The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

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.

Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.