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  • Previously RA and FGF have been used together to induce

    2018-10-22

    Previously, RA and FGF-2 have been used together to induce neuronal differentiation. FGF-2 has been introduced not only during the early stages of neurogenesis but also to regulate processes that occur in the later stages, such as the caudalization of neuronal tissues, motor neuron specification and the enhancement of the proliferation of neuronal progenitor phosphodiesterase inhibitors (Baharvand et al., 2007). The external addition of RA to trophoblast stem cells augments the formation of giant cells and causes a reduction in spongiotrophoblasts via the expression of RARĪ² in trophoblast cell fate specification during placentation (Yan et al., 2001). Homogeneous expression of FGF and its receptors was observed in bovine trophoblast giant cells, and the external addition of growth factors in the presence of extra cellular matrix on blastocysts synergistically promotes trophoblast outgrowth and could influence implantation (Haimovici and Anderson, 1993; Pfarrer et al., 2006). The ExE lineage which is essential for the survival of the embryo in the uterine environment arises from trophectoderm and primitive endoderm. These two types of cell lineage largely contribute to the ExE membranes, such as the placenta and the yolk sac. A number of signaling pathways and transcription factors are implicated in the perpetuation of these lineages (Rossant, 1995). During pre-implantation and conceptus development, bovine trophoblast cell migration, reorganization and morphogenesis are influenced by the FGF-2 and FGF-10. The interaction between FGF and RA has been successfully utilized previously for anteriorā€“posterior neural patterning, endoderm patterning and the initiation of differentiation in vertebrate body axis formation in extending limbs using different in vitro methods (Johannesson et al., 2009; Olivera-Martinez and Storey, 2007; Shiotsugu et al., 2004). We hypothesized that the synergistic effect of two mutually opposing morphogens in early embryonic development in hESCs may at least partially elucidate the mechanism of ExE lineage development. To elucidate the combinatorial effect of RA and FGF-2 on the lineage decisions of hESCs and to identify mechanisms by which RA might bypass the self-renewal signaling pathway induced by FGF-2 in hESCs to thereby promote ExE lineage differentiation, we investigated the effect of RA in the presence and absence of FGF-2 using global expression microarrays.
    Methods
    Results
    Discussion The FGF-2 signaling pathway is involved in several processes, such as self-renewal, survival, and adhesion and depending on additional factors, induces differentiation toward certain lineages, including trophoblast outgrowth (Eiselleova et al., 2009; Haimovici and Anderson, 1993). The differentiation of hESCs toward trophoblasts lineage in the absence of FGF-2 can be induced by the knockdown of genes such as SOX2 and NANOG (for review see Schulz et al., 2008). The siRNA silencing of POU5F1 and conditions such as the absence of FGF-2 result in the expression of ExE markers, such as BMP4, CGA, CGB, EOMES, ID2 and GATA2 (Hay et al., 2004; Matin et al., 2004). NANOG is a vital gene for the maintenance of pluripotency and is responsible for deciding lineage outcome. The knockdown of NANOG combined with the addition of FGF-2 has previously been reported to induce the differentiation of hESCs into the ExE lineage (Vallier et al., 2005; Yu et al., 2011). An analogous study reported that the downregulation of NANOG results in an upregulation of ExE lineage markers (Hyslop et al., 2005). TDGF1 (CRIPTO), a co-receptor of NODAL, is expressed in explants treated with BMP4. Thus, NODAL induction by BMP4 may induce TDGF1 in the ExE lineage (Beck et al., 2002). Previously, it was shown that exogenous addition of RA in trophoblast stem cells enhances the expression of RAR family markers and augments the differentiation of these cells into trophoblast giant cells (Yan et al., 2001). In the current study, instead of the genetic manipulation of pluripotent genes, we applied RA as a differentiation inducer of hESCs in the presence of FGF-2 to induce ExE lineage differentiation. Downregulation of pluripotency genes such as NANOG, TDGF1, SOX2, FOXD3, LIN28, and PODXL and upregulation of ExE markers such as CGA, MSX2, GATA3, KRT7, and MUC15 were found upon RA treatment in the presence of FGF-2, providing evidence for the exit of pluripotency and the initiation of the ExE differentiation pattern. These findings suggest that exogenous FGF-2, which is essential for maintaining pluripotency, cannot sustain the self-renewal potential of hESCs in the presence of RA. Therefore, we hypothesize that RA affects the FGF-2 signaling pathway and directs hESC differentiation toward the ExE lineage.