Similar to mouse embryonic stem cells (ESCs) and the more recently derived mouse epiblast stem cells (EpiSCs) [1
], human ESCs proliferate without limit and maintain the potential to differentiate into advanced derivatives of all three embryonic germ layers: ectoderm, mesoderm and endoderm [3
]. In mice, ESCs closely resemble the inner cell mass from which they are derived, and EpiSCs closely resemble the later postimplantation epiblast from which they are derived [1
]. Human ESC growth in polarized, epithelial colonies, and the particular growth factors that promote human ESC self-renewal, such as transforming growth factor β/nodal/activin and basic fibroblast growth factor [6
], suggest that human ESCs more closely resemble the primitive ectoderm than the earlier inner cell mass stage. However, although generally similar, the gene expression profiles and growth factor responses of human ESCs do differ in some significant ways from mouse EpiSCs [2
Interestingly, under some culture conditions, the addition of bone morphogenetic proteins (BMPs) cause human ESCs to differentiate into cells that uniformly express trophoblast-specific genes, secrete progesterone and estradiol into the culture medium, and form syncytia with hundreds of nuclei with a concomitant upregulation of chorionic gonadotropin, suggesting a homogeneous trophoblast differentiation [8
]. This is somewhat surprising because if human ESCs more closely resemble the primitive ectoderm than the earlier inner cell mass stage, they should have lost the capability of trophoblast differentiation. A recent study calls into question the identity of the in vitro produced trophoblast-like cells, and an extraembryonic mesoderm cell has been proposed as a more likely in vivo counterpart [10
]. However, although that study raises significant questions about the identity of the BMP-induced cells, because it has not been possible to study the molecular signatures of these peri-implantation human lineages in appropriately staged intact embryos, the actual in vivo counterpart still remains somewhat in doubt. Thus, further elucidating the developmental status and differentiation potential of human ESCs remains important to understand the stage of their in vivo counterpart. Here, we show that activation of the protein kinase C (PKC) pathway causes human ESCs to differentiate to a second extraembryonic lineage, the primitive endoderm, which provides another example suggesting that human ESCs can differentiate into not only three germ layers but also some extraembryonic tissues.
PKC is a serine-threonine kinase family that consists of 11 different isotypes [11
]. These PKC isozymes are further divided into three subclasses based on their second messenger requirements: conventional, novel, and atypical. PKC isozymes have different tissue distribution, subcellular localization, cofactor dependence, and substrate specificity; therefore, they exert various roles in cell proliferation, differentiation, apoptosis, and angiogenesis. Phorbol ester 12-O
-tetradecanoylphorbol 13-acetate (TPA) activates conventional and novel PKCs, but not atypical PKCs, by mimicking the function of the endogenous secondary messenger, diacylglycerol. Interestingly, in some situations, TPA can also reduce PKC activity by enhancing the degradation of PKC [12
]. By increasing or decreasing overall PKC activity, TPA induces a wide range of biological effects in vitro and in vivo, including the induction of epithelial-mesenchymal transition (EMT) [13
EMT is a process in which cells transition from a polarized, tightly connected epithelial phenotype to an individualized, nonpolarized, and migratory mesenchymal phenotype. EMT is a fundamental process for both normal development and carcinoma progression. In the development of the mouse embryo, the earliest EMTs lead to the formation of the three germ layers through gastrulation and differentiation of parietal endoderm from primitive endoderm [14
]. Parietal endoderm is one of the two sublineages of primitive endoderm, which exhibits distinct differences in morphology and marker gene expression compared with the other lineage, visceral endoderm [15
]. In recent studies of induced pluripotent stem cells, it was also found that EMT blocks reprogramming while the opposite process of EMT, mesenchymal-epithelial transition, not only facilitates but also is required for the reprogramming of fibroblasts [16
]. Previously, it has also been shown that TPA disrupts gap junction communication in human ESCs [17
], an early event in EMT.
To examine the effect of PKC activation on human ESCs, we treated the cells with TPA and examined the gene expression profile along a 7-day time course. TPA induced fast EMT of human ESCs, accompanied by dramatic downregulation of pluripotency gene OCT4
and upregulation of multiple endoderm markers without previously expressing primitive streak markers such as brachyury [18
]. We further identified that only certain PKC subtype plays essential role in this TPA-induced differentiation, which suggests that the 11 PKC isotypes may have distinct functions in human ESCs. Overall, this and some previous reports [9
] suggest that human ESCs have the ability to differentiate to both extraembryonic lineages normally differentiating from the embryo prior to implantation, the trophoblast and the extraembryonic endoderm.