Akel Salem, Petrow-Sadowski Cari, Laughlin Mary J, Ruscetti Francis W
Leukocyte Biology Section, Basic Research Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702-1201, USA.
Stem Cells. 2003;21(5):557-67. doi: 10.1634/stemcells.21-5-557.
Transforming growth factor (TGF)-beta1 exerts autocrine and paracrine effects on hematopoiesis. Here, we have attempted to evaluate the effect of endogenous TGF-beta1 on early erythroid development from primitive human hematopoietic stem cells (HSCs) and to assess the effects of TGF-beta1 on different phases of erythropoiesis. Cord blood CD34(+)CD38(-) lineage-marker-negative (Lin(-)) cells were cultured in serum-free conditions using various combinations of stem cell factor (SCF), erythropoietin (Epo), and TGF-beta-neutralizing antibody. Generation of erythroid progenitors was assessed using colony assay and flow cytometry. Terminal erythroid differentiation was examined when SCF/Epo-stimulated cells were recultured in the presence of Epo with and without TGF-beta1. Anti-TGF-beta augmented the proliferation of CD34(+)CD38(-)Lin(-) cells (day 21) in SCF-stimulated (6.4-fold +/- 1.5-fold) and SCF/Epo-stimulated (2.9-fold +/- 1.2-fold) cultures. Cells stimulated by SCF/Epo underwent similar levels of erythroid differentiation with and without anti-TGF-beta. While SCF alone stimulated the production of tryptase-positive mast cells, cells stimulated by SCF/anti-TGF-beta were predominantly erythroid (CD36(+)CD14(-) and glycophorin A positive). A distinct expansion of erythroid progenitors (CD34(+)CD36(+)CD14(-)) with the potential to form erythroid colonies was seen, revealing early Epo-independent erythroid development. In contrast, the kinetics of erythroid progenitor generation from primitive HSCs indicate that TGF-beta1 is not inhibitory in late erythropoiesis, but it accelerated the conversion of large BFU-E into colony-forming units-erythroid. Finally, TGF-beta1 accelerated Epo-induced terminal erythroid differentiation and resulted in a greater level of enucleation (22% +/- 6% versus 7% +/- 3%) in serum-free conditions. Serum addition stimulated enucleation (54% +/- 18%), which was lower (26% +/- 14%) with anti-TGF-beta, suggesting that optimal erythroid enucleation is Epo dependent, requiring serum factors including TGF-beta1.
转化生长因子（TGF）-β1对造血作用发挥自分泌和旁分泌效应。在此，我们试图评估内源性TGF-β1对原始人类造血干细胞（HSCs）早期红系发育的影响，并评估TGF-β1对红系生成不同阶段的作用。脐血CD34(+)CD38(-) 谱系标志物阴性（Lin(-)）细胞在无血清条件下，使用干细胞因子（SCF）、促红细胞生成素（Epo）和TGF-β中和抗体的各种组合进行培养。使用集落测定法和流式细胞术评估红系祖细胞的生成。当SCF/Epo刺激的细胞在有或无TGF-β1的Epo存在下重新培养时，检查终末红系分化。抗TGF-β增强了SCF刺激（6.4倍±1.5倍）和SCF/Epo刺激（2.9倍±1.2倍）培养物中CD34(+)CD38(-)Lin(-)细胞（第21天）的增殖。有无抗TGF-β时，SCF/Epo刺激的细胞经历相似水平的红系分化。虽然单独的SCF刺激色氨酸酶阳性肥大细胞的产生，但SCF/抗TGF-β刺激的细胞主要是红系细胞（CD36(+)CD14(-)和血型糖蛋白A阳性）。可见具有形成红系集落潜力的红系祖细胞（CD34(+)CD36(+)CD14(-)）明显扩增，揭示了早期不依赖Epo的红系发育。相反，原始HSCs产生红系祖细胞的动力学表明，TGF-β1在晚期红系生成中无抑制作用，但它加速了大型爆式红系集落形成单位（BFU-E）向红系集落形成单位（CFU-E）的转化。最后，TGF-β1加速了Epo诱导的终末红系分化，并在无血清条件下导致更高水平的去核（22%±6%对7%±3%）。添加血清刺激去核（54%±18%），抗TGF-β时较低（26%±14%），表明最佳红系去核依赖Epo，需要包括TGF-β1在内的血清因子。
