Protein Kinase C Establishes the Signaling Boundary Between Mesodermal and Extraembryonic Lineages in Human Embryonic Stem Cell Differentiation. Protein Kinase C Establishes the Signaling Boundary Between Mesodermal and Extraembryonic Lineages in Human Embryonic Stem Cell Differentiation

The interplay among mitogenic signaling pathways is crucial for proper embryogenesis. These pathways collaboratively act through intracellular master regulators to determine specific cell fates. Identifying the master regulators is critical to understanding embryogenesis and to developing new applications of pluripotent stem cells. In this report, we demonstrate protein kinase C (PKC) as an intrinsic master switch between embryonic and extraembryonic cell fates in the differentiation of human pluripotent stem cells (hPSCs). PKCs are essential to inducing the extraembryonic lineage downstream of various mitogenic modulators. PKC-alpha (PKCα) suppresses BMP4-induced mesoderm differentiation, and PKC-delta (PKCδ) is required for extraembryonic trophoblast cell fate. PKC activation overrides mesoderm induction conditions and leads to extraembryonic fate. In contrast, PKC inhibition leads to β-catenin activation, switching cell fate from extraembryonic to mesoderm lineages. This study establishes PKC as a central player directing the segregation of extraembryonic and embryonic lineages. The manipulation of intrinsic PKC activity could greatly enhance cell differentiation under mitogenic regulation in stem cell applications. Overall design: Total RNA obtained from H1 hESC cultured in E6 medium supplemented with 20 ng/ml BMP4, 100ng/ml FGF2, 5 μM GF109203X, 3 mM 2DG, 100 nM LDN193189, 5 μM CHIR99021, 50 nM TPA, 1 μM PD0325901, 10 μM SB431542 and 5 μM DAPT. Samples were collected after 6 days.