SummaryTrophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

Embryonic hematopoietic stem and progenitor cells (HSPCs) robustly proliferate while maintaining multilineage potential in vivo; however, an incomplete understanding of spatiotemporal cues governing their generation has impeded robust production from human induced pluripotent stem cells (iPSCs) in vitro. Using the zebrafish model, we demonstrate that NLRP3 inflammasome-mediated interleukin-1-beta (IL1β) signaling drives HSPC production in response to metabolic activity. Genetic induction of active IL1β or pharmacologic inflammasome stimulation increased HSPC number as assessed by in situ hybridization for runx1/cmyb and flow cytometry. Loss of inflammasome components, including il1b, reduced CD41 + HSPCs, and prevented their expansion in response to metabolic cues. Cell ablation studies indicated that macrophages were essential for initial inflammasome stimulation of Il1rl1 + HSPCs. Significantly, in human iPSC-derived hemogenic precursors, transient inflammasome stimulation increased multilineage hematopoietic colony-forming units and T-cell progenitors. This work establishes the *

In recent years, knowledge regarding the genetic and epigenetic programmes governing specification, maintenance and differentiation of the extraembryonic lineage has advanced substantially. Establishment and analysis of mice deficient in genes implicated in trophoblast lineage and the option to generate and manipulate murine stem cell lines from the inner cell mass and the trophectoderm in vitro represent major advances. The activating enhancer binding protein 2 (AP2) family of transcription factors is expressed during mammalian development and in certain malignant diseases. This article summarizes the data regarding expression and function of murine Tcfap2 and human TFAP2 in extraembryonic development and differentiation. It also presents a model integrating Tcfap2c into the framework of trophoblast development and highlights the requirement of Tcfap2c to maintain trophoblast stem cells. With regard to human trophoblast cell-lineage restriction, the role of TFAP2C in lineage specification and maintenance is speculated upon. Furthermore, an overview of target genes of AP2 in mouse and human affecting placenta development and function is provided and the evidence suggesting that defects in regulating TFAP2 members might contribute to placental defects is discussed.

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In mammals, the first cell fate decision is initialized by cell polarization at the 8-to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.Life starts with a single totipotent cell, followed by several cleavages and the specification of the earliest cell lineages at the blastocyst stage. The trophectoderm (TE) represents the extraembryonic trophoblast lineage and envelops the inner cell mass (ICM), which gives rise to the embryonic tissues and the primitive endoderm. Embryonic stem cells (ESCs) derived from the ICM are pluripotent because they differentiate into all embryonic tissues, including the germ line, but have lost the ability to form trophoblast derivatives (10). Likewise, trophoblast stem cells (TSCs) derived from the TE layer exclusively recapitulate the developmental potency of their parental lineage and contribute to all trophoblast cell types of the conceptus (40, 50).The first cell fate decision is initiated by cell polarization at the 8-to 16-cell stage, where inner and outer cells are differentiated from each other. Outer cells adopt the trophoblast fate, whereas the inner cell population gives rise to the ICM. Differentiation is established by the expression of lineage-specific transcription networks and fixed by epigenetic asymmetries at the late blastocyst stage (4,42,43,54).The caudal-related homeobox 2 factor, Cdx2, is regarded as one key transcription factor for TE specification and maintenance. Cdx2 is expressed in the outer blastomeres and later in TE (7, 32). Cdx2-null embryos lose trophectoderm integrity, and TSCs cannot be derived (48). The ectopic expression of Cdx2 in ESCs leads to lineage conversion to the extraembryonic trophoblast fate by the direct downregulation of pluripotency-associated transcription factors, such as Oct4 and Nanog, and the activation of the trophectodermal transcription network (5, 35). Oct4 (...

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extraembryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA + progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a ( previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway.

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