Angiotensin II type 1 and bradykinin 132 receptors expressed in early stage epithelial cells derived from human embryonic stem cells

When human embryonic stem (hES) cells were placed into suspension culture followed by culture on BID matrigel (TM) coated plates in the presence of medium conditioned by NIH-3T3 cells, they differentiated into cells of which more than 95% stained positive for keratin 8 by day 14, demonstrating that the hES cells had committed to an epithelial lineage. Approximately 50% of the keratin 8 staining cells became positive for cytokeratin 14 after 26 days. Binding experiments supported by real time PCR showed that the expression of bradykinin B2 (BKB2) and angiotensin II type I (AT I) receptors accompanied this differentiation. Neither receptor was expressed in the pluripotent H9 stem cells. However, transduction of the hES cells with lentivirus containing BKB2 or ATIR cDNA resulted in ligand binding and ERK 1/2 activation but not in G alpha i or G alpha q coupled signaling. In the differentiated cells, both BKB2R and ATIR were expressed constitutively and effected typical Gai and Gaq coupled signaling characterized by the release of arachidonate, generation of inositol phosphates, and Ca2+ mobilization. These signals were abolished by the receptor antagonists, losartan, and HOE 140. Angiotensin II and bradykinin also stimulated the phosphorylation of ERK 1/2, JNK 1/2, and p70S6 in the differentiated cells. Our results demonstrate that human embryonic stem cells can be differentiated effectively into the epithelial lineage and that when differentiated express functional, signaling ATI and BKB2 receptors.