Bcr-Abl kinase promotes cell cycle entry of primary myeloid CML cells in the absence of growth factors

Cell cycle control of both immature and differentiated primary myeloid normal and chronic-phase chronic myeloid leukaemia (CML) cells to growth factor deprivation was studied. CD34⁺ cells were cultured in liquid culture. After removal of growth factors for 48 h normal cells were very efficiently arrested with the fraction of cells in S phase reduced by 70.8 ± 6.5% in CD34⁺ and 50.5 ± 4.2% in CD34⁻ cells. In contrast, a significantly higher proportion of leukaemic cells remained in S phase. The fraction of S-phase cells was reduced by only 29.3 ± 5.7% in CD34⁺ CML cells and 21.2 ± 3.8% in CD34⁻ cells. This abnormal negative cell cycle control in leukaemic cells was specific for growth factor deprivation. Reaction to IFN-α and TNF-α treatment was identical both in normal and CML cells. Equal quantities of the cytokines TNF-α, IL-1α, IL-1RA and IL-6 were produced by CML and normal cells. However, production of GM-CSF, with a median of 11 ± 5 pg/ml, was found only in the supernatants of CML cells. But antibodies to GM-CSF did not restore growth factor dependence of the leukaemic cells. The defect was completely corrected by the abl-specific tyrosine kinase inhibitor CGP 57148 without effecting cell cycle control of normal cells. Our results demonstrate a directly Bcr-Abl-dependent defective response of both immature and differentiated primary myeloid CML cells to growth factor deprivation.     

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon- γ (IFN- γ ) and tumour necrosis factored (TNF- α ), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34⁺ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN- γ , increased TNF- β expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34⁺ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34⁺ cells showed markedly increased percentages of Fas receptor-expressing CD34⁺ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients'BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34⁺ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN- γ and/or TNF-α and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.     

The effect of interleukin-12 in ex-vivo expansion of human haemopoietic progenitors

Summary. We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL-12. In cultures of low density cells, cytokine combinations including IL-12 were associated to a greater proliferation (up to 6.7 ± 2.5 CFU-GM fold expansion). However, in cultures of purified CD34⁺ cells the more efficient cytokine combination (147 ± 49 CFU-GM fold expansion) was SCF, IL-3, IL-11 and MlP-la, and the addition of IL-12 did not further enhance expansion of progenitors.
These results indicate that accessory cells, lost in CD34⁺ cell purification, could be in part responsible for IL-12 effect on progenitor cell proliferation. In CD34⁺ cell cultures the addition of IL-12 led to CD19 mRNA generation, suggesting that IL-12 acts on haemopoietic cells with both myeloid and lymphoid potential.     

HIGH-DOSE THERAPY WITH PERIPHERAL BLOOD STEM CELL TRANSPLANTATION RESULTS IN A SIGNIFICANT REDUCTION OF THE HaEMOPOIETIC PROGENITOR CELL COMPARTMENT

In order to study the effect of high-dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)-derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6–27 months after transplantation. Following PBSCT, the proportion of CD34⁺ cells was significantly smaller than before mobilization (BM 1.99±0.24 versus 0.8±0.09, P <0.001), and no change was observed at several follow-up visits thereafter.
The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34⁺/DR⁻ and CD34⁺/Thy-1⁺ cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples.
No relationship was found between the decrease in the proportion of CD34⁺ cells and any particular patient characteristics, kind of high-dose therapy or the CD34⁺ cell content in the autograft.
In conclusion, high-dose therapy with PBSC transplantation is associated with a long-term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post-transplantation were normal in the majority of patients.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

Transforming growth factor-beta 1 delays formation of granulocyte-macrophage colony-forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haemopoietic progenitor cells

Ex vivo culture and expansion of autologous haemopoietic transplants has been developed to improve tumour cell purging and accelerate haemopoietic reconstitution by transplantation of increased progenitor cell numbers. We studied the effect of the negative haemopoietic regulator, transforming growth factor beta-1 (TGF-beta 1) on primitive precursors during ex vivo expansion of CD34⁺ cells. When added directly to methylcellulose colony-forming assays, TGF-beta 1 potently suppressed the development of granulocyte-macrophage colonies from CD34⁺ enriched peripheral blood progenitor cells (80–90% inhibition). In contrast, expansion of total nucleated cells and granulocyte-macrophage colony-forming cells (GM-CFC) from CD34⁺ progenitors in liquid culture in the presence of stem cell factor (SCF), interleukin (IL)-1 beta, IL-3, IL-6 and erythropoietin (EPO) was inhibited to 32–65% of control culture levels within 14 d when TGF-beta 1 was added, and still produced an average 3.3-fold absolute amplification of GM-CFC. The inhibitory effect of TGF-beta 1 on GM-CFC generation was reversed when it was washed out on day 6 of ex vivo expansion cultures, and total numbers of GM-CFC generated from expansion cultures then reached levels of untreated controls by day 16. Long-term bone marrow culture-initiating cell (LTCIC) numbers were preserved, at least at input levels, over a culture period of 14 d both in control and TGF-beta-1-treated expansion cultures. These findings suggest that TGF-beta 1, a cytokine which induces apoptosis or terminal differentiation in a number of malignant cell types, may be added to ex vivo expansion cultures without loss of primitive cells from autologous haemopoietic transplants.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients ( P  = 0.039), which are absent in both CD34⁻ MDS cells ( P  = 0.53) and also CD34⁺ cells from normal subjects ( P  = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34⁻ cells ( P  = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34⁻ bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.     

SLEx expression of normal CD 34 positive bone marrow haemopoietic progenitor cells

Summary. We investigated sialylated Lewis x (sLe^x) antigen expression on CD 34 positive (CD 34⁺) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD 34⁺ bone marrow progenitors strongly expressed the sLe^x antigen. This contradicts previous publications which reported sLe^x expression on malignant blast cells but not on normal CD 34⁺ progenitor cells.     

Thrombopoietin-independent effect of interferon-γ on the proliferation of human megakaryocyte progenitors

Flow cytometric study revealed that almost all CD34⁺ cells in human umbilical cord blood expressed interferon-γ receptor (IFN-γR). To clarify the precise functional roles of IFN-γR in human CD34⁺ cells, we examined the effect of IFN-γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34⁺ cells using a serum-free clonal culture. Surprisingly, IFN-γ alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-γ. IFN-γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 10³ CD34⁺ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-γ monoclonal antibody (mAb) to the IFN-γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-γ, suggesting that the observed effect of IFN-γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-γ may have regulatory roles in human megakaryocytopoiesis.     

Combined negative and positive selection of mobilized CD34+ blood cells

We tested four negative and two positive selection methods for separation of CD34⁺ cells from mobilized blood cells, and analysed fold-enrichment, purity and recovery of CD34⁺ cells after selection procedures. The elimination of mature CD34⁻ cells was achieved by adhesion to nylon-wool fibre (5.9 ± 1.0 mean fold-enrichment and 65.2 ± 2.3 mean recovery of CD34⁺ cells). Standard or modified Ficoll-Hypaque and Percoll density gradients, as well as phagocytosis with magnetic beads, were less effective in eliminating CD34⁻ cells, both purity and fold-enrichment of CD34⁺ cells being lower than those obtained with separation by nylon-wool. Both positive selection methods tested, Ceprate and MiniMacs System, generated highly purified CD34⁺ cell populations ranging from 80% to 90%. The recovery of CD34⁺ cells was optimal with MiniMacs (77.9±3.6) and low with Ceprate (28.8±2.8). Based on these results, in two large-scale experiments we combined nylon-wool fibre and MiniMacs System in a two-step separation procedure obtaining a 36.9±2.6 mean fold-enrichment and a 50.5±0.3 mean recovery of CD34⁺ cells. In this way we achieved optimal enrichment and recovery of CD34⁺ cells, with a substantial saving of cost compared to either selection method alone.     

Interactions between purified human cord blood haemopoietic progenitor cells and accessory cells

Summary. The role of accessory cells in haemopoiesis remains confused. This appears in large part to reflect the use of impure populations of accessory cells and progenitor cells in previous studies. In this study, cell sorter purified populations of both accessory cells and haemopoietic progenitor cells were used to examine interactions between these cell types. We used a double culture protocol in which purified CD34⁺ cells were cultured with purified NK, T or monocytic cells in the first liquid culture phase after which the cells were transferred to secondary agar cultures to determine the number of colony forming cells (CFC). NK cells co-cultured with CD34⁺ cells resulted in an increased number of erythroid progenitors with no effect on the number of non-erythroid progenitors. In contrast, there were increased numbers of erythroid and non-erythroid CFC when the CD34⁺ cells were co-cultured with either purified T cells or monocytes. CD34⁺ cells cultured with cell-conditioned media derived from NK cells, T cells or monocytes in transwells where the CD34⁺ cells and the accessory cells were separated by a 0·2 μm membrane, showed no enhancement in CFC. These results suggest that intimate cell–cell contact is required for these events.     

High-resolution cell division tracking demonstrates the Flt3-ligand-dependence of human marrow CD34+CD38− cell production in vitro

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34⁺ (CD45RA/CD71)⁻, but not CD34⁺(CD45RA/CD71)⁺, human marrow cells initially recruited into division in vitro , shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34⁺CD38⁻ population through several (up to four) cell generations. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34⁺(CD45RA/CD71)⁻ cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

Generation of human natural killer cells from peripheral blood CD34+ cells mobilized by granulocyte colony-stimulating factor

We studied the generation of human natural killer (NK) cells from CD34⁺ cells that were isolated from peripheral blood stem cells (PBSC) mobilized by granulocyte colony-stimulating factor (G-CSF). The isolated CD34⁺ cells were cultured in the presence of a combination of interleukin-1 (IL-1α), IL-2, and stem cell factor for 5 weeks without marrow stroma. We found that the CD34⁺ cells isolated from G-CSF-mobilized PBSC (G-CSF/PBSC) could differentiate into a population of NK cells which were CD56^+(bright)/CD3⁻ and showed morphologic characteristics of large granular lymphocytes. Immunophenotypic analysis of the NK cells thus generated showed that a small proportion of them expressed CD2, CD8 and CD16 surface markers and approximately half of them coexpressed CD7. This NK population exhibited cytotoxic activity against a NK-sensitive cell line, K562. These observations suggest that CD34⁺ cells from G-CSF/PBSC contain precursors of NK cells that can differentiate into functional NK cells.     

Megakaryocyte progenitors derived from bone marrow or G-CSF-mobilized peripheral blood CD34+ cells show a distinct phenotype and responsiveness to interleukin-3 (IL-3) and PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)

In the present study we investigated the proliferative response of megakaryocyte progenitor cells (CFU-MK) derived from peripheral blood stem cell (PBSC) collections of patients with haematological malignancies and normal donors. Highly purified CD34⁺ cells and mononuclear cell fractions were assayed in the presence of recombinant interleukin-3 (IL-3) and pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), alone or in combination, and megakaryocyte colony formation was evaluated in the plasma clot. In comparison, steady-state bone marrow samples from normal donors were highly enriched in CD34⁺ cells and tested with the cytokines studied. Our results showed that IL-3 was able to stimulate CFU-MK colony formation from bone marrow and peripheral blood CD34⁺ cells. Similarly, PEG-rHuMGDF stimulated, in a dose–response manner, CD34⁺ cells from the bone marrow. However, normal mobilized peripheral blood CD34⁺ cells were not induced to generate CFU-MK colonies by PEG-rHuMGDF. The same lack of response was observed when patients peripheral blood CD34⁺ cells primed with chemotherapy plus G-CSF or with G-CSF alone were assessed. In contrast, PEG-rHuMGDF stimulated CFU-MK growth when mononuclear cells, either from the bone marrow or from mobilized peripheral blood, were grown in plasma clot. Moreover, we analysed by flow cytometry the expression of Mpl receptor on the cell membrane of normal mobilized peripheral blood and normal steady-state bone marrow CD34⁺ cells. Our results showed a reduced expression of Mpl receptor on mobilized peripheral blood progenitor cells in comparison with bone marrow cells.     

Chronic myelogenous leukaemia CD34+ cells exit G0/G1 phases of cell cycle more rapidly than normal marrow CD34+ cells

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34⁺ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34⁺ cells were cycling than those from normal BM. The HLA-DR⁻ compartment of CML CD34⁺ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34⁺ HLA-DR⁺ fraction. When the activation of CD34⁺ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34⁺ cells exited G₀/G₁ more rapidly than normal CD34⁺ cells. Interestingly, although normal BM CD34⁺ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34⁺ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34⁺ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.     

Regulation of colony forming cell generation by flt-3 ligand

The recently cloned ligand for the flt-3/flk-2 receptor was examined for its effect on colony formation by subpopulations of CD34⁺ cells including the least mature CD34⁺lin⁻CD38⁻ small-medium lymphocyte-sized cell population. Flt-3 ligand (flt-3l) had little or no effect when added alone to cells. Isolated CD34⁺lin⁺ cells formed increased numbers of colony-forming cells (CFC) when flt-3l was added together with IL-3, IL-6, G-CSF, GM-CSF or c-kit ligand (KL), or with the combination of IL-3 and KL. Significant increases in CFC formation from CD34⁺lin⁻ cells were consistently seen when flt-3l was added to the IL-3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34⁺lin⁻ cells in liquid cultures showed that cultures containing IL-3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL-3, KL and flt-3l produced few CFC past 2 weeks of culture. Flt-3l alone or the combination of IL-3 and KL did not stimulate significant growth of CD34⁺lin⁻CD38⁻ small-medium lymphocyte-sized cells, although these cells reproducibly generated CFC when grown in the combination of IL-1β, IL-3, IL-6, G-CSF, GM-CSF and KL. Addition of flt-3l to either IL-3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt-3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34⁺ cells.     

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis , on prolonged maintenance and expansion of cord blood CD34⁺ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34⁺ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34⁺, CD34⁺CD38⁻, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45⁺ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34⁺ cells for transplantation.     

Direct and reversible inhibition of platelet factor 4 on megakaryocyte development from CD34+ cord blood cells: comparative studies with transforming growth factor β1

Mechanisms of the action of platelet factor 4 (PF4) on the growth of megakaryocyte (MK) progenitor cells in CD34⁺ cord blood (CB) cells were studied in comparison with transforming growth factor β1 (TGFβ1). Development of MK from CD34⁺ CB cells in both plasma clot culture and liquid culture was significantly inhibited by either purified human PF4 and by recombinant human TGFβ1. Inhibition of MK colony formation by PF4 was reversible, because CD34⁺ cells preincubated with PF4 could regenerate colonies after washing and replating into secondary cultures. In contrast, TGFβ1-preincubated CD34⁺ cells gave rise to few colonies following replating. Moreover, incubation of CD34⁺ cells with PF4 in liquid culture caused the increased number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells. In addition, PF4-preincubated CD34⁺ cells exibited a higher potential in MK colony formation in the presence of 5-fluorouracil (5FU). These results demonstrate that both PF4 and TGFβ1 inhibit MK development from CD34⁺ CB cells by different mechanisms, and suggest that PF4, unlike TGFβ1, exerts its inhibitory effect on the growth of the target cells in a reversible manner which results in a preservation of a more immature and 5FU-resistant cell population.