Burst-forming Unit-erythroid Colonies Research Articles

Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species

Several compounds, including butyrate and trichostatin A, have been shown to activate gamma-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H(2)O(2)) in drug-mediated fetal hemoglobin (HbF) induction. H(2)O(2) levels were measured using 2',7'-dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H(2)O(2) in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block gamma-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation. Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H(2)O(2)-dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated gamma-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF. The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A.

Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development

To find a human cell line that could support differentiation of human embryonic stem cells (hESCs) into hematopoietic cells. To determine in detail the expression profiles of the beta-like globin genes in hESC-derived erythroid cells. FH-B-hTERT, a human fetal liver-derived cell line, and S17, a mouse bone marrow stromal cell line, were used as stromas to induce the differentiation of hESC into hematopoietic cells. The number of hematopoietic progenitors and surface antigen expression were monitored during time-course experiments using colony assays and flow cytometry. Globin expression patterns in individual erythroid colonies were determined by real-time quantitative reverse transcriptase polymerase chain reaction. Comparison of coculture of hESCs with FH-B-hTERT or S17 cells revealed that the fraction of CD34(+) cells and the number of clonogenic progenitors per 250,000 cells plated were higher with FH-B-hTERT than with S17. Analysis of beta-like globin expression in individual burst-forming unit erythroid and colony-forming unit erythroid colonies revealed that erythroid cells derived from hESC cocultured for 8 to 21 days on either FH-B-hTERT or S17 produced epsilon- and gamma-globin mRNAs in similar amounts. With increasing time in coculture, the mean ratio of gamma/epsilon increased by more than 10-fold on both S17 and FH-B-hTERT stroma. Importantly, beta-globin expression was barely detectable at all time point examined. FH-B-hTERT can induce hESCs differentiation into hematopoietic cells more efficiently than S17. In vitro differentiation of hESCs recapitulates the epsilon-globin to gamma-globin switch but not the gamma-globin to beta-globin switch that occurs around birth. This experimental system will be useful for studying the regulation of globin gene expression during early human hematopoiesis.

Essential Role of Synoviolin in Embryogenesis

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

The Critical Role of Src Homology Domain 2–Containing Tyrosine Phosphatase-1 in Recombinant Human Erythropoietin Hyporesponsive Anemia in Chronic Hemodialysis Patients

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.

Suboptimal Erythropoietic Response to Hypoxemia in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory process characterized by severe derangement of gas exchange in the advanced stages of disease. However, erythrocytosis is infrequent in IPF. The aim of this study was to investigate the potential relation between the blunted erythropoietic response and the chronic inflammation. Nine patients (6 men and 3 women) with IPF and profound hypoxemia (PO(2) < 65 mm Hg) and 34 sex- and age-matched healthy volunteers participated in the study. We evaluated the hematologic parameters, serum erythropoietin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 levels. We also studied the development of burst-forming unit-erythroid (BFU-E)-derived colonies in semisolid methylcellulose cultures in blood samples from all patients. Hemoglobin and serum erythropoietin levels were almost comparable between the two studied groups. On the contrary, serum TNF-alpha, IL-6, and IL-8 values were significantly higher in patients with IPF (p < 0.05, p < 0.01, and p < 0.001, respectively). IPF sera induced a significant growth inhibition of erythroid bursts arising from mononuclear cells of either patients or control subjects compared with heat-inactivated AB serum (p < 0.05 and p < 0.01, respectively). Moreover, there was an apparent increment in the number of BFU-E colonies when patients' mononuclear cells were cultured in comparison with those of healthy subjects (p < 0.05). Our findings suggest that in IPF there is an increased number of primitive erythroid progenitors, which fail to proliferate and differentiate in vivo, suggesting a kind of ineffective erythropoiesis. As a consequence, hemoglobin levels do not rise in proportion to the severity of hypoxemia. Cytokines released from alveolar macrophages seem to have not only local but also systemic effects, since the serum of these patients directly suppressed erythropoiesis; however, the suboptimal erythropoietic response to hypoxia cannot be entirely attributed to this suppression. It is possible that several other factors interfere, synergistically or additively.

In vitro induction of fetal hemoglobin in human erythroid progenitor cells.

Clinical heterogeneity among patients with sickle cell anemia (SCA) is influenced by the amount of fetal hemoglobin (HbF) within circulating erythrocytes. Current pharmacotherapy focuses on increasing HbF in order to reduce hemolysis and help prevent acute vaso-occlusive events. Hydroxyurea, a known S-phase-specific cytotoxic ribonucleotide reductase (RR) inhibitor, is an effective agent for HbF induction in patients with SCA, but the mechanisms by which hydroxyurea induces HbF in vivo have not been elucidated. We adapted an in vitro assay for HbF induction, growing burst-forming unit erythroid (BFU-E) colonies in methylcellulose from peripheral blood of children with SCA and extracting the hemoglobin for high-performance liquid chromatography analysis of HbF. Hydroxyurea and other known RR inhibitors, along with cytotoxic agents that are not RR inhibitors, were tested for the ability to induce HbF using this in vitro assay. Hydroxyurea decreased the number of BFU-E colonies that grew in culture and significantly increased HbF from 13.6%+/-6.2% to 25.4%+/-8.0% at 50 microM HU (p=0.012). Three other known RR inhibitors also significantly induced HbF: 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (p=0.025), guanazole (p=0.008), and gemcitabine (p=0.028). Cytarabine and alkylating agents BCNU and 4-hydroperoxycyclophosphamide, which are cytotoxic agents but not RR inhibitors, reduced BFU-E colony number but did not significantly induce HbF. Hydroxyurea and other RR inhibitors significantly induce HbF in vitro in human erythroid progenitor cells. Inhibition of RR may be a critical mechanism by which hydroxyurea increases HbF in vivo in patients with SCA.

Expression of genes regulating angiogenesis in human circulating hematopoietic cord blood CD34+/CD133+ cells

Human CD34+ cells represent a heterogeneous population of immature cells which may differentiate to various cell types. The aim of the study was to determine angiogenesis regulating genes expression in CD34+ cells, their subpopulations, and during their differentiation induced by hematopoietic growth factors. We have measured the expression of angiogenesis regulating genes angiopoietin-1 (Ang-1), angiopoietin-1 (Ang-2) and their receptor Tie-2, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 in sorted population of CD34+ and CD34+/CD133+ cells from human cord blood and bone marrow, and in their differentiating progeny, using real time reverse transcriptase polymerase chain reaction. The hematopoietic differentiation of CD34+ cells was induced in semisolid or liquid differentiation supporting media containing appropriate hematopoietic growth factors. A higher expression of Ang-1, Ang-2, and Tie-2 mRNAs was detected in CD34+/CD133+ cord blood cells as compared with CD34-/CD133- fraction, but no expression of these genes was detected in burst-forming unit erythroid (BFU-E) nor colony-forming unit granulocyte macrophage (CFU-GM) colonies. The level of Ang-1 and Tie-2 mRNAs, but not that of Ang-2 mRNA gradually decreased during a 14-d incubation of cord blood CD34+ cells in a liquid culture. A significantly higher expression of VEGF mRNA was in BFU-E as compared with CFU-GM cell colonies and CD34+/CD133+ cells. VEGFR-1 mRNA was equally expressed in CD34+/CD133+ and CD34-/CD133- cells as well as in BFU-E and CFU-GM colonies. Expression of VEGFR-2 mRNA was detected at the borderline of method sensitivity only in CD34+/CD133+ cells. CD34+/CD133+ cord blood cells express Ang-1, Ang-2 and VEGF as well as their receptor mRNAs, suggesting a role of these cells in regulation both angiopoiesis and hematopoiesis.

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes.

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity. Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.

Effects of cyclic polylactate (CPL) on the growth of cloned leukemic cells in vitro.

A novel supramolecular oligomer, cyclic polylactate (CPL), was first discovered in the culture medium of HeLa-S tumour cells, and was reported to inhibit the growth of FM3A ascites tumour cells by inhibiting the activities of pyruvate kinase (PK) and lactic dehydrogenase (LDH). We have now synthesized CPL-containing oligomers with polymerization numbers ranging from 9 to 19, by prolonged heating and rapid mixing of a carbohydrate compound of the L-lactic acid monomer (C(3)H(6)O(3)) under decreased pressure and have studied its effects on the growth of leukemic cells. Treatment with 0.02 mg/ml CPL inhibited the growth of HL60 and TF-1 cells, while the growth of K562 cells was inhibited by 0.2 mg/ml CPL. A concentration of 2 mg/ml CPL was required to inhibit granulocyte-macrophage progenitor cell (CFU-GM) and burst-forming unit erythroid (BFU-E) precursor colony formation among normal bone marrow cells. Furthermore, 7A6 antigen expression and DNA ladder formation were observed in leukemic cells cultured with CPL, indicating that CPL induces apoptotic changes in these cells. These findings suggest that CPL might be a useful chemotherapeutic agent for leukemia.

Role of Members of the Wnt Gene Family in Human Hematopoiesis

AbstractThe hematopoietic system is derived from ventral mesoderm. A number of genes that are important in mesoderm development have been identified including members of the transforming growth factor-β (TGF-β) superfamily, the fibroblast growth factor (FGF) family, and the Wnt gene family. Because TGF-β plays a pleiotropic role in hematopoiesis, we wished to determine if other genes that are important in mesoderm development, specifically members of theWnt gene family, may play a role in hematopoiesis. Three members of the Wnt gene family (Wnt-5A, Wnt-2B, and Wnt-10B) were identified and cloned from human fetal bone stromal cells. These genes are expressed to varying levels in hematopoietic cell lines derived from T cells, B cells, myeloid cells, and erythroid cells; however, only Wnt-5A was expressed in CD34+Lin− primitive progenitor cells. The in vitro biological activity of these Wnt genes on CD34+Lin− hematopoietic progenitors was determined in a feeder cell coculture system and assayed by quantitating progenitor cell numbers, CD34+ cell numbers, and numbers of differentiated cell types. The number of hematopoietic progenitor cells was markedly affected by exposure to stromal cell layers expressing Wnt genes with 10- to 20-fold higher numbers of mixed colony-forming units (CFU-MIX), 1.5- to 2.6-fold higher numbers of CFU-granulocyte macrophage (CFU-GM), and greater than 10-fold higher numbers of burst-forming units-erythroid (BFU-E) in the Wnt-expressing cocultures compared with the controls. Colony formation by cells expanded on theWnt-expressing cocultures was similar for each of the three genes, indicating similar action on primitive progenitor cells; however, Wnt-10B showed differential activity on erythroid progenitors (BFU-E) compared with Wnt-5A and Wnt-2B. Cocultures containing Wnt-10B alone or in combination with all three Wnt genes had threefold to fourfold lower BFU-E colony numbers than the Wnt-5A– or Wnt-2B–expressing cocultures. The frequency of CD34+ cells was higher inWnt-expressing cocultures and cellular morphology indicated that coculture in the presence of Wnt genes resulted in higher numbers of less differentiated hematopoietic cells and fewer mature cells than controls. These data indicate that the gene products of theWnt family function as hematopoietic growth factors, and that they may exhibit higher specificity for earlier progenitor cells.© 1998 by The American Society of Hematology.

Role of Members of the Wnt Gene Family in Human Hematopoiesis

The hematopoietic system is derived from ventral mesoderm. A number of genes that are important in mesoderm development have been identified including members of the transforming growth factor-β (TGF-β) superfamily, the fibroblast growth factor (FGF) family, and the Wnt gene family. Because TGF-β plays a pleiotropic role in hematopoiesis, we wished to determine if other genes that are important in mesoderm development, specifically members of theWnt gene family, may play a role in hematopoiesis. Three members of the Wnt gene family (Wnt-5A, Wnt-2B, and Wnt-10B) were identified and cloned from human fetal bone stromal cells. These genes are expressed to varying levels in hematopoietic cell lines derived from T cells, B cells, myeloid cells, and erythroid cells; however, only Wnt-5A was expressed in CD34+Lin− primitive progenitor cells. The in vitro biological activity of these Wnt genes on CD34+Lin− hematopoietic progenitors was determined in a feeder cell coculture system and assayed by quantitating progenitor cell numbers, CD34+ cell numbers, and numbers of differentiated cell types. The number of hematopoietic progenitor cells was markedly affected by exposure to stromal cell layers expressing Wnt genes with 10- to 20-fold higher numbers of mixed colony-forming units (CFU-MIX), 1.5- to 2.6-fold higher numbers of CFU-granulocyte macrophage (CFU-GM), and greater than 10-fold higher numbers of burst-forming units-erythroid (BFU-E) in the Wnt-expressing cocultures compared with the controls. Colony formation by cells expanded on theWnt-expressing cocultures was similar for each of the three genes, indicating similar action on primitive progenitor cells; however, Wnt-10B showed differential activity on erythroid progenitors (BFU-E) compared with Wnt-5A and Wnt-2B. Cocultures containing Wnt-10B alone or in combination with all three Wnt genes had threefold to fourfold lower BFU-E colony numbers than the Wnt-5A– or Wnt-2B–expressing cocultures. The frequency of CD34+ cells was higher inWnt-expressing cocultures and cellular morphology indicated that coculture in the presence of Wnt genes resulted in higher numbers of less differentiated hematopoietic cells and fewer mature cells than controls. These data indicate that the gene products of theWnt family function as hematopoietic growth factors, and that they may exhibit higher specificity for earlier progenitor cells.© 1998 by The American Society of Hematology.

Human Hematopoietic Progenitors Express Erythropoietin

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34+lin− hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34+ cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34+ cells represent a heterogeneous cell population, we analyzed individual burst-forming units–erythroid (BFU-E) and nonerythroid colony-forming unit–granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Human Hematopoietic Progenitors Express Erythropoietin

AbstractErythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34+lin− hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34+ cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34+ cells represent a heterogeneous cell population, we analyzed individual burst-forming units–erythroid (BFU-E) and nonerythroid colony-forming unit–granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Relative sedimentation of hematopoietic progenitors in human cord blood, peripheral blood, and bone marrow as determined by counterflow centrifugal elutriation.

The current use of cord blood (CB) and peripheral blood (PB) stem cells as alternatives or adjunctives to bone marrow (BM) for hematopoietic reconstitution in the treatment of various diseases prompted an examination of the progenitors of these tissues by counterflow centrifugal elutriation (CCE). The cells, obtained from normal donors not primed with colony-stimulating factors, were centrifuged at 3000 rpm in a Beckman Sanderson Chamber. Fractions (Frs.) were collected at (1) 18 ml/min, (2) 25 ml/min, (3) 32 ml/min, (4) 40 ml/min, and (5) the rotor-off fraction. Clonogenic assays revealed differences in the fraction localizations for CB and PB when compared to BM, i.e., recovery of the colony-forming units for CB and PB was greater in the small-medium cell size CCE fractions, and those from BM were found primarily among the medium-large cell size fractions. Thus, although colony-forming unit granulocyte/macrophage colonies were distributed throughout Frs. 2-5 of BM, CB and PB showed 80% of the total to be in Frs. 2 and 3. Further, although burst-forming unit erythroid colonies of BM were distributed equally in Frs. 2 and 3, greater than 70% of the total burst-forming unit erythroid colonies in CB and PB were found in Fr. 2. Distribution of the CD34 cells in the fractions correlated with the colony-forming units in that these were found primarily in Frs. 2 and 3 of CB and PB, whereas they were present in significant numbers throughout Frs. 1-5 of BM. We interpret these findings to indicate CB and PB to be qualitatively similar in their hematopoietic lineage development and to contain a greater proportion of early versus late progenitors relative to those found in BM.

Flt3high and Flt3low CD34+Progenitor Cells Isolated From Human Bone Marrow Are Functionally Distinct

We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin− marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38− marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34−CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit–granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3highcells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin−Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin−Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.

Flt3high and Flt3low CD34+Progenitor Cells Isolated From Human Bone Marrow Are Functionally Distinct

AbstractWe generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin− marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38− marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34−CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit–granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3highcells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin−Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin−Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.

BFU-E colony growth in response to hydroxyurea: correlation between in vitro and in vivo fetal hemoglobin induction.

Patients with sickle-cell anemia treated with hydroxyurea may have significant reduction in frequency and severity of pain episodes. However, previous clinical trials show a variable response to hydroxyurea. Criteria which can be used to select patients who are likely to respond to hydroxyurea treatment would be useful. Our laboratory has previously demonstrated an inverse linear relationship between the total number of burst-forming unit-erythroid (BFU-E) colonies and fetal hemoglobin levels in sickle-cell patients treated with hydroxyurea. In the present report, an in vitro cell culture system was established to evaluate the effects of hydroxyurea on BFU-E colony growth and induction of fetal hemoglobin production. Five Hb SS patients who were not previously treated with hydroxyurea and three Hb SS patients who failed to respond to hydroxyurea treatment were included in the study. The results show that the number of BFU-E colonies is decreased from 153.7 to 7.2 per 3 x 10(5) mononuclear cells, whereas fetal hemoglobin levels were increased from 5.1 to 19.4% in the presence of hydroxyurea in vitro in cultured erythroid progenitors, which were derived from 5 patients before treatment. The number of BFU-E colonies decreased from 153.7 to 2.0 per 3 x 10(5) mononuclear cells in the in vitro cultures obtained from serial peripheral blood samples over a 9- to 20-week period of oral hydroxyurea therapy. A simultaneous rise in fetal hemoglobin level from 10.2 to 28.6% in the peripheral blood over the same period of hydroxyurea therapy was also observed. Our results demonstrate that the increase in fetal hemoglobin levels in cells treated with hydroxyurea in vitro is comparable to the rise of fetal hemoglobin production following hydroxyurea therapy in these patients. On the contrary, these findings were not observed in three previously non-responsive sickle-cell patients. These results suggest that the changes in number of BFU-E colonies and fetal hemoglobin levels after in vitro exposure to hydroxyurea may be a useful approach to select sickle-cell patients who will respond to hydroxyurea therapy.

Globin Gene Silencing in Primary Erythroid Cultures: AN INHIBITORY ROLE FOR INTERLEUKIN-6

There are numerous similarities between the erythroid and megakaryocytic lineages which suggest that commitment to either lineage occurs relatively late in hematopoiesis. Commitment toward megakaryocyte development requires obligatory silencing of erythroid-specific genes. Therefore, we investigated the effects of interleukin-6, a known inducer of thrombocyte production, on globin gene expression during erythroid differentiation. Studies in K562 cells demonstrated inhibition of gamma globin gene mRNA production and chain biosynthesis in the presence of exogenous interleukin-6 which was abrogated by anti-interleukin-6 monoclonal antibody. Similar studies in primary erythroid progenitors showed inhibition of burst-forming unit-erythroid colony formation when interleukin-6 was added late in cultures with decreased gamma and beta globin gene mRNA production. Protein binding studies demonstrated an increase in activator protein-1 binding to its consensus sequence by 24 h of interleukin-6 treatment. Inhibition of activator protein-1 gene activity had no effect on gamma gene silencing by interleukin-6. A potential interleukin-6 response element was identified in the gamma globin gene. Interleukin-6 treatment led to a rapid increase in protein binding to the target DNA sequence. These results suggest that interleukin-6 may play an important role in globin gene silencing during megakaryocytic lineage commitment.

Bone Marrow Stroma-Derived Prolactin Is Involved in Basal and Platelet-Activating Factor–Stimulated In Vitro Erythropoiesis

Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.

Bone Marrow Stroma-Derived Prolactin Is Involved in Basal and Platelet-Activating Factor–Stimulated In Vitro Erythropoiesis

AbstractCooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.