells (CFU-S) and granulocyte-macrophage progenitor cells (GM-CFC) was significantly delayed in the spleen of N2O-exposed mice after 150 rad irradiation, compared to that of control mice. However, in bone marrow, there was a significant but slight difference in the recovery of GM-CFC and no significant difference in the recovery of CFU-S between the two groups. These results suggest that N2O augments the damage of splenic hemopoiesis in irradiated mice, and this may be responsible for the increased hemopoietic death.

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia.

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.

Myeloid progenitor cells in the peripheral blood of patients with hairy cell leukemia and other “leukemic” lymphoproliferative disorders

We assayed granulocyte-macrophage committed progenitor cells (CFU-GM), erythroid committed progenitor cells (BFU-E) and pluripotent hemopoietic progenitor cells (CFU-MIX) in the peripheral blood of patients with hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL). In 8 HCL patients retaining their spleens, the number of circulating CFU-GM, BFU-E and CFU-MIX were under the lower limits of normal controls in 6, 6 and 5 cases, respectively, and were in the lower normal ranges in the remaining cases. Six splenectomized HCL patients had generally more circulating progenitor cells than their nonsplenectomized counterparts. In the peripheral blood of 2 patients with ALL and 3 patients with CLL, progenitor cells of all types were markedly increased compared to their respective values in the blood of control subjects. Hairy cells from 2 HCL patients failed to inhibit CFU-GM, BFU-E and CFU-MIX derived colony growth from control peripheral blood mononuclear cells. In 3 HCL patients previously low circulating progenitor cells did not rise 5–7 months after RC-α 2-IFN treatment despite normalization of peripheral blood counts. Our results suggest that a reduction of the committed and pluripotent progenitor cell compartment might be at least in part responsible for the pancytopenia in the majority of patients with HCL.

Studies of Sub‐human Primate (Marmoset) Pluripotent Hemopoietic Stem Cells (CFU‐GEMM) In Vitro

Pluripotent hemopoietic progenitor cells (CFU-GEMM) grow in vitro from marmoset bone marrow using a modified human CFU-GEMM assay. Characteristics of growth are similar to those reported in the human CFU-GEMM assay. The number of CFU-GEMM/10(5) marrow cells from marmoset bone marrow is approximately four times that grown in human marrow in our laboratory. Data concerning EPO and other requirements for growth of CFU-GEMM demonstrate an assay for the pluripotent hemopoietic progenitor in the marmoset. This assay may be useful in designing preclinical primate bone marrow transplant experiments.

Two classes of primitive pluripotent hemopoietic progenitor cells: separation by adherence.

Methylcellulose cultures containing mouse marrow cells at low densities and partially purified preparations of erythropoietin and Interleukin-3 were scored after 2 weeks for the presence of macroscopic multilineage colonies (from "primary" CFU-macro GEMM). Whole cultures were then harvested and replanted to assess the number of "secondary" CFU-macro GEMM produced, but not detected, during the primary culture period. In such experiments adherent marrow cells yielded significantly higher numbers of secondary CFU-macro GEMM than did either fresh or nonadherent marrow cells. Removal of macroscopic colonies prior to replating showed that most secondary CFU-macro GEMM were not derived from primary CFU-macro GEMM. In vivo studies also revealed a differential effect of adherence separation on the frequency of day 10 CFU-S, which decreased, by comparison to cells capable of long-term repopulation, which increased. Primary adherent CFU-macro GEMM from 5-fluorouracil (5-FU) treated mice showed an 18-fold higher self-renewal capacity than their counterparts in normal marrow. Nevertheless the majority of secondary CFU-macro GEMM obtained from primary cultures of adherent 5-FU cells were again not derived from primary CFU-macro GEMM. Cells capable of immediately generating large multilineage colonies thus appear to represent an intermediate compartment of pluripotent progenitors whose self-renewal properties, may, however, vary over a considerable range. Our results further suggest that these progenitors are derived ultimately from a more primitive adherent cell whose tendency to begin to divide in vitro is low and whose presence correlates with cells capable of long-term myeloid repopulation in vivo.

The effect of the leukemic cell line HL60 and acute myeloblastic leukemic cells before and after induction of differentiation on normal pluripotent hematopoietic progenitors (CFU-GEMM)

A leukemic cell line (HL60) and acute myeloblastic leukemia (AML) cells from six patients were co-cultured with normal marrow cells to assess their effects on growth of normal CFU-GEMM. The effects of the following inducers: 12- O-tetradecanoyl-phorbol-13-acetate (TPA), retinoic acid (RA), dimethylsulphoxide (DMSO), 1–25 (OH) D, (Vitamin D 3) and PHA-LCM on both the HL60 and AML cells, were studied. Inhibition of growth of normal CFU-GEMM was observed in the co-cultures in the presence of 1 × 10 4 HL60 or AML leukemic cells/ml. This inhibition was reversed by pretreating the HL60 line with vitamin D 3, TPA and RA. No effect on growth of CFU-GEMM was noted when DMSO and PHA-LCM were used. AML cells were morphologically induced to differentiate by TPA or RA in all six cases. In three cases, reversal of inhibition of growth of normal pluripotent hemopoietic progenitors occurred and in three the inhibition of growth persisted. Regulation of inhibition by different inducers did not seem to correlate in all cases with morphological differentiation.

Flexible association of hemopoietic differentiation programs in multilineage colonies.

Pluripotent hemopoietic progenitors lose potentialities during the process of differentiation. We have examined events that lead to lineage restriction by determining the cellular composition of 785 multilineage colonies grown from peripheral blood samples of glucose-6-phosphate-dehydrogenase (G-6-PD) heterozygous volunteers. Of these colonies, 762 contained only one isoenzyme type and were considered to be of clonal origin. A considerable heterogeneity was observed. Some colonies were composed of cells belonging to two different lineages, while other colonies contained three or more different cell types. A small number of colonies consisted--in addition to myeloid cells--of T-lymphocytes. The variable association within individual colonies of members belonging to different hemopoietic lineages suggests a flexible determination and expression of differentiation programs by early progenitors.

Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.

Enrichment of Pluripotent Hemopoietic Progenitor Cells From Human Bone Marrow

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.