Berthier R, Prandini M H, Schweitzer A, Thevenon D, Martin-Sisteron H, Uzan G
CEA, Laboratoire d'Hématologie, INSERM U217, Grenoble, France.
Exp Hematol. 1997 Jun;25(6):481-90.
Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.
通过使用适当的培养条件，小鼠胚胎干细胞能够分化为红系、肥大细胞系以及粒单核细胞系细胞。由于我们对血小板糖蛋白IIb基因的组织特异性表达调控感兴趣，因此我们研究了培养条件，旨在从胚胎干细胞中可重复地产生包括巨核细胞（MKs）在内的髓系细胞。我们发现，即使是多种生长因子（干细胞因子、白细胞介素3、白细胞介素6、白细胞介素11、粒细胞集落刺激因子和促红细胞生成素）的复杂组合，也无法在第12天的胚状体中诱导显著的髓系分化。MS-5基质细胞与胚胎干细胞的共培养比生长因子的效果稍好。当我们将MS-5细胞与生长因子组合使用时，观察到对髓系集落和巨核细胞的生长有强烈的协同作用。来自MS-5细胞的条件培养基也与生长因子组合协同作用，产生大量含有巨核细胞的混合集落。在生长因子组合加上MS-5的基础上添加成纤维细胞生长因子-2（FGF-2），包括含有巨核细胞的髓系祖细胞数量几乎增加了一倍。单独使用血小板生成素（TPO）或与MS-5或生长因子以任何组合使用，都不会增加含巨核细胞集落的数量。然而，当将TPO添加到生长因子组合+FGF-2+MS-5中时，液体培养物和混合集落中的巨核细胞数量增加，并且许多呈现出类似伪足状前血小板形成的“多毛”外观。在确定了能够产生包括巨核细胞在内的所有髓系谱系的胚胎干细胞培养条件后，我们得出结论，胚胎干细胞的造血分化模型对于研究早期造血过程中任何重要基因的表达调控特别有用。
