de Wolf J T, Muller E W, Hendriks D H, Halie R M, Vellenga E
Department of Hematology, University Hospital Groningen, The Netherlands.
Blood. 1994 Jul 1;84(1):59-64.
To study the differentiation process of erythroid progenitors from normal human bone marrow and peripheral blood, CD34/CD36 sorted cells were cultured in the presence of Erythropoietin (Epo) and Epo plus mast cell growth factor (MGF). The CD34+/CD36- cell fraction from bone marrow supported 74 +/- 33 erythroid burst forming units (BFU-E)/10(4) cells (mean +/- SD, n = 4) in the presence of Epo, which increased 2.1-fold by coculturing with MGF. However, erythroid colony-forming units (CFU-E) were not cultured from the CD34+/CD36- cell fraction. In contrast, the CD34-/CD36+ cell fraction supported CFU-Es in the presence of Epo (152 +/- 115/10(5)) or Epo plus MGF (180 +/- 112/10(5)), whereas BFU-Es were hardly noticed. However, the transition of the BFu-E to CFU-E was observed by incubating CD34+/CD36- cells (10(4)/100 microL) in suspension with Epo plus MGF for 7 days followed by Epo in the colony assay. This was reflected by the appearance of CD34-/CD36+/Glycophorin A+/CD14- cells. In addition high numbers of CFU-Es (1,000 +/- 150, n = 4) were cultured from this cell fraction. In contrast to bone marrow erythroid progenitors, no peripheral blood CFU-Es were cultured from either the CD36+ or CD36- fraction, whereas BFU-Es were predominantly present in the CD36+ fraction. However, the CD34+ progenitor cell from peripheral blood did have intrinsic capacity to differentiate to CFU-Es because CD34+/CD36- cells incubated with Epo plus MGF for 7 days and followed by Epo in the colony assay, supported high numbers of CFU-Es (1,200 +/- 400, n = 3). To study whether additional growth factors have similar effects on erythroid progenitors, experiments were performed with interleukin 1 (IL-1), IL-3, and IL-6. IL-1 and IL-6 did not modulate the Epo supported proliferation and differentiation. In contrast, IL-3 in the presence of Epo did support CFU-Es, from CD34+/CD36- cells after 7 days in suspension culture. However, flow cytometry analysis showed that Epo plus IL-3 not only supported CD34-/CD36+/Glycophorin A+ cells but also CD36+/CD14+ cells, indicating the differentiation along different cell lineages. In summary, the data show a phenotypic distinction between bone marrow and peripheral blood erythroid progenitors with regard to CD36 expression. In addition, the results suggest that Epo plus MGF or IL-3 and preincubation in suspension culture are prerequisites for the transition of the BFU-E to the CFU-E.
为研究正常人骨髓和外周血中红系祖细胞的分化过程，将经CD34/CD36分选的细胞在促红细胞生成素（Epo）以及Epo加肥大细胞生长因子（MGF）存在的条件下进行培养。在Epo存在时，骨髓来源的CD34⁺/CD36⁻细胞亚群每10⁴个细胞可支持74±33个红系爆式集落形成单位（BFU-E）（均值±标准差，n = 4），与MGF共培养后增加2.1倍。然而，CD34⁺/CD36⁻细胞亚群未能培养出红系集落形成单位（CFU-E）。相反，CD34⁻/CD36⁺细胞亚群在Epo存在时（152±115/10⁵）或Epo加MGF时（180±112/10⁵）可支持CFU-E形成，而BFU-E几乎未被检测到。然而，通过将CD34⁺/CD36⁻细胞（10⁴/100 μL）与Epo加MGF悬浮培养7天，随后在集落测定中加入Epo，可观察到BFU-E向CFU-E的转变。这表现为CD34⁻/CD36⁺/血型糖蛋白A⁺/CD14⁻细胞的出现。此外，从该细胞亚群培养出了大量CFU-E（1000±150，n = 4）。与骨髓红系祖细胞不同，外周血中无论是CD36⁺还是CD36⁻亚群均未培养出CFU-E，但BFU-E主要存在于CD36⁺亚群中。然而，外周血来源的CD34⁺祖细胞具有分化为CFU-E的内在能力，因为将CD34⁺/CD36⁻细胞与Epo加MGF培养7天，随后在集落测定中加入Epo，可支持大量CFU-E形成（1200±400，n = 3）。为研究其他生长因子对红系祖细胞是否有类似作用，使用白细胞介素1（IL-1）、IL-3和IL-6进行了实验。IL-1和IL-6未调节Epo支持的增殖和分化。相反，在悬浮培养7天后，Epo存在时IL-3确实可支持CD34⁺/CD36⁻细胞形成CFU-E。然而，流式细胞术分析显示，Epo加IL-3不仅支持CD34⁻/CD36⁺/血型糖蛋白A⁺细胞，还支持CD36⁺/CD14⁺细胞，表明细胞沿着不同细胞谱系分化。总之，数据显示骨髓和外周血红系祖细胞在CD36表达方面存在表型差异。此外，结果表明Epo加MGF或IL-3以及悬浮培养预孵育是BFU-E向CFU-E转变的前提条件。
