Ketamine enhances structural plasticity in mouse mesencephalic and human iPSC-derived dopaminergic neurons via AMPAR-driven BDNF and mTOR signaling.

Among neurobiological mechanisms underlying antidepressant properties of ketamine, structural remodeling of prefrontal and hippocampal neurons has been proposed as critical. The suggested mechanism involves downstream activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which trigger mammalian target of rapamycin (mTOR)-dependent structural plasticity via brain-derived neurotrophic factor (BDNF) and protein neo-synthesis. We evaluated whether ketamine elicits similar molecular events in dopaminergic (DA) neurons, known to be affected in mood disorders, using a novel, translational strategy that involved mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. Sixty minutes exposure to ketamine elicited concentration-dependent increases of dendritic arborization and soma size in both mouse and human cultures as measured 72 hours after application. These structural effects were blocked by mTOR complex/signaling inhibitors like rapamycin. Direct evidence of mTOR activation by ketamine was revealed by its induction of p70S6 kinase. All effects of ketamine were abolished by AMPA receptor antagonists and mimicked by the AMPA-positive allosteric modulator CX614. Inhibition of BDNF signaling prevented induction of structural plasticity by ketamine or CX614. Furthermore, the actions of ketamine required functionally intact dopamine D3 receptors (D3R), as its effects were abolished by selective D3R antagonists and absent in D3R knockout preparations. Finally, the ketamine metabolite (2R,6R)-hydroxynorketamine mimicked ketamine effects at sub-micromolar concentrations. These data indicate that ketamine elicits structural plasticity by recruitment of AMPAR, mTOR and BDNF signaling in both mouse mesencephalic and human induced pluripotent stem cells-derived DA neurons. These observations are of likely relevance to the influence of ketamine upon mood and its other functional actions in vivo.

Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17).

The human embryonic stem cell line RCe021-A (RC-17) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe013-A (RC-9).

The human embryonic stem cell line RCe013-A (RC-9) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11).

The human embryonic stem cell line RCe015-A (RC-11) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Developmental differences in the expression of FGF receptors between human and mouse embryos.

INTRODUCTION: Fibroblast growth factor (FGF) signaling is essential for early trophoblast expansion and maintenance in the mouse, but is not required for trophectoderm specification during blastocyst formation. This signaling pathway is stably activated to expand the trophoblast stem cell compartment in vivo, while in vitro, FGFs are used for the derivation of trophoblast stem (TS) cells from blastocysts and early post-implantation mouse embryos. However, the function of FGFs during human trophoblast development is not known. METHODS: We sought to derive TS cells from human blastocysts in a number of culture conditions, including in the presence of FGFs and stem cell factor (SCF). We also investigated the expression of FGF receptors (FGFRs) in blastocysts, and the expression of FGFR2 and activated ERK1/2 in first trimester human placentae. RESULTS: We found that SCF, but not FGF2/4, improved the quality of blastocyst outgrowths, but we were unable to establish stable human TS cell lines. We observed CDX2 expression in the trophectoderm of fully blastocysts, but rarely observed transcription of FGFRs. FGFR2 protein was not detected in human blastocysts, but was strongly expressed in mouse blastocysts. However, we found robust FGFR2 expression and activated ERK1/2 in the cytotrophoblast layer of early human placenta. DISCUSSION: Our data suggests that initiation of FGF-dependent trophoblast expansion may occur later in human development, and is unlikely to drive maintenance of a TS cell compartment during the peri-implantation period. These findings suggest that cytotrophoblast preparations from early placentae may be a potential source of FGF-dependent human TS cells.

Early trophoblast determination and stem cell maintenance in the mouse--a review.

The first priority of a mammalian embryo is to establish an intimate relationship with its mother. This is accomplished by precocious differentiation of the trophoblast lineage, which mediates uterine implantation and initiates the process of placentation. Surprisingly little is known about the molecular mechanisms that drive trophectoderm differentiation from the equipotent blastomeres of the morula. Somewhat more is known about the maintenance of trophoblast stem cells, once this lineage has been established. The first half of this review will focus on determination of the mouse trophoblast lineage and the second half will discuss the maintenance of trophoblast stem cells.

Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta.

The orphan nuclear receptor ERR beta is expressed in undifferentiated trophoblast stem cell lines and extraembryonic ectoderm, and genetic ablation of ERR beta results in abnormal trophoblast proliferation and precocious differentiation toward the giant cell lineage. Here, we show that the synthetic estrogen diethylstilbestrol (DES) promotes coactivator release from ERR beta and inhibits its transcriptional activity. Strikingly, treatment of trophoblast stem cells with DES led to their differentiation toward the polyploid giant cell lineage. In addition, DES-treated pregnant mice exhibited abnormal early placenta development associated with an overabundance of trophoblast giant cells and an absence of diploid trophoblast. These results define a novel pathway for DES action and provide evidence for steroidlike control of trophoblast development.

cis-Determinants in the cytoplasmic domain of CEACAM1 responsible for its tumor inhibitory function.

CEACAM1, also known as C-CAM, BGP and CD66a, is a member of the carcinoembryonic antigen (CEA) family which is itself part of the immunoglobulin supergene family. CEACAM1 is involved in intercellular adhesion, signal transduction and tumor cell growth regulation. CEACAM1 is down-regulated in colon and prostate carcinomas, as well as in endometrial, bladder and hepatic tumors, and 30% of breast cancers. We have shown in a mouse colon tumor model that CEACAM1 with a long cytoplasmic domain inhibited the development of tumors whereas a splice variant lacking the cytoplasmic domain did not. In this study, we define the subregions of the long cytoplasmic domain participating in the tumor inhibition phenotype of CEACAM1. We show that a single point mutation of Tyr488, conforming to an Immunoreceptor Tyrosine Inhibition Motif (ITIM), was sufficient to reverse the in vivo tumor cell growth inhibition. Substitution or deletion of residues in the C-terminal region of the CEACAM1 cytoplasmic domain also led to reversal of tumor cell growth inhibition. This result is in agreement with our previous studies demonstrating the C-terminal region of the cytoplasmic domain influences the levels of CEACAM1 Tyr phosphorylation and its association with the protein Tyr phosphatases SHP-1 and SHP-2. Furthermore, removal of the N-terminal domain of CEACAM1, essential for intercellular adhesion, did not impair the tumor inhibitory effect. These results suggest that Tyr phosphorylation or dephosphorylation of the CEACAM1 cytoplasmic domain represents a crucial step in the control of epithelial cell proliferation.

The carboxyl-terminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells.

Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptor tyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position +3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues +4, +5, and +6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.

Promotion of trophoblast stem cell proliferation by FGF4.

The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.

Optimal ratios of biliary glycoprotein isoforms required for inhibition of colonic tumor cell growth.

Rodent biliary glycoprotein (Bgp), also known as C-CAM, has recently been shown to function as a tumor suppressor in colon, prostate, and bladder cancers. This glycoprotein is a member of the carcinoembryonic antigen family and is one of the only proteins in this family to encode either a long (71-73 amino acids) or short (10 amino acids) cytoplasmic domain. We and others have shown that the growth-inhibitory properties of Bgp depend upon the expression of its long cytoplasmic domain. However, the two Bgp isoforms normally coexist in most cell types surveyed; the longer variant is usually present in lower amounts than the shorter one. In this study, we have examined the in vitro and in vivo growth properties of both mouse Bgp variants separately and in combination. To determine the physiologically relevant expression levels and ratios of the two Bgp variants, we have quantified the amount of the longer variant in normal colonic epithelial cells and showed that it constitutes 15-20% of total Bgp expressed in this tissue. To mimic the in vivo situation, we have generated double transfectant cell lines expressing the longer and shorter Bgp isoforms coordinately in tumorigenic CT51 mouse colonic carcinoma cells and demonstrated that the longer Bgp isoform exhibits a dominant tumor growth inhibition phenotype over that of the shorter variant within physiological levels of expression of Bgp. Unexpectedly, significant overexpression of the longer Bgp isoform alone led to reversal of the tumor inhibition phenotype. These results, therefore, suggest that there may be a limiting threshold of Bgp expression or Bgp-associating proteins mediating the tumor inhibition phenotype.

Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells.

Biliary glycoprotein (Bgp) is a member of the immunoglobulin superfamily and the carcinoembryonic antigen family. Previous studies have shown that Bgp functions as an intercellular adhesion molecule and a canalicular bile salt transporter. Moreover, we and others demonstrated that Bgp can inhibit colonic and prostatic tumor cell growth in vivo, through a mechanism which depends on sequences present in its cytoplasmic domain. In this study, we have examined the possibility that the cytoplasmic domain of Bgp can interact with signal transduction molecules. We showed that tyrosine phosphorylated Bgp, expressed in mouse colon carcinoma CT51 cells, could reversibly associate with protein tyrosine phosphatase SHP-1. Mutation of either of two tyrosine residues present in the cytoplasmic domain of Bgp abrogated SHP-1 binding, suggesting that this association was mediated by both tyrosine residues. Similarly, we noted that either of the two SH2 domains of SHP-1 could bind tyrosine phosphorylated Bgp in vitro. It is therefore conceivable that some of the functions of Bgp are mediated through its ability to induce intracellular protein tyrosine dephosphorylation.

Inhibition of colonic tumor cell growth by biliary glycoprotein.

Biliary glycoproteins (BGPs) are members of the carcinoembryonic antigen (CEA) family. These glycoproteins function in vitro as intercellular adhesion molecules and, in vitro as intercellular adhesion molecules and, in the mouse, serve as receptors for the mouse hepatitis viruses. In previous studies, BGP expression has been reported to be generally downregulated in colon and liver carcinomas of human, rat and mouse origins. We now demonstrate that introduction of murine Bgp1 cDNA isoforms into a mouse colonic carcinoma cell line, negative for endogenous Bgpl expression, significantly alters the growth properties of these cells. Cells bearing two Bgp1 isoforms were growth-retarded and exhibited a reduced ability to form colonies in an in vitro transformation assay, when compared to parental or control neor cells. Furthermore, tumor formation was inhibited by 80% when cells bearing a full-length Bgp1 isoform were injected into BALB/c syngeneic mice, while cells expressing a Bgp1 isoform lacking most of the intracytoplasmic domain produced tumors as readily as the parental cells. There results indicate that a biliary glycoprotein isoform is involved in negative regulation of colonic tumor cell growth, by a process which requires its intracytoplasmic domain. The precise mechanisms causing Bgp-dependent tumor growth inhibition remain, however, to be defined.