Induction of megakaryocyte colonies with platelet formation in vitro

McLeod, Donald W.; Shreeve, Mona M.; Axelrad, Arthur A.

doi:10.1038/261492a0

Cited by 177 publications

(69 citation statements)

References 4 publications

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“…The agar method of 38) was used in early experiments. The culture medium consists of agar dissolved in aMEM (GIBCO) and added to medium at final concentration of 0.37%/20% fetal bovine serum (FBS)/1% bovine serum albumin (BSA) (fraction V, Miles)/20 ,Ag of L-asparagine per ml/25 ,ug of CaCl2 per ml in NCTC 109 (GIBCO).…”

Section: Methodsmentioning

confidence: 99%

Interleukin 3 promotes erythroid burst formation in "serum-free" cultures without detectable erythropoietin.

Goodman¹,

Hall²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Erythroid burst-forming units (BFU-E) from mouse bone marrow were grown for 7 days in agar or serumfree methylcellulose cultures in the presence or absence of erythropoietin (Ep) and/or interleukin 3 (IL-3). It was found that IL-3, even in the absence of serum and detectable Ep, was able to stimulate the full development of many erythroid bursts. This IL-3 effect was cell-dose dependent and did not appear to correlate with Ep dose. Spontaneous bursts and those stimulated by Ep only were rare and when seen were very small relative to those produced by IL-3 or IL-3 plus Ep. When addition of IL-3 or Ep to 7-day cultures was delayed, IL-3 but not Ep was shown to maintain BFU-E. No evidence was found by radioimmunoassay that Ep was produced or released in 7-day, "serum-free" cultures of bone marrow nor was Ep activity detected in culture media except those to which it had been added deliberately.Interleukin 3 (IL-3) began its interesting history as a factor believed to regulate early T-cell differentiation. This was based on the observation by Ihle that a factor in conditioned medium from activated T cells had the ability to induce expression of 20a-hydroxysteroid-dehydrogenase, an enzyme uniquely associated with the T-cell lineage, in splenic lymphocytes from athymic nude mice (1, 2). Ihle et al. (3) proposed that this factor, a glycoprotein of Mr 28,000, be called IL-3 and has since purified it to homogeneity (4). Activated T cells are believed to be the major source of IL-3, although WEHI-3, classified as a myelomonocytic leukemic cell line, produces it constitutively. In the short time since WEHI-3 was shown to produce IL-3, it has been found that the factor also has the ability to stimulate the growth in vitro of many kinds of hemopoietic cells (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and it has been called by some a multispecific colony-stimulating factor (20).It has been shown to be identical with Schrader's P-cellstimulating factor (21) and with stem cell-activating factor (22).It has been recognized for some time that early erythroid progenitors require a second factor, burst-promoting activity (BPA), in addition to the hormone erythropoietin (Ep) for their full development in culture (23-26). The ability of IL-3 to stimulate erythropoiesis has been attributed to its activity as a burst promoter in the production of differentiated erythrocytes in vitro from the burst-forming progenitor or erythroid burst-forming units (BFU-E) (17, 27). It has been reported that under some circumstances "Ep-independent" BFU-E can develop (27-34). Evaluation of the site and nature of action of both BPA and Ep have, until recently, been burdened by the lack of highly purified material and by the presence of unknown quantities of BPA and Ep in constituents of culture media. Some crude preparations of Ep have been found to contain BPA as a contaminant (35). Now that a completely "serum-free" system has been devised (36) for the in vitro growth of erythroid bursts, it is possible to seek experimental answ...

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Section: Methodsmentioning

confidence: 99%

Interleukin 3 promotes erythroid burst formation in "serum-free" cultures without detectable erythropoietin.

Goodman¹,

Hall²,

Miller³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Bone marrow and splenic cell morphology were examined microscopically, following cytocentrifugation and Wright's Giemsa staining with, or without, neutral benzidine staining (McLeod et al, 1974). Flow cytometry was employed to assess cell surface expression of Ter119 (phycoerythrin-conjugated antimouse Ter119, BD Biosciences) and c-kit (fluorescein isothiocyanate-conjugated anti-mouse CD117, BD Biosciences), as detailed previously (Sarna et al, 2003) using an Epics XL/ MCL flow cytometer (Beckman-Coulter, Palo Alto, CA, USA).…”

Section: Cell Culturementioning

confidence: 99%

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

et al. 2004

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In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn À/À mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn À/À erythroblasts, and the phenotype of Lyn À/À animals was remarkably similar to GATA-1 low animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn À/À animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn À/À mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

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“…The intensities of the protected 237nt PU.1 RNA fragment and the PU.1 protein at the indicated times were determined relative to those obtained from untreated (0 h) cells which were assigned a value of 100. c-myc mRNA hybridization intensities were normalized to the intensities of hybridization with a 28S rRNA probe in each lane and Myc mRNA levels were calculated relative to that obtained with RNA from untreated (0 h) cells which was assigned a value of 100. Cell commitment to terminal di erentiation was determined by plasma clot assay (McLeod et al, 1974). Brie¯y, MEL cells treated with HMBA for the times indicated were plated in plasma clots at 100 ± 200 cells/well in microtiter plates without HMBA.…”

Section: Expression Of Pu1 Is Down Regulated As Mel Cells Commit To mentioning

confidence: 99%

Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation

et al. 1997

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Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are blocked from completing the late stages of erythroid di erentiation. A frequent event in the generation of these malignant cells is deregulation of the hematopoietic-speci®c transcription factor PU.1 (Spi-1) by retroviral insertion of the spleenfocus-forming virus component of Friend virus. During chemically induced reinitiation of MEL cell terminal di erentiation, expression of PU.1 is rapidly downregulated, suggesting that PU.1 might interfere with processes required for terminal di erentiation of erythroid precursors. To investigate the role of PU.1 in erythroid di erentiation we transfected MEL cells with a PU.1 cDNA controlled by the eucaryotic translation elongation factor EF1a promoter. Deregulated expression of PU.1 blocked chemically induced di erentiation and terminal cell division. Deregulated expression of two other protooncogenes, c-myc and c-myb, also has been shown to block MEL di erentiation. We present evidence that PU.1 inhibits terminal di erentiation at an earlier step than c-Myc and c-Myb. Thus reinitiation of MEL cell terminal di erentiation appears to be controlled by an ordered program of turning o several protooncogenes. Down-regulation of PU.1 may be a very early step in this program.

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Induction of megakaryocyte colonies with platelet formation in vitro

Cited by 177 publications

References 4 publications

Interleukin 3 promotes erythroid burst formation in "serum-free" cultures without detectable erythropoietin.

Interleukin 3 promotes erythroid burst formation in "serum-free" cultures without detectable erythropoietin.

Lyn deficiency reduces GATA-1, EKLF and STAT5, and induces extramedullary stress erythropoiesis

Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation

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