Clare M. Heyworth

The Role of Hemopoietic Growth Factors in Self-Renewal and Differentiation of IL-3-Dependent Multipotential Stem Cells

A multipotent hemopoietic cell line has been employed to assess the influence of the hemopoietic growth factors, IL-3, GM-CSF, G-CSF, and CSF-1 on the processes of self-renewal, the generation of lineage restricted progenitor cells and the production of mature neutrophils and macrophages. At a high concentration of IL-3, the cells undergo self-renewal and demonstrate little or no ability to undergo differentiation in the presence of the other growth factors. In the absence of IL-3, the cells show minimal (GM-CSF) or no (G-CSF or CSF-1) ability to respond to these other growth factors. When combined with a low concentration of IL-3, the ability of the cells to respond to GM-CSF, G-CSF, and CSF-1 is enhanced and a selective preference for the neutrophil or macrophage lineage is seen depending on the combination used, i.e., the presence of CSF-1 preferentially promotes macrophage development and G-CSF preferentially promotes neutrophil development. Conditions optimal for neutrophil development were seen using a combination of low IL-3 concentrations plus GM-CSF plus G-CSF. In such conditions, the cells undergo extensive proliferation and progressively lose their clonogenic potential (i.e., differentiation much greater than self-renewal) and acquire the biochemical markers characteristic of fully mature phagocytes.

Molecular signatures of self-renewal, differentiation, and lineage choice in multipotential hemopoietic progenitor cells in vitro.

ABSTRACT The molecular mechanisms governing self-renewal, differentiation, and lineage specification remain unknown. Transcriptional profiling is likely to provide insight into these processes but, as yet, has been confined to “static” molecular profiles of stem and progenitors cells. We now provide a comprehensive, statistically robust, and “dynamic” analysis of multipotent hemopoietic progenitor cells undergoing self-renewal in response to interleukin-3 (IL-3) and multilineage differentiation in response to lineage-affiliated cytokines. Cells undergoing IL-3-dependent proliferative self-renewal displayed striking complexity, including expression of genes associated with different lineage programs, suggesting a highly responsive compartment poised to rapidly execute intrinsically or extrinsically initiated cell fate decisions. A remarkable general feature of early differentiation was a resolution of complexity through the downregulation of gene expression. Although effector genes characteristic of mature cells were upregulated late, coincident with morphological changes, lineage-specific changes in gene expression were observed prior to this, identifying genes which may provide early harbingers of unilineage commitment. Of particular interest were genes that displayed differential behavior irrespective of the lineage elaborated, many of which were rapidly downregulated within 4 to 8 h after exposure to a differentiation cue. These are likely to include genes important in self-renewal, the maintenance of multipotentiality, or the negative regulation of differentiation per se.

The phorbol ester, TPA inhibits glucagon‐stimulated adenylate cyclase activity

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour‐promoting phorbol ester TPA (12‐O‐tetradecanoyl phorbol‐13‐acetate). The half‐maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon‐stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 μg/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon‐stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

Transforming growth factor-β1 (TGF-β1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-β1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-β1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-β1 induced apoptosis. The proapoptotic effects of TGF-β1 occur via a p53-independent pathway, since p53null FDCP-Mix demonstrated the same responses to TGF-β1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-xL expression. In FDCP-Mix cells, neither IL-3 nor TGF-β1 induced any change in Bcl-xL protein levels or the proapoptotic proteins Bad or Bax. However, TGF-β1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-β1-induced apoptosis but was incapable of inhibiting TGF-β1-mediated growth arrest. We conclude that TGF-β1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-xL. The significance of these results for stem cell survival in bone marrow are discussed.

AC133+ G0 Cells from Cord Blood Show a High Incidence of Long‐Term Culture–Initiating Cells and a Capacity for More Than 100 Million‐Fold Amplification of Colony‐Forming Cells In Vitro

AC133+ cells may provide an alternative to CD34+ cells as a target for cell expansion and gene therapy protocols. We examined the differences in proliferative potential between cord blood selected for AC133 or CD34 in serum‐free, stroma cell–free culture for up to 30 weeks. Because most hemopoietic stem cells reside within the G0/G1 phase of the cell cycle, we combined enrichment according to AC133 or CD34 expression with G0 position in the cell cycle to identify populations enriched for putative stem cells. Our results show that AC133+ G0 cells demonstrated a long‐term culture‐initiating cell incidence of 1 in 4.2 cells, had a colony‐forming cell incidence of 1 in 2.8 cells, were capable of producing 660 million‐fold expansion of nucleated cells and 120 million‐fold expansion of colony‐forming units–granulocyte‐macrophage over a period of 30 weeks, and were consistently superior to CD34+ G0 cells according to these parameters. Furthermore, we have shown that AC133+CD34− cells have the ability to generate CD34+ cells in culture, which suggests that at least some AC133+ cells are ancestral to CD34+ cells. We conclude that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34 and that, in combination with isolation according to G0 phase of the cell cycle, AC133 isolation identifies a highly enriched population of putative stem cells.

Erythroid development of the FDCP-Mix A4 multipotent cell line is governed by the relative concentrations of erythropoietin and interleukin 3

Summary .Conditions are described which promote the erythroid development of the FDCP‐Mix A4 (A4) cell line with accompanying proliferation of the cells. The requirements for this development are low concentrations of interleukin 3 (IL‐3) plus the presence of erythropoietin (epo) and haemin. When high concentrations of IL‐3 are added with erythropoietin and haemin the cells do not differentiate and maintain their blast cell morphology. Addition of haemin, in the absence of erythropoietin, does not promote erythroid development, but the presence of haemin with erythropoietin promotes increased proliferation and maturation. The morphological maturation of A4 cells along the erythroid lineage is accompanied by a gradual loss of clonogenic potential, loss of A4 cell multipotency, increased erythropoietin receptor expression, and an increased expression of the β‐globin gene. An initial increase in mitogenic responsiveness to erythropoietin is followed by a decrease as the cells become refractory to all mitogenic stimuli with the acquisition of a postmitotic, mature erythroid cell phenotype.

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity.

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity. Over the concentration range that ethanol activates adenylate cyclase, it also increases bilayer fluidity as indicated by a decrease in the values of the order parameters for an incorporated fatty acid spin probe. At high concentrations forskolin does increase bilayer fluidity. However, it only begins to do so at concentrations above those where forskolin has already exerted its maximal effect in activating adenylate cyclase. Forskolin can still activate, albeit to a reduced extent, detergent-solubilized adenylate cyclase whereas ethanol cannot. Forskolin elicits a pronounced rise in hepatocyte intracellular cyclic AMP concentrations, whereas ethanol does not. Both forskolin and ethanol reduce the temperature of onset of the lipid phase separation occurring in rat liver plasma membranes. This is detected in Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and order parameters of an incorporated fatty acid spin probe, where we find that forskolin is particularly potent in decreasing the temperature at which this lipid phase separation occurs. Our results are consistent with the notion that forskolin exerts its effect on adenylate cyclase primarily by a direct action on the catalytic unit of the enzyme. However, as forskolin is a potent perturber of the organisation of the lipid bilayer it is possible that this could modulate its effect on adenylate cyclase and might be expected to affect the activity of other membrane enzymes.

Insulin: in search of a mechanism

Several properties of Ln 3+ suggest a potential therapeutic role in conditions involving inflammation and excessive breakdown of connective tissue. These properties include the inhibition of col- lagenase, the stabilization of collagen fibrils, and the inhibition of lymphocyte activation, stimulus-mediated cell secre- tion and neutrophil chemotaxis and aggregation. Arthritis is well suited for further investigation of this potential, as Ln 3+ can be introduced by local intra- articular injection, thereby obviating the problems of toxicity when administered by the intravenous route, and poor oral absorption. Furthermore, Ln 3+ bind tightly to cartilage, thus retaining the agent in the desired location and minimizing pos- sible systemic complications. Preliminary results from experimental arthritis in rabbits are encouraging. References A brief reference list is a requirement for

Biological effects of stroma-derived factor-1α on normal and CML CD34+ haemopoietic cells

We compared the biological effects of the CXC chemokine SDF-1α on immunomagnetically purified CD34+ cells isolated from human normal bone marrow (NBM), leukapheresis products (LP) and patients with chronic myeloid leukaemia (CML). LP CD34+ cells showed a significantly stronger migration response to SDF-1α (100 ng/ml) than CD34+ cells isolated from the peripheral blood (PB) of CML patients (P < 0.05). the chemotactic response to sdf-1α was also reduced in cml bm cd34+ cells in comparison to NBM CD34+ cells but the observed differences were not statistically significant. In analogy to normal CD34+cells circulating CML PB CD34+ cells were less responsive to SDF-1α than their BM counterparts (P < 0.05). furthermore, sdf-1α elicited similar concentration-dependent growth suppressive effects on normal and cml cd34+cells (P > 0.05) in colony-forming cell assays. We then demonstrated that SDF-1α triggers intracellular calcium increases in CD34+ cells and there were no differences in the time course and dose response characteristics of normal and CML CD34+ cells. The reduced migration response to SDF-1α in CML CD34+ cells was not due to a down-regulation of the SDF-1α receptor CXCR-4 as flow cytometric analysis revealed similar CXCR-4 expression levels on NBM, LP, CML PB and CML BM CD34+cells (P > 0.05). Finally, no differences in the modulation of CXCR-4 levels in response to SDF-1α and serum were observed in CML and normal CD34+ cells. Our data suggest that the impaired chemotactic response of CML CD34+ cells to SDF-1α is not caused by a lack or complete uncoupling of CXCR-4, but may be due to an intracellular signalling defect downstream of the receptor. Leukemia (2000) 14, 1652–1660.

Cord Blood G0 CD34+ Cells Have A Thousand‐Fold Higher Capacity For Generating Progenitors In Vitro Than G1 CD34+ Cells

We examined the functional differences between G0 and G1 cord blood CD34+ cells for up to 24 weeks in serum‐free suspension culture, containing Flt‐3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000‐fold difference in granulocyte, macrophage‐colony‐forming cells (GM‐CFC) cumulative production between the two populations, with cultures initiated from G0 demonstrating an amplification of 1.1 × 105‐1.8 × 106 of GM‐CFC compared to 45‐2.7 × 103 for the G1 cells. Cells from the initial G0 population are able to produce about 250‐fold higher numbers of BFU‐E than those from G1 which translates to 3 × 103‐1.1 × 105‐fold expansion and 25‐390‐fold expansion for G0 and G1, respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G0 population are CD34+, resulting in a 600‐fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G0 and G1 cultures with a mean fold expansion of 2 × 107 at 24 weeks.