Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D04996 (Methylcellulose)
 116 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Normal human bone marrow cells, highly enriched for burst-forming units-erythroid (BFU-E), were cultured in serum-free medium, in the presence and absence of various factors, to investigate the mechanisms involved in regulating erythroid differentiation. In cultures containing interleukin 3 (IL-3), Steel factor (SF), and erythropoietin (Ep), benzidine-positive erythroblasts first became detectable on day 6. Their numbers then rapidly increased until, by day 16, > 99% of the cells, which were 20,000-fold amplified over input numbers, were benzidine-positive. It is interesting to note that omission of either SF or Ep from this assay markedly enhanced the rate of differentiation and reduced total cell numbers, whereas omission of IL-3 had no effect on the rate of differentiation and only slightly reduced cell numbers. Of various agents tested, the most potent erythroid differentiation inducer (and inhibitor of cell proliferation) was found to be transforming growth factor beta 1 (TGF-beta 1). This cytokine stimulated both the rapid appearance of hemoglobin-positive cells and an early cessation of cell proliferation. Using fluorescently tagged antibodies to glycophorin A and fluorescence-activated cell sorter (FACS) analysis, this phenomenon was shown to be due to an early induction of erythroid differentiation rather than an aberrant production of hemoglobin. Methylcellulose assays indicated that the well-documented reduction of BFU-E colony numbers observed with TGF-beta 1 may actually be due to a TGF-beta 1-induced "conversion" of BFU-E into colony-forming units-erythroid (CFU-E). Thus, in vivo, TGF-beta 1 might serve, in part, to decrease the number of mature erythrocytes by stimulating BFU-E to skip a number of cell divisions and differentiate early.
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PMID:Transforming growth factor beta 1 is an inducer of erythroid differentiation. 752 Apr 75

We tested the ability of a constitutively activated erythropoietin receptor [EpoR(R129C)] to alter the growth requirements of primary hematopoietic precursors that terminally differentiate in culture. Two recombinant retroviruses expressing EpoR(R129C), spleen focus-forming virus (SFFVc-EpoR) and myeloproliferative sarcoma virus (MPSVcEpoR), were used to infect fetal liver cells that served as a source of hematopoietic progenitors. Methylcellulose cultures were incubated in the absence of any added growth factors or in combination with selected growth factors. EpoR(R129C) completely abrogated the Epo requirement of erythroid colony-forming units to form erythrocytes after 2-5 days in culture and did not interfere with the differentiation program of these cells. In the absence of added growth factors EpoR(R129C) did not enhance erythroid burst-forming unit development. In contrast to experiments in heterologous cell lines, EpoR(R129C) did not render progenitor cells independent of interleukin 3 or granulocyte/macrophage colony-stimulating factor (GM-CSF). However, when progenitors were cultured with added steel factor, but not with interleukin 3 or GM-CSF, EpoR(R129C) augmented the growth and differentiation of erythroid bursts, mixed erythroid/myeloid, and granulocyte/macrophage (GM) colonies. Furthermore, both viruses were capable of expressing EpoR(R129C) in erythroid, mixed erythroid/myeloid, and GM colonies. Thus an aberrantly expressed and constitutively activated EpoR can stimulate proliferation of some GM progenitors. The ability of EpoR(R129C) to abrogate the Epo requirement of primary hematopoietic cells, but not the requirement for other cytokines, is consistent with the induction of erythroblastosis in vivo.
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PMID:Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth. 767 18

The J2E erythroid cell line terminally differentiates in response to recombinant human erythropoietin (rHu-epo). Here we demonstrate that retrovirally produced monkey erythropoietin (Zen-epo) is also capable of inducing the final maturation steps of J2E cells in a dose-dependant manner. The hormone committed J2E cells to a pathway of accelerated proliferation, haemoglobin synthesis and morphological changes including enucleation. However, only 6 h exposure to Zen-epo was required to initiate maximum haemoglobin synthesis 72 h later. Significantly, the synthesis of haemoglobin was achieved by concomitantly activating globin production, haem synthesis and iron uptake. Methylcellulose assays revealed that the vast majority of clonogenic cells were able to respond to Zen-epo. These data show that retrovirally manufactured erythropoietin is an effective stimulus for the erythropoietic development of J2E cells in vitro.
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PMID:Retrovirally-produced erythropoietin effectively induces differentiation and proliferation of J2E erythroid cells. 821 20

We investigated the effect of the human ligand for flt-3 (FL) on the committed progenitor colony formation of normal bone marrow (BM) (n = 9) and BM from four aplastic anaemia (AA) and three Diamond-Blackfan anaemia (DBA) patients. Methylcellulose committed progenitor cell assays were carried out using FL alone and in combinations with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and c-kit ligand (KL). FL alone had a limited, though significant, effect on the production of granulocyte-macrophage colony-forming unit (CFU-GM) colonies from normal BM and showed an additive effect with IL-3 and GM-CSF separately, but not in combination. FL did not increase the stimulation of KL and did not have an effect on the production of erythroid progenitor colonies. FL had no effect on the AA and DBA BMs studied.
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PMID:The effect of human flt-3 ligand on committed progenitor cell production from normal, aplastic anaemia and Diamond-Blackfan anaemia bone marrow. 855 52

Haemopoietic cultures may experience pH variations of as much as 0.5 units depending on culture duration and cell density. Since pH is a potent modulator of cellular proliferation and differentiation, we examined its effects on the performance of both semisolid and liquid haemopoietic cultures. Culture pH was found to have substantial effects both on progenitor cloning efficiency (as measured in liquid cultures) and on progenitor cell differentiation (as measured in methylcellulose cultures). Liquid cultures were conducted with both peripheral blood (PB) mononuclear cells (MNCs) and cord blood (CB) MNCs using growth factor combinations that promote either erythroid expansion (IL-3/IL-6/SCF/Epo) or granulocyte/macrophage expansion (IL-3/IL-6/SCF/G-CSF/GM-CSF). Reduced pH was found to have either a positive or neutral effect on the expansion and cloning efficiency of progenitors in ex vivo liquid cultures. Cloning efficiencies of PB BFU-E in the erythroid combination were 9-fold higher at low pH (7.1) when compared to high pH (7.6). A small pH increase of 0.2 units over physiological values consistently produced significant reductions (42-85%) in cloning efficiencies for all cell types and cytokine combinations tested. Methylcellulose cultures conducted using CB MNC and PB MNC indicated that differentiation of CFU-GM into progeny was optimal between pH 7.2 and 7.4. The differentiation of erythroid progenitors (BFU-E) progressively increased as pH was increased from 6.95 (no colonies detected) to 7.4 (maximum colonies detected), to 7.6 (maximum haemoglobin content). Methylcellulose cultures using PB CD34+ cells exhibited similar patterns to the MNC cultures. We conclude that even small variations in pH substantially affected the performance of human haemopoietic cultures. The erythroid lineage was particularly sensitive, with its extent of differentiation increasing with increasing pH. PB progenitors are more sensitive to pH variations than CB progenitors.
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PMID:Variations in culture pH affect the cloning efficiency and differentiation of progenitor cells in ex vivo haemopoiesis. 921 93

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.
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PMID:Purification, amplification and characterization of a population of human erythroid progenitors. 1051 92

One important question in stem cell biology of childhood acute lymphoblastic leukemia (ALL) is whether immature CD34+CD19- cells are part of the leukemic cell clone. CD34+CD19- cells from the bone marrow of 9 children with TEL/AML1-positive ALL were purified by flow sorting and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization, and methylcellulose cultures. In 3 of 8 patients analyzed by RT-PCR, no TEL/AML1-positive cells could be detected in the CD34+CD19- cell fraction. Altogether, the percentage of TEL/AML1-positive cells was low: 1.6% (n = 8; SD 2.2%) by nested real-time RT-PCR and 2.5% (n = 5; SD 2.6%) by fluorescence in situ hybridization. This correlated with the percentage of contaminating CD19+ leukemic cells in the CD34+CD19- cell fraction in 6 control sorts (mean 4.6%, SD 3.6%), indicating that the low levels of leukemic cells detected in the CD34+CD19- cell fraction could be attributed to sorter errors. Methylcellulose cultures in 3 patients provided further evidence that CD34+CD19- cells represent a candidate normal cell population. The clonogenicity of the CD34+CD19- cell fraction was similar to normal progenitors, including growth of primitive granulocyte, erythroid, macrophage, megakaryocyte colony-forming units. Each of 92 colonies from cultures with CD34+CD19- cells tested negative for TEL/AML1. In conclusion, our data support the hypothesis that the leukemia in TEL/AML1-positive childhood ALL originates in a CD19+ lymphoid progenitor. This has many therapeutic implications, eg, for purging of autologous stem cell products, flow cytometric monitoring of minimal residual disease, and targeting immunotherapy against the leukemic cell clone.
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PMID:Immature CD34+CD19- progenitor/stem cells in TEL/AML1-positive acute lymphoblastic leukemia are genetically and functionally normal. 1209 59