A Subset of Monocytic Cells Derived From Human Embryonic Stem Cells Can Give Rise to Mesenchymal Stromal Cells,

Abstract 3416

The relationship between monocytes and mesenchymal stromal cells remains a controversial issue. During embryonic development, the two cell types emerge early and share a large pattern of tissue expression. Using human embryonic stem cells (hES), we recently reported that embryonic monocytes/macrophages were endowed mainly with anti-inflammatory and remodeling functions. Here, we show that a subset of embryonic monocytic cells can give rise to stromal cells.

Mesoderm and hematopoietic specification of hES were achieved from embryoid bodies in Iscove's Modified Dulbecco's medium (IMDM) supplemented with 15% fetal bovine serum (FBS) in presence of Bone Morphogenetic Protein 4 (BMP-4, 10 ng/ml) and Vascular Endothelial Growth Factor (VEGF, 5 ng/ml), followed by fetal liver tyrosine kinase 3 ligand (FLT3-ligand, 10 ng/ml), stem cell factor (SCF, 50 ng/ml), interleukin-3 (IL-3, 100 U/ml) and thrombopoietin (TPO, 10 ng/ml). Between day 14–21 of culture, CD45⁺14⁺ cells were sorted, cultured for 4 days in presence of Monocyte-Colony Stimulating Factor (M-CSF, 50 ng/ml), Granulocyte-Macrophage CSF (GM-CSF, 20 ng/ml) and IL-3, and subsequently seeded on fibronectin. After culture in Endothelial Cell Growth Medium supplemented with Endothelial GF (EGF), VEGF (25 ng/ml) and bFibroblast GF (bFGF) (1 ng/ml) for 14 days, clones of adherent cells with typical fibroblast-like morphology emerged at a frequency of 2/10⁴ plated embryonic monocytic cells. In order to eliminate contaminating stromal cells before seeding on fibronectin, CD34^low43⁺ hematopoietic cells were sorted at day 10 and CD45⁺14⁺ cells were sorted 7 days later. These cells were cultured with the previously described growth factors, but in serum free medium that does not support stromal cell proliferation.

In these conditions, we observed that stromal cell developed from CD45⁺CD14⁺ embryonic monocytes. These EM-SCs shared several phenotypic and functional characteristics with adult mesenchymal stem cells (MSCs). They could be expanded in vitro in complete alpha–modified Eagle's medium (MEMa) supplemented with 10% FBS, by successive cycles of dissociation. At a density of 500 and 1000 per cm², EM-SCs formed small colonies of CFU-F. EM-SCs did not express the hematopoietic surface markers CD14 and CD45, nor the endothelial markers CD31 and KDR, and strongly expressed CD105, CD73, CD13 and CD90. In contrast with adult MSCs, they expressed CD133 and low levels of CD34. EM-SCs could not elicit a proliferative response in the presence of allogeneic lymphocytes, and exhibited a suppressive effect on T-cell proliferation in mixed lymphocyte reaction. Under appropriate conditions, EM-SCs displayed osteogenic, chondrogenic and adipogenic differentiation. They could also adopt a smooth muscle cell but not an endothelial or a cardiac phenotype.

Compared to adult MSCs, EM-SCs did not expressed telomerase reverse transcriptase, but demonstrated longer telomeres and enhanced expression of genes encoding growth factors, adhesion proteins, tissue degrading enzymes, and anti-inflammatory chemokines. EM-SCs also secreted high amounts of proteins involved in tissue remodeling and angiogenesis.

Thus, a rare subset of embryonic monocytic cells can give rise to a population of stromal cells with high immunosuppressive and remodeling functions. A large body of evidence shows that macrophages and stromal cells are involved in tumor development. It remains to be explored whether stromal cells with remodelling potential could derive from tumor-infiltrating macrophages as it can derive from embryonic macrophages. hES cells offer a valuable experimental model for in vitro studies of these differentiation pathways.