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Summary. We examined the influence of cytokines on erythroid- and myeloid-lineage development of AC133+ cells during primary and secondary cultures. Cells cultured for 14 d in liquid medium containing erythropoietin (EPO) were amplified 831-fold with 98·2% erythroid cells. A similar culture exposed to granulocyte colony-stimulating factor (G-CSF) grew 1350-fold with 97·4% myeloid cells. To assess whether the cells with EPO inducement could respond at this point to G-CSF signal, or vice versa, the EPO-stimulated population was re-grown with G-CSF, constituting 95·2% myeloid, of 5075-fold, cells after 14 d of re-culture. Conversely, reculture of the G-CSF-stimulated population with EPO resulted in a 4083-fold growth with 81·4% erythroid cells. Semisolid culture containing EPO orG-CSF showed that some individual colonies had self- renewal potential after 14 d culture and could be induced todevelop into a different lineage. Analysis of primitive markers, CD34 and Notch1, or lineage markers, EPO-R and CD13, by single-cell reverse transcription polymerase chain reaction showed that individual colonies of 2–16 cells contained at least one CD34-positive cell with expression ofNotch1 and co-expression of EPO-R and CD13 appeared on either CD34-positive or CD34-negative cells. In situ hybridization with the same cell surface markers in cell populations confirmed the asymmetric cell division and co-expression from single cell data. The study provides a useful model for the analysis of multipotential progenitor development, and indicates that progenitor cells co-express genes from different lineage pathways before commitment and that cytokines influence lineage commitment. |
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