Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors  Yael Zermati, Serge Fichelson, Françoise Valensi, Jean Marc Freyssinier, Philippe Rouyer-Fessard, Elizabeth Cramer, Josette Guichard, Bruno Varet, Olivier Hermine  Experimental Hematology  Volume 28, Issue 8, Pages 885-894 (August 2000) DOI: 10.1016/S0301-472X(00)00488-4

Figure 1 TGF-β1 inhibits proliferation of adult CD36+ cells. CD36+ cells (105/ml) were grown in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL). Bottom left inset shows growth curve during the first 4 days of culture. Viable cells were assessed daily by blue trypan staining. Cells were diluted every 2 or 3 days to 105 viable cells/ml. Means of cumulative cell numbers of five representative experiments are shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 2 TGF-β1 accelerates and increases expression of glycophorin A (GPA) and hemoglobin of adult CD36+ cells. Adult CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL) and analyzed for GPA expression and hemoglobin at various times as indicated. (A) Expression of GPA on cell surface was assessed by fluorescence cytometric analysis as described in the Materials and methods section. Irrelevant IgG1 antibody was used as the negative control. A representative experiment of five experiments is shown. Percentages of highly positive cells are indicated. (B) The relative number of hemoglobinized cells was assessed by benzidine staining. Means of percentages of benzidine-positive cells of five representative experiments are shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 3 TGF-β1 accelerates and allows full terminal erythroid differentiation including enucleation of adult CD36+ cells. Cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL), and May-Grünwald-Giemsa stained cytospins were analyzed at various times as indicated. The starting population (day 0) consisted of immature blasts cells. Mature erythroid cells appeared earlier in the presence of TGF-β1 Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 4 Ultrastructural analysis of erythroblasts generated from CD36 progenitors in the presence of SCF+IL-3+Epo+-TGF-β1. CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) for 7 days. After washing, the cells were incubated in the medium of Graham and Karnovsky to enhance the electron density of hemoglobin. (A) Some erythroblasts (E) are still in the process of maturing, having reached different maturation stages with various sizes, chromatin clumping, and charge in hemoglobin proportional to electron density [basophilic (left), polychromatophilic (bottom), orthochromatic (top) normoblasts]. Other culture cells seem to have expelled their nucleus (arrowheads), and naked nuclei (N) were found occasionally in the culture dish. No macrophages were observed (magnification ×2970). (B) Naked nuclei (N) are observed in the vicinity of cells deprived of nuclei and highly charged in hemoglobin as assessed by a dense benzidine staining similar to red blood cells (RBC) (magnification ×13,220) Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 5 Effect of TGF-β1 on absolute number of mature erythroblasts. Adult CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL). The absolute number of mature erythroid cells (polychromatic normoblasts, orthochromatic normoblasts, and erythrocytes) was determined after May-Grünwald-Giemsa staining. Means of five representative experiments are shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 6 TGF-β1 increases hemoglobin production per cell and does not change pattern of hemoglobin synthesis of adult and fetal CD36+ cells. CD36+ cells derived from CD34+ adult peripheral blood cells (PBMC) or cord blood cells (CB) were cultured for 7 days in the presence of SCF (50 ng/ml) + IL-3 (10 ng/ml) + Epo (2 UI/ml) with or without TGF-β1 (2 ng/ml). In all conditions of culture, 5 × 105 cells were pelleted and lysed, and hemoglobin pattern was analyzed by HPLC as described in the Materials and methods section. Profile of one of three representative experiments is shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 7 TGF-β1 does not induce massive apoptosis of adult CD36+ cells. Adult CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL). Apoptosis was assessed by annexin-V binding at different times as indicated in the Materials and methods section. Means of five representative experiments are shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 8 TGF-β1 induces cell cycle arrest of adult CD36+ cells. Adult CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL). Percentages of cell in G0/G1 or S phases were assessed at different times as indicated by analysis of the DNA content after propidium iodid staining and plotted. A representative experiment of five is shown Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)

Figure 9 TGF-β1 reduces the number of erythroid precursors in cycle. Adult CD36+ cells were cultured in the presence of SCF (50 ng/mL) + IL-3 (10 ng/mL) + Epo (2 UI/mL) with or without TGF-β1 (2 ng/mL). Cell in cycle was assessed by Ki-67–labeled cytospin, at various times as indicated. At day 0, immature cells weakly expressed Ki-67 antigen. Without TGF-β1, immature cells reentered rapidly in cycle. At day 3, while immature cells were still strongly positive, the few mature erythroblasts did not express Ki-67 antigen. In contrast, TGF-β1 decreased expression of Ki-67 antigen on immature cells and increased the number of mature erythroblasts not expressing Ki-67 antigen Experimental Hematology 2000 28, 885-894DOI: (10.1016/S0301-472X(00)00488-4)