Development Of Haemopoietic Cells Research Articles

The role of mTOR-mediated signals during haemopoiesis and lineage commitment.

The serine/threonine protein kinase mechanistic target of rapamycin (mTOR) has been implicated in the regulation of an array of cellular functions including protein and lipid synthesis, proliferation, cell size and survival. Here, we describe the role of mTOR during haemopoiesis within the context of mTORC1 and mTORC2, the distinct complexes in which it functions. The use of conditional transgenic mouse models specifically targeting individual mTOR signalling components, together with selective inhibitors, have generated a significant body of research emphasising the critical roles played by mTOR, and individual mTOR complexes, in haemopoietic lineage commitment and development. This review will describe the profound role of mTOR in embryogenesis and haemopoiesis, underscoring the importance of mTORC1 at the early stages of haemopoietic cell development, through modulation of stem cell potentiation and self-renewal, and erythroid and B cell lineage commitment. Furthermore, the relatively discrete role of mTORC2 in haemopoiesis will be explored during T cell development and B cell maturation. Collectively, this review aims to highlight the functional diversity of mTOR signalling and underline the importance of this pathway in haemopoiesis.

NF‐κB subunit specificity in hemopoiesis

Although the diverse functions served by the nuclear factor-κB (NF-κB) pathway in virtually all cell types are typically employed to deal with stress responses, NF-κB transcription factors also play key roles in the development of hemopoietic cells. This review focuses on how NF-κB transcription factors control various aspects of thymic T-cell and myeloid cell differentiation that include its roles in hemopoietic precursors, conventional αβ T cells, CD4(+) regulatory T cells, natural killer T cells, γδ T cells, macrophages, and dendritic cells.

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

The reparative properties of bone marrow stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors. We isolated typical human BMSCs by plastic adherence and compared them with BMSC sub-populations isolated by magnetic-activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) or CD271 [p75 low-affinity nerve growth factor receptor (p75LNGFR), p75BMSCs]. Microarray assays of expressed genes, and enzyme-linked immunosorbent assays (ELISAs) of selected growth factors and cytokines secreted under normoxic and hypoxic conditions demonstrated that the three transit-amplifying progenitor cell populations were distinct from one another. CD133BMSC-conditioned medium (CdM) was superior to p75BMSC CdM in protecting neural progenitor cells against cell death during growth factor/nutrient withdrawal. Intracardiac (arterial) administration of concentrated CD133BMSC CdM provided neuroprotection and significantly reduced cortical infarct volumes in mice following cerebral ischemia. In support of the paracrine hypothesis for BMSC action, intra-arterial infusion of CD133BMSC CdM provided significantly greater protection against stroke compared with the effects of CD133BMSC (cell) administration. CdM from CD133BMSCs also provided superior protection against stroke compared with that conferred by CdM from p75BMSCs or typically isolated BMSCs. CD133 identifies a sub-population of nonhematopoietic stem/progenitor cells from adult human bone marrow, and CD133BMSC CdM may provide neuroprotection for patients with stroke.

The role of granulocyte macrophage-colony-stimulating factor in acute intestinal inflammation

An imbalance of mucosal pro- and anti-inflammatory cytokines is crucial in the pathogenesis of inflammatory bowel disease (IBD). GM-CSF influences the development of hemopoietic cells. The precise role of GM-CSF in IBD remains to be elucidated. GM-CSF gene knockout (GM-CSF(-/-)) and wild-type (Wt) mice were challenged with 2.5% dextran sulfate sodium (DSS) for 7 days. The ensued clinical and pathological changes, macrophage infiltration, colonic cytokine production, and bacterial counts were examined. DSS-treated GM-CSF(-/-) mice developed more severe acute colitis than DSS-treated Wt mice, reflected by a greater body weight loss, more rectal bleeding, and aggravated histopathological changes. More infiltrating macrophages were observed in GM-CSF(-/-), compared with Wt mice following DSS challenge, correlating with monocyte chemoattractant protein-1 (MCP-1) production. The levels of colonic IL-17 and TNF-alpha were increased significantly in GM-CSF(-/-) mice, but not in Wt mice, following DSS administration. The level of IL-6 was increased by 1.5- and 2-fold in the colon of GM-CSF(-/-) and Wt mice, respectively, following DSS challenge. No significant changes in IL-4 and IFN-gamma were detected in Wt and GM-CSF(-/-) mice following DSS treatment. The bacteria recovery from colon was increased about 15- and 5-fold, respectively, in Wt mice and GM-CSF(-/-) mice following DSS challenge. These results suggest that GM-CSF(-/-) mice are more susceptible to acute DSS-induced colitis, possibly because of an impaired gut innate immune response as a result of diminished GM-CSF.

You say ITAM and I say ITIM, let's call the whole thing off: the ambiguity of immunoreceptor signalling

Paradigms of protein tyrosine kinase (PTK)-mediated immunoreceptor signalling have developed largely from studies of molecules containing immunoreceptor tyrosine-based activation motifs (ITAM) and immunoreceptor tyrosine-based inhibition motifs (ITIM). In some circumstances, ITAM can mediate inhibition and ITIM can propagate activation signals. In addition to classical immune cells, some ITAM- and ITIM-encoding proteins are more widely expressed and are crucial to the development of haemopoietic cells, like osteoclasts and natural killer cells, as well as tissues such as bone and brain. Closer analysis of activating proteins reveals that some potentially encode ITIM within an ITAM. These 'closet' ITIM could, under conditions of partial ITAM phosphorylation, mediate inhibitory signalling.

Progenitor cells divide symmetrically to generate new colony-forming cells and clonal heterogeneity.

Self-renewal is the most fundamental property of haemopoietic stem and progenitor cells. However, because of the need to produce differentiated cells, not all cell divisions involve self-renewal. We have used a colony replating assay to follow the fates of individual haemopoietic progenitor cell clones. For this, human myeloid colony-forming cells (CFCs) were cultured by standard methodology. Onset of proliferation and growth rates were established by a video recording method. Individual colonies were replated several times to document the rate of clonal extinction, and the numbers of secondary, tertiary and quaternary CFCs. The clonogenic population exhibited similar kinetics in terms of onset of proliferation and growth rate. Clonal extinction was progressive so that only 30 +/- 7% (mean +/- standard error of the mean; n = 4) of the original primary colonies formed quaternary colonies after the third replating step. However, individual primary CFCs that produced colonies throughout the experiment generated, on average, 40 +/- 8 secondary and tertiary CFCs overall. The values obtained in standard culture conditions were modified when granulocyte colony-stimulating factor (G-CSF) or G-CSF plus interleukin 3 were used to stimulate colony growth, showing that the kinetics of colony formation respond to extrinsic regulation. Examination of the replating potential of individual secondary colonies in the clones demonstrated that they generated different numbers of tertiary colonies. The data best fit a stochastic model of haemopoietic cell development where event probabilities can be modified by extracellular factors.

Transcriptional regulation of hemopoiesis

The regulation of blood cell formation, or hemopoiesis, is central to the replenishment of mature effector cells of innate and acquired immune responses. These cells fulfil specific roles in the host defense against invading pathogens, and in the maintenance of homeostasis. The development of hemopoietic cells is under stringent control from extracellular and intracellular stimuli that result in the activation of specific downstream signaling cascades. Ultimately, all signal transduction pathways converge at the level of gene expression where positive and negative modulators of transcription interact to delineate the pattern of gene expression and the overall cellular hemopoietic response. Transcription factors, therefore, represent a nodal point of hemopoietic control through the integration of the various signaling pathways and subsequent modulation of the transcriptional machinery. Transcription factors can act both positively and negatively to regulate the expression of a wide range of hemopoiesis-relevant genes including growth factors and their receptors, other transcription factors, as well as various molecules important for the function of developing cells. The expression of these genes is dependent on the complex interactions between transcription factors, co-regulatory molecules, and specific binding sequences on the DNA. Recent advances in various vertebrate and invertebrate systems emphasize the importance of transcription factors for hemopoiesis control and the evolutionary conservation of several of such mechanisms. In this review we outline some of the key issues frequently identified in studies of the transcriptional regulation of hemopoietic gene expression. In teleosts, we expect that the characterization of several of these transcription factors and their regulatory mechanisms will complement recent advances in a number of fish systems where identification of cytokine and other hemopoiesis-relevant factors are currently under investigation.

The genetic regulation of fucosylated and sialylated antigens on developing myeloid cells.

The first part of this article reviews the stages of normal development of haemopoietic cells committed to the myeloid lineage, properties of leukaemic cell lines that are arrested at specific maturation stages along the granulocytic pathway, the structures of carbohydrate antigenic markers that appear on myeloid cell surfaces, with especial reference to sialyl-Le(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc), and the role of this antigen on mature granulocytes as a ligand for selectin molecules. The families of fucosyl- and sialyltransferase genes encoding enzymes responsible for the biosynthesis of sialyl-Le(x), and the pathways leading to the formation of this antigen, and more complex related structures, are described. The second part of the article outlines the work carried out in the authors' laboratory with leukaemic cell lines in an attempt to ascertain the biochemical and genetic basis of the lowering of sialyl-Le(x) expression that occurs at intermediate stages of normal haemopoietic development. Analysis of enzyme levels and mRNA expression of the fucosyl- and sialyltransferase genes has led to the conclusion that depletion of substrate resulting from high levels of enzyme activity from co-expressed genes FUT4 and ST6Gal1 probably accounts for the dip in expression of sialyl-Le(x), rather than a change in the level of expression of FUT7, the gene in myeloid cells encoding the enzyme ultimately responsible for the synthesis of sialyl-Le(x). The possible significance of this change in relation to normal cell maturation is discussed.

Getting closer to the haemopoietic stem cell utilising internal CD34 expression

CD34 surface molecules has been exhaustively used as surrogate markers for primitive haemopoietic cells, but, increasing immunophenotyping combined with SCID mice / sheep repopulation evidence since 1997 suggests extremely primitive haemopoietic cells exist that do not express external (e)CD34. CD38 is expressed in increasing amounts on (e)CD34+ cells as they mature. Most primitive (≅10%) (e)CD34+ cells, are also characterized by lack of CD38. AC133 (a novel 5-transmembrane glycoprotein), is expressed on early (e)CD34+ subsets. CD34 can be quickly upregulated (1 min) independently of transcription / translation, probably from preformed intracellular stores. Our study investigated internal CD34, determining its significance in haemopoietic hierarchy, via triple flow cytometry examining (i)CD34 & (e)CD34, with one other haemopoietic cell marker CD38 or AC133).Such statistically different staining patterns suggests the CD34 molecule translocates from internal stores to surface during haemopoietic cell development. (i)CD34+/(e)CD34neg cell expression profiles are markedly different to (e)CD34+ cells indicating they are a separate distinct subset. Although (i)CD34 is not a feasible “positive” marked for harvest/selection, it's analysis allows a new haemopoietic developmental stage to be determined. Further immunophenotyping of these (i)CD34+ cells is being performed to elucidate the significance of internal CD34 expression, and its relationship to reversible CD34 expression.

The role of IL-2 in allograft rejection--a lesson learned from experimental work.

The role of IL-2 in allograft rejection--a lesson learned from experimental work.

INTERLEUKIN 3 (IL-3), BUT NOT STEM CELL FACTOR (SCF) INCREASES SELF-RENEWAL BY HUMAN ERYTHROID BURST-FORMING UNITS (BFU-E) IN VITRO

Interleukin 3 (IL-3) and stem cell factor (SCF) are both important regulators of early haemopoietic cell development. Here, we have compared their effects on the kinetics of erythroid burst formation by BFU-E in normal adult bone marrow. We grew the BFU-E in the presence of erythropoietin (Epo) alone, Epo+IL-3 or Epo+SCF and scored the numbers of subcolonies in individual bursts after 14 days. The data were plotted as the cumulative distribution of the numbers of subcolonies per erythroid burst then linearised by logarithmic transformation. Analysis of the data revealed that IL-3 increases the numbers of subcolonies in BFU-E whilst SCF increases the size of the subcolonies themselves. Experiments involving combinations of Epo+IL-3+SCF and the delayed addition of Il-3 or SCF indicated that the actions of IL-3 and SCF are largely independent of one another. We conclude that: (1) Il-3 acts at an earlier stage of erythroid differentiation than SCF, and (2) it may be possible to classify haemopoietic growth factors according to their effects on cell kinetics in vitro.

General Classes and Functions of Four-Helix Bundle Cytokines

The 4α-helical cytokines are the dominant regulators of hemopoietic cell development and the state of activation of the effector cells of innate and acquired resistance to infections. They also play major roles in the regulation of whole body metabolism, physiology, and the response to pregnancy. Finally, some members of the family are also active on cells derived from the neural crest, as well as the central neurons, muscle, and bone. The pleiotropy and apparent redundancy of many of these cytokines raise many issues about the regulation of the production and activity of these cytokines, most of which remain unresolved. They also raise issues regarding how the cell integrates the complex information encoded in its cytokine milieu and responds appropriately. Reductionist approaches have given great insight into the potential biological activities of cytokines and how they may act individually at biochemical and structural levels.

Effect of hepatocyte growth factor on early human haemopoietic cell development.

Hepatocyte growth factor (HGF) stimulates cell proliferation, differentiation and migration by binding to its receptor, MET R. Whether the HGF/MET R axis plays an important regulatory role in human haemopoietic cell growth is an unresolved issue. To investigate this situation, we employed several complementary strategies including RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). We found that very primitive, FACS sorted, CD34+ Kit+ marrow mononuclear cells (MNC) failed to express RT-PCR detectable MET R mRNA. In contrast, MET R expression was easily detectable by RT-PCR in marrow stroma fibroblasts, in cells isolated from BFU-E and CFU-GM colonies, and in unselected normal MNC. Subsequent FACS analysis revealed that MET R protein was detectable on approximately 5% of the latter cells. HGF, at concentrations of 1-50 ng/ml, had no demonstrable effect on survival or cloning efficiency of normal CD34+ MNC in serum-free cultures. Antisense ODN mediated perturbation of MET R mRNA expression in normal CD34+ MNC, with FACS documented decline in protein expression, had no effect on the ability of these cells to give rise to haemopoietic colonies of any lineage. We also examined the biology of HGF/MET R expression in malignant haemopoietic cells. Using the strategies described above, we found that MET R mRNA was expressed in many human haemopoietic cell lines, and that the protein was expressed at high levels on HTLV transformed T lymphocytes. Wild-type CML and AML blast cells also expressed MET mRNA, and HGF was able to co-stimulate CFU-GM colony formation in approximately 20% of cases studied. Therefore, although the HGF/MET R axis appears to be dispensable for normal haemopoietic cell growth, it may play a role in the growth of malignant haemopoietic progenitor cells.

Fibroblast Growth Factors and Early Hemopoietic Cell Development

The role of fibroblast growth factors (FGFs) in the regulation of the human hematopoiesis is controversial. Older publications suggest that FGFs play an important role in regulating the proliferation of human hematopoietic progenitor and stem cells. Such studies should be interpreted with caution because they were typically carried out in the presence of serum which not only contains significant amounts of FGFs, but other cytokines as well. The development of greatly improved techniques for isolating purified populations of primitive hematopoietic cells, for culturing such cells in serum free conditions, and for analyzing the molecular consequences of exposing these cells to FGF cytokines allow the conclusions of these earlier studies to be tested more rigorously. Our investigations, and those of others, suggest that FGFs may not play an important direct role in regulating the development of human hematopoietic stem cells. These data, and our view of their implications, are discussed in this review.

Effect of basic (FGF-2) and acidic (FGF-1) fibroblast growth factors on early haemopoietic cell development.

Basic fibroblast growth factor (FGF-2) and acidic fibroblast growth factor (FGF-1) are mitogens for a variety of cell types. Many reports suggest that haemopoietic cells are among these. Nevertheless, when we examined the effect of recombinant human FGF-1 or 2 on normal human marrow cell proliferation in vitro, only minimal stimulatory activity could be detected. In this regard, the addition of either growth factor to cultures of ancillary cell depleted marrow mononuclear cells (MNC), or to highly enriched CD34+ MNC, failed to enhance haemopoietic colony number and induced only a slight increase in colony size. Perturbation of FGF receptor (FGF-R) expression on CD34+ MNC with antisense (AS) oligodeoxynucleotides (ODN) was also without apparent effect on cell growth. Neither could we demonstrate any effect of FGF-1 or 2 on survival of early progenitor cells in serum-free culture. To explain these findings, we examined progenitor cells for expression of the FGF-R at the mRNA and protein level using RT-PCR and flow cytometry. Primitive CD34+/KIT+ MNC had no detectable FGF-R (FGF-R1, 2, 3 or 4) mRNA or protein expression. In fact, direct immunofluorescence labelling of MNC for CD34 antigen and FGF-R1 demonstrated that expression of these markers was mutually exclusive in the populations examined. FGF-R1 expression was detected on subpopulations of MNC and on cells derived from day-6 CFU-GM and BFU-E colonies. Accordingly, FGF-R1 is either absent, or present at very low levels, on primitive haemopoietic cells. This fact, combined with our in vitro culture data, suggest that receptors are unlikely to play a significant role in the development of these early cells. Nevertheless, the development of mature cells may be influenced by the FGFs since the FGF-Rs are expressed on more mature cells.

The role of stromal cell heparan sulphate in regulating haemopoiesis.

Intimate contact between haemopoietic progenitor cells and elements of the bone marrow stroma is required for progenitor cell proliferation and differentiation. It is believed that the stroma provides particular niches for the development of haemopoietic cells of different lineages. Cytokines, stromal cell surface molecules and molecules of the stromal extracellular matrix all contribute to defining these microenvironmental niches. Data obtained using an in vitro model of haemopoiesis support the view that progenitor cell adhesion to stroma is mediated by multiple receptor-ligand interactions. The possibility of a tethering step, mediated by the engagement of stromal cell heparan sulphate with its ligands on the progenitor cells, preceding stable cell adhesion is discussed. The role of stromal cell heparan sulphate is likely to include cytokine presentation to progenitors as well as the tethering of progenitors to stroma. It is proposed that intracellular signals induced by progenitor cell adhesion to stroma act in association with cytokine induced signals to regulate progenitor cell proliferation and differentiation.

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.

Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C.

The Kit/stem cell factor receptor (Kit/SCF-R) is a transmembrane tyrosine kinase receptor of importance for the normal development of hemopoietic cells, melanoblasts, and germ cells. We recently reported that protein kinase C (PKC) is involved in a negative feedback loop regulating the Kit/SCF-R by direct phosphorylation on serine residues in the receptor. Inhibition of PKC led to increased SCF-induced tyrosine kinase activity and mitogenicity, but PKC was necessary for SCF-induced motility. In this report we have further examined the modulatory role of PKC on SCF-induced signaling. The ligand-activated Kit/SCF-R associated weakly with GRB2 and induced only little tyrosine phosphorylation of phospholipase C-gamma in porcine aortic endothelial cells transfected with Kit/SCF-R. In contrast, the SCF-stimulated Kit/SCF-R associated efficiently with, and induced tyrosine phosphorylation of, the p85 alpha regulatory subunit of phosphatidyl inositide-3'-kinase (PI-3'-kinase). Both receptor association and tyrosine phosphorylation of p85 alpha were increased after inhibition of PKC, while its serine phosphorylation was decreased. Concomitantly, the specific activity of receptor-associated PI-3'-kinase activity was increased. Inhibition of PI-3'-kinase with wortmannin inhibited SCF-induced mitogenicity. SCF-induced phosphorylation of Raf-1 and activation of ERK2 still occurred after PKC inhibition but was not increased. In conclusion, SCF-induced PI-3'-kinase activation paralleled the increased SCF-induced mitogenicity after inhibition of PKC.

The molecular control of haemopoiesis and leukaemia

The establishment of a cell culture system for the clonal development of haemopoietic cells made it possible to discover the proteins that regulate cell viability, growth and differentiation of different haemopoietic cell lineages and the molecular basis of normal and abnormal development in blood-forming tissues. These regulators include cytokines now called colony stimulating factors (CSFs) and interleukins (ILs). Different cytokines can induce cell viability, multiplication and differentiation, and haemopoiesis is controlled by a network of cytokine interactions. The multigene network includes positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor β and tumour necrosis factor. The cytokine network which has arisen during evolution allows considerable flexibility depending on which part of the network is activated and the ready amplification of response to a particular stimulus. The CSFs and ILs induce cell viability by inhibiting programmed cell death (apoptosis). Programmed cell death is also regulated by the genes wild-type and mutant p 53, c-myc and bcl-2, and suppression or induction of this programme can result in tumour promotion or tumour suppression. Cytokines that regulate normal haemopoiesis can control the abnormal growth of certain types of leukaemic cells and suppress malignancy by inducing differentiation. Genetic abnormalities that give rise to malignancy in these leukaemic cells can be by-passed and their effects nullified by inducing differentiation and programmed cell death. The haemopoietic cytokines discovered in culture are active in vivo and are being used clinically to correct defects in haemopoiesis.

Stochastic effects in hemopoiesis.

Stochastic effects underlie all hemopoietic cell responses but can only be observed at the level of one or a very few cells. Here we have considered the relevance of stochastic effects to aspects of hemopoietic cell development.