Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability- promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum- supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca- 1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.     

The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.     

Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro

Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU- Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.     

FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units

The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony- stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell- depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU-GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth- promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages.     

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL- 3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13- 7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G- CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.     

Transforming growth factor-beta potently inhibits the viability- promoting activity of stem cell factor and other cytokines and induces apoptosis of primitive murine hematopoietic progenitor cells

In contrast with the extensively characterized effects of transforming growth factor-beta (TGF-beta) on proliferation and differentiation of hematopoietic progenitors, little is known about the effects of TGF- beta on viability of normal hematopoietic progenitors. In the present report, we demonstrate that TGF-beta potently counteracts hematopoietic growth factor (HGF)-induced survival of individually cultured primitive Lin-Sca-1+ bone marrow progenitors. Specifically, 74% of single Lin-Sca- 1+ cells cultured for 40 hours in the presence of stem cell factor (SCF) survived, whereas only 16% survived in the presence of SCF plus TGF-beta. Similarly, the enhanced survival of primitive hematopoietic progenitors in response to granulocyte colony-stimulating factor (G- CSF), interleukin (IL)-1, IL-6, or IL-11 was also potently opposed by TGF-beta. Furthermore, it is demonstrated that neutralization of endogenous TGF-beta present in the cultures enhances survival of Lin- Sca-1+ progenitors in the absence, as well as in the presence, of HGFs such as SCF and IL-6. The reduced HGF-induced survival of primitive hematopoietic progenitors in the presence of TGF-beta was associated with increased apoptosis, as detected by an in situ terminal deoxynucleotidyl transferase (TdT) assay. After 16 hours of incubation in the absence of HGFs, 61% +/- 6% of the hematopoietic progenitors had DNA strand breaks characteristic of apoptosis. The presence of SCF reduced the frequency of apoptic cells to 27% +/- 5%, whereas 55% +/- 3% of the cells had signs of apoptosis in the presence of SCF plus TGF- beta.     

Combined signaling through interleukin-7 receptors and flt3 but not c- kit potently and selectively promotes B-cell commitment and differentiation from uncommitted murine bone marrow progenitor cells

Multiple cytokines can synergize to stimulate the in vitro proliferation and exclusive myeloid differentiation of multipotent bone marrow progenitor cells. The ligand for c-kit (stem cell factor [SCF]) plays a key role in stimulating myeloid and erythroid cell production of primitive hematopoietic progenitors. SCF in combination with interleukin-7 (IL-7) can also stimulate the combined myeloid and B-cell differentiation of uncommitted hematopoietic progenitor cells as well as the growth of early B-cell progenitor cells, although the involvement of c-kit in early B lymphopoiesis remains controversial. In the present study, the flt3-ligand (FL), which, in combination with other cytokines, has overlapping activities with SCF on myeloid cell production from uncommitted progenitors, was investigated for its ability to induce selective stroma-independent B-cell commitment from uncommitted Lin-Sca-1+ bone marrow progenitor cells. IL-7 alone did not induce any clonal growth and FL alone gave rise to a few clusters (< 50 cells) but no colonies (> 50 cells), whereas the combined stimulation with FL and IL-7 resulted in clonal growth of 10% of Lin-Sca-1+ bone marrow cells. After 12 days of incubation of Lin-Sca-1+ cells in FL + IL-7, an almost 400-fold increase in cell production was observed. Phenotyping showed that greater than 99% of the cells produced were of the B-cell lineage, in that they expressed B220, but not cell surface markers specific for myeloid, erythroid, or T-cell lineages. Furthermore, the cells did not express cytoplasmic mu-heavy chain (cmu) or surface IgM, but were positive for CD24 (heat stable antigen [HSA]) and CD43 (leukosialin), suggesting that the cells produced were blocked at a late pro-B-cell stage. Interestingly, although all FL + IL-7- responsive Lin-Sca-1+ progenitor cells and the resulting pro-B cells expressed c-kit, FL + IL-7 was much more potent (62-fold) than SCF + IL- 7 in stimulating production of cells of the B-cell lineage. In addition, whereas FL + IL-7 selectively stimulated the production of pro-B cells, SCF + IL-7 predominantly stimulated the production of mature granulocytes. Replating studies showed that FL + IL-7-responsive Lin-Sca-1+ progenitors were not committed to the B-cell lineage, because after 2 days of incubation in FL + IL-7, 80% of the progenitors retained a myeloid potential. As much as 27% of the FL + IL-7- responsive progenitors remained uncommitted after 7 days of incubation, but all had committed to the B-cell lineage after 10 days of incubation in FL + IL-7. These results show that FL much more potently and selectively than SCF synergizes with IL-7 to enhance B-cell commitment and development from uncommitted progenitor cells.     

Thrombopoietin stimulates murine lineage negative, Sca-1+, C-Kit+, CD34- cells: comparative study with stem cell factor or interleukin-3

It has recently been reported that human thrombopoietin (TPO) acts on early hematopoietic progenitor cells. Consequently, we investigated the effects of TPO on murine hematopoietic progenitor cells using lineage negative (Lin-), Sca-1+, c-Kit+ marrow cells from 5-fluorouracil-treated mice. One hundred enriched cells were cultured in suspension with various single cytokines for 5 days. When cultured with the single cytokines as stem cell factor (SCF), TPO, or interleukin (IL)-3, these cells were maintained or had increased by day 5, whereas only a few cells survived in cultures with granulocyte colony stimulating factor, IL-11, or IL-6. We extended the study in serum-free or serum-containing suspension cultures with SCF or TPO. Anti-TPO antibodies did not inhibit the effects of SCF on enriched cells but did inhibit the effects of TPO on those cells. We further examined the effects of TPO, SCF, and IL-3 on other populations of murine hematopoietic progenitor cells. Either TPO or SCF as a single cytokine could maintain murine Lin-, Sca-1+, c-Kit+, CD34- marrow cells, which are the most dormant cells. In addition, IL-3 increased Lin-, Sca-1-, c-Kit+ cells more than did SCF and TPO but did not stimulate Lin-, Sca-1+, c-Kit+, CD34- cells more. These results indicate that TPO as well as SCF may be key regulators in the proliferation of murine hematopoietic early progenitor cells.     

Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34(+) cells through glycoprotein (gp)130 signaling

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.     

Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor-beta and tumor necrosis factor-alpha

The recently cloned flt3 ligand (FL) stimulates the growth of primitive hematopoietic progenitor cells through synergistic interactions with multiple other cytokines. The present study is the first demonstrating cytokines capable of inhibiting FL-stimulated hematopoietic cell growth. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta l) potently inhibited the clonal growth of murine Lin-Sca-l+ bone marrow progenitors stimulated by FL alone or in combination with granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin (IL)-3, IL-6, IL-11, or IL-12. TGF-beta 1 inhibited more than 96% of the myeloid colony formation in response to these cytokine combinations, whereas TNF-alpha reduced the number of colonies by 58% to 96% depending on the cytokine by which FL was combined. In addition, both TNF-alpha and TGF-beta 1 inhibited more than 90% of B220+ cell production from B220- bone marrow cells stimulated by FL + IL-7. The effects of TNF-alpha and TGF-beta 1 appeared to be due to a direct effect and on the early progenitors because the inhibition was observed at the single cell level, and because delayed addition of the two inhibitors for only 48 hours dramatically reduced their inhibitory effects. A neutralizing anti-TGF- beta antibody showed the presence of endogenous TGF-beta in the cultures and potently enhanced the ability of FL to stimulate progenitor cell growth in the absence of other cytokines. Agonistic antibodies specifically activating the p75 TNF receptors were more efficient than wild type murine TNF-alpha in signaling growth inhibition of Lin-Sca-l+ progenitor cells, whereas the p55 agonist had less effect than murine TNF-alpha. Finally, TGF-beta increased the number of FL + IL-11-stimulated Lin-Sca-1+ cells in the G1 phase of the cell cycle with 76%, whereas TNF-alpha only had a marginal effect on cell cycle distribution. Thus, TGF-beta, TNF-alpha, and p75 TNF receptor agonists are potent direct inhibitors of FL-stimulated progenitor cell growth in vitro.     

FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver

The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL- cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.     

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.     

Recombinant rat stem cell factor stimulates the amplification and differentiation of fractionated mouse stem cell populations.

The role of recombinant rat stem cell factor (rrSCF) was studied on defined primitive bone marrow cell populations. In agar culture of 500 lineage-negative/Sca-1-positive (Lin-/Sca-1+) cells, rrSCF alone stimulates small colonies of predominantly granulocytic cells. The combinations of rrSCF plus interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage CSF (CSF-1) stimulated primitive progenitor cells defined as high proliferative potential colony-forming cells (HPP-CFC). Synergistic increases in total colony numbers were obtained with rrSCF plus GM-CSF, granulocyte CSF (G-CSF), CSF-1, or IL-6, but not IL-1 or IL-3. Lin-/Sca-1+ cells were incubated in liquid culture at 3,000 cells/mL for 6 days in the presence of rrSCF alone or in combination with other growth factors. The total number of cells was increased twofold in the presence of rrSCF, with the progeny primarily myeloid in nature. The greatest increase in cell number was obtained with rrSCF plus IL-3, where the cell number increased 40-fold. These factors also stimulated an increase in HPP-CFC (10-fold) and GM-CFC (500-fold). To determine if these interactions were direct, single Lin-/Sca-1+ cells were sorted into microtiter wells and the cell proliferation scored 6 days later. RrSCF synergized with IL-3, IL-6, and G-CSF to stimulate the proliferation of single cells. The cells in positive wells were subcultured into colony-forming assays and up to 400 CFC per well were obtained after 14 days incubation of the secondary cultures. These data demonstrate that rrSCF acts in combination with various growth factors to directly stimulate the amplification potential of hematopoietic primitive precursors, resulting in differentiation of these precursors.     

Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth

Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.     

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL- responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.     

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions

Summary. CD34⁺ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells.
The number of the different haemopoietic progenitors/1 × 10³ CD34⁺ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10–15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently syner-gized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.     

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO(2)) than the core of the BM where stem and progenitor cells reside, we investigated whether pO(2) influences Mk maturation. Mks were generated from CD34(+) cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O(2). At day 15, CD41(+) Mk expansion in 20% and 5% O(2) cultures was 85-fold and 31-fold respectively. Twenty percent O(2) cultures also had higher levels of high ploidy (> or = 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O(2) cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O(2) promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41(+) Mks, indicating that pO(2) directly affects Mks. The change from 20% to 5% O(2) on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34(+) cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.     

The in vitro response of phenotypically defined mouse stem cells and myeloerythroid progenitors to single or multiple growth factors.

Pluripotential stem cells (Thylo Lin- Sca+; referred to as Sca+) and primitive myeloerythroid progenitor cells (Thylo Lin- Sca-; referred to as Sca-), defined by their in vivo repopulating properties, have been purified from mouse bone marrow. In this study, the growth factor requirements of these two subsets were compared in colony-forming assays. Sca- progenitor cells grew well in interleukin (IL) 3 alone and showed maximum growth when two factors, IL-3 plus IL-1 or IL-3 plus IL-6, were combined. In contrast, Sca+ stem cells were generally not responsive to any single factor tested. Some colony formation was found when IL-3 was paired with either IL-1 or IL-6, and this was significantly enhanced as additional factors were included. A remarkable frequency of as much as 1 colony per 1.7 input Sca+ cells was achieved when IL-1, IL-3, IL-6, and colony-stimulating factors were used together. These differences in factor requirements presumably reflect the need for multiple factor signaling in the more primitive stem cell population. In most other aspects of colony formation, Sca+ and Sca- cells were very similar. They generated colonies that had equivalent distributions in size and cellular composition. One notable difference was found in the kinetics of their response. Whereas nearly all Sca- cells formed colonies within 7 days, a significant fraction of Sca+ cells delayed colony formation for greater than 1 week. During this quiescent period, cell survival was absolutely dependent on the presence of factors in the medium.     

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein

Abstract: We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34⁺ blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte-colony stimulating factor (G–CSF) by peripheral blood (PB)-derived CD34⁺c-kit^? cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)-3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34⁺c-kit^high cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM–CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34⁺c-kit^{high or low} cells in the presence of Epo. Replating of E-Mix colonies derived from CD34⁺c-kit^high cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34⁺c-kit^low cells which represent a more immature population than CD34⁺c-kit^high cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.