Bone Morphogenetic Protein-2/-4 Upregulation Promoted by Endothelial Cells in Coculture Enhances Mouse Embryoid Body Differentiation

Abstract

Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insulin1 (endoderm), nestin, neurofilament light (ectoderm), CD31, cardiotin, and cardiac troponin I (mesoderm) in EBs cultured alone (controls) or with ECs. A significant increase of these markers was observed in EBs cocultured with ECs compared to controls. The cocultured EBs also exhibited more robust vascular networks similar to those EBs treated with bone morphogenetic protein-2 or -4 (BMP-2 or -4). Therefore, the role of these peptides in the differentiation was investigated. We found a significant upregulation of BMP-2/-4 and BMP receptor 1A in EBs treated with EC conditioned medium (EC-CM) at early or middle stages of EB development. Recombinant human BMP-2 and BMP-4 exerted similar effects than EC-CM in the expression of BMPs or in the upregulation of the three germ layer specific markers. BMP-2/-4 antagonists, such as noggin and chordinlike-1, respectively inhibited the EC-CM inductive effects. These results demonstrate that ECs enhance the differentiation in vitro of cells that derived from the three germ layers and that BMP-2/-4 play a central role in this process.

Endothelium-derived essential signals involved in pancreas organogenesis

“…In the model using mouse EBs, we observed that ECs promote up-regulation of BMPs within EBs [53] . However, the target cells that produce these BMPs are still unknown.…”

“…The role of BMPs in pancreas development has also been explored previously [72,93] . We demonstrated that HMECs or mouse dermis as well as mouse AECs expressed BMPs and that BMP-2 and BMP-4 increased the phosphorylation levels of SMAD1,5,8 in pancreatic progenitors and beta-like cells derived from mouse ESCs [52,53,94] . These findings together with previous works pointed out the important role of ECs in beta-cell differentiation in vitro.…”

“…For instance, high signaling of the canonical WNT/β-catenin pathway promotes endoderm posterior pattern with foregut-derived structures such as pancreas and liver [68] . Interestingly it has been found that some of these factors are produced by ECs [13,42,53,[69][70][71] .…”

“…Although some epithelial cells appeared, no cells derived from endoderm were identified [90] . Then, studies using co-cultures between human microvascular ECs (HMECs) and mouse EBs were performed [52,53,91] . In these studies, ECs from human dermis were able to induce differentiation of mouse EBs to pancreatic progenitors and insulin-producing cells [52] .…”

“…BMPs are members of the TGF-b superfamily [92] . In addition to the effects of BMP antagonists, we explored the levels of phosphorylation of SMAD1, 5, 8 in cells that expressed proinsulin [52,53] . The role of BMPs in pancreas development has also been explored previously [72,93] .…”