Enhancement of embryonic stem cell differentiation promoted by avian chorioallantoic membranes.

Avian chorioallantoic membrane (CAM) has been used as a model to explore angiogenesis and to study the microvasculature of transplanted tissues. Because CAM provides a vascular bed, cells can be implanted, and their development can be monitored and modified. We used the CAM model to study the differentiation process of embryoid bodies (EBs) derived from mouse embryonic stem cells (ESCs) influenced by the CAM vascular bed. After EBs were incubated in CAM for 5 days, they underwent further differentiation and became tissue masses (TMs) of different morphologies from those that grew outside CAM. Immunohistochemical analysis of TMs demonstrated tissue-specific markers such as neurofilament light, CD34, collagen IV, cardiac myosin heavy chain (MHC), and cardiotin. Differentiated mouse blood vessels stained with anti-CD31 were found within the TMs, as well as blood vessels stained positive for QH1 and QCPN, markers for quail endothelial cells and perinuclear quail antigen, respectively. Quail erythrocytes inside mouse blood vessels suggested a connection between existing quail vessels and blood vessels growing inside the TMs as a result of EB differentiation. Therefore, CAM could be a suitable model to trigger and study the differentiation of EBs in close interaction with surrogate quail blood vessels.