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Summary Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs., Highlights • A Runx1 and Ly6a dual reporter system identifies intra-arterial HECs • Lymphoid and HSC potential is restricted to dual reporter double-positive cells • The placenta is the first major niche for HSC maturation and expansion • Single-cell RNA-seq analyses reveal early acquisition of a quiescent HSC phenotype, Chen and colleagues generate a Runx1 and Ly6a dual reporter mouse that marks pre-HSCs from specification of hemogenic endothelial cells to their migration to placenta, where they mature and expand, before colonization of the fetal liver as mature HSCs. Single-cell RNA-seq analyses of maturing HSCs reveal progressive acquisition of a quiescent state reminiscent of adult bone marrow long-term repopulating HSCs.

Intercellular communication is important for tissue development and homeostasis, and when dysregulated contributes to a multitude of pathobiological processes. Cells communicate with each other by several mechanisms, including direct cell-cell contacts between membrane-tethered ligands and receptors on the cell surface, through secreted molecules that bind their cognate receptor on the receiving cell, or alternative modalities such as exosomes. The study of cell-cell communication networks using single-cell genomic approaches is now under intensive investigation, and innovative algorithms to interpret the data, infer how cells interact and identify the downstream effectors of a putative binding of a ligand to its cognate receptor are critically needed. Here, we describe a protocol to run CellComm, a data-driven systems biology algorithm that integrates single-cell RNA-sequencing, protein interaction networks, and gene regulatory networks to infer which cells within a heterogeneous tissue are actively communicating, as well as their downstream transcriptional programs. When spatial transcriptomics data is available, CellComm additionally identifies spatially-resolved cell-cell interactions within the tissue. This protocol is associated with our Nature Cell Biology paper describing our algorithm: CellComm infers cellular crosstalk that drives hematopoietic stem and progenitor cell development.

Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell–cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta–gonad–mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7–independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

Introduction The first lineage separation in mammalian development occurs when totipotent cells of the zygote give rise to the inner cell mass and the trophectoderm. The lineages are strictly separated by an epigenetic barrier. In vitro derivatives of these lineages embryonic stem cells (ESC) and trophoblast stem cells (TSC) are used to study the requirements needed to overcome the barrier in ESC to TSC conversion approaches. Methods Different combinations of TSC transcription factors were induced in ESC for three days. Cells were kept in TS medium with fetal bovine serum (FBS) or the chemically defined TX medium. Obtained cells were analysed for OCT4 levels, TSC surface marker levels, expression of TSC markers and methylation status of Elf5, Oct4 and Nanog promoters. Further, long-term culture stability and in vitro and in vivo differentiation was tested. Results Overexpression of Gata3, Eomes, Tfap2c, Ets2 and Cdx2 in ESC resulted in induction of TSC fate. Overexpression of Cdx2 or four factors (Gata3, Eomes, Tfap2c and Ets2) resulted in complete conversion only when cells were cultured in TX medium. The obtained induced TSC (iTSC) display characteristics of bona fide TSC in terms of marker expression and promoter methylation patterns. The generated converted cells were shown to display self-renewal and to be capable to differentiate into TSC derivatives in vitro and in vivo. Conclusion Gata3, Eomes, Tfap2c, Ets2 and Cdx2 overexpression in ESC resulted in stable iTSC fate independent of culture conditions. For four factors or Cdx2 alone, TX medium is required for complete TSC conversion.

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαβ T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy.

SummaryBlastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that synthetic embryo-like structures are generated solely based on transcription factor-mediated molecular reprogramming of embryonic stem cells in a simple 3D co-culture system. ESCs in these cultures self-organize into elongated, compartmentalized synthetic embryo-like structures over the course of reprogramming exhibiting anterior visceral endoderm formation and symmetry breaking. Single-cell RNA-Seq reveals transcriptional profiles resembling epiblast, visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5–E5.5. Within the epiblast, compartment marker gene expression supports primordial germ cell specification. After transplantation, synthetic embryo-like structures implantin uteriand initiate the formation of decidual tissues. This system allows for fast and reproducible generation of synthetic embryo-like structures, providing further insights into synthetic embryology.

Activating mutations in the human KIT receptor is known to drive severe hematopoietic disorders and tumor formation spanning various entities. The most common mutation is the substitution of aspartic acid at position 816 to valine (D816V), rendering the receptor constitutively active independent of ligand binding. As the role of the KIT receptor in placental signaling cascades is poorly understood, we analyzed the impact of KITD816V expression on placental development using a humanized mouse model. Placentas from KITD816V animals present with a grossly changed morphology, displaying a reduction in labyrinth and spongiotrophoblast layer and an increase in the Parietal Trophoblast Giant Cell (P-TGC) layer. Elevated differentiation to P-TGCs was accompanied with reduced differentiation to other Trophoblast Giant Cell (TGC) subtypes and by severe decrease in proliferation. The embryos display growth retardation and die in utero. KITD816V-trophoblast stem cells (TSC) differentiate much faster compared to wild type (WT) controls. In undifferentiated KITD816V-TSCs, levels of Phosphorylated Extracellular-signal Regulated Kinase (P-ERK) and Phosphorylated Protein Kinase B (P-AKT) are comparable to wildtype cultures differentiating for 3&ndash, 6 days. Accordingly, P-TGC markers Placental Lactogen 1 (PL1) and Proliferin (PLF) are upregulated as well. The results reveal that KIT signaling orchestrates the fine-tuned differentiation of the placenta, with special emphasis on P-TGC differentiation. Appropriate control of KIT receptor action is therefore essential for placental development and nourishment of the embryo.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.

Many prior studies have focused on the role of mutations in epigenetic factors in cancer (e.g., EZH2, CREBBP, EP300, KMT2C, KMT2D) but drugs targeting these mutations have shown only modest activity in patients. Here we identify overexpression of KDM4A and KDM4C, two erasers of histone H3K9 methylation in multiple cancers, and demonstrate that their misexpression in lymphoma and colorectal cancer results in low H3K9me2/3 over pro-oncogenic transcription factor binding sites (TFBS) present in the vicinity of endogenous retro-elements (EREs). Consistently, KDM4A/4C inhibition results in the reallocation of H3K9me2/3 and H3K27Ac over enhancers, leading to activation of intracellular nucleic acid-sensing, and repression of the Wnt/beta-catenin transcriptional program. Our integrative epigenomic and transcriptomic analysis revealed a geneset that risk-stratified both lymphoma and colorectal cancer patients. We propose that high levels of H3K9me2/3 in somatic cells maintain genome stability and lineage-fidelity by repressing EREs and associated oncogenic enhancers, and that reduction of H3K9me2/3 from these regions is a prerequisite for oncogenesis. Our study establishes KDM4A and KDM4C as relevant anti-cancer targets in lymphoma and Wnt-dependent cancers and suggests that modulating innate immunity through dynamic regulation of H3K9 methylation can be leveraged for anti-cancer therapy.

Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

Summary 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 inflammasome as a conserved metabolic sensor that expands HSPC production in vivo and in vitro.

With age, HSCs accumulate somatic mutations that can increase their relative fitness and cause clonal expansion, triggering sporadic age-related clonal hematopoiesis (CH). In most people, CH is of little or no clinical significance as mature blood cell output is not typically affected. However, some individuals will accumulate further mutations that drive evolution to HSC failure, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Moreover, in patients with bone marrow failure syndromes caused by germline mutations that impair HSC function, this process of CH is markedly accelerated, with an amplified risk of MDS and AML in young adulthood. Here, we aimed to use gene editing and directed differentiation of human induced pluripotent stem cells (IPSCs) to model sporadic and predisposed CH. We found that hematopoietic stem and progenitor cells (HSPCs) derived from IPSCs and deficient for the Fanconi anemia gene FANCA showed poor hematopoietic potential compared to isogenic FANCA-expressing cells. Multiplexed editing of 10 CH-associated genes in FANCA-deficient HSPCs resulted in reversal of this hematopoietic failure and implementation of an aberrant program of sustained self-renewal. To model sporadic CH, we introduced a heterozygous P95H mutation in the SRSF2 gene - a mutation commonly observed in human CH and MDS. We found that in HSPCs, this mutation globally disrupted splicing, mildly impaired differentiation, and enhanced clonogenesis. Together, our data demonstrate that we can capture hallmarks of predisposed and sporadic CH in IPSC-based models as a basis for ongoing studies on mechanisms of clonal evolution in HSCs.

While the generation of hematopoietic progenitor cells from pluripotent stem cells (hiPSCs) can readily be accomplished in a dish, transgene free generation of long term reconstituting hematopoietic stem cells (HSCs) remains a major challenge to the field. In order to recapitulate definitive hematopoiesis in vitro, we need a better understanding of the developmental processes occurring during HSC emergence in vivo. Despite having identified the origin of HSCs to be specialized endothelium, termed hemogenic endothelium, we have yet to fully characterize all distinct cell types which may play a role during the process of endothelial-to-hematopoietic transition. Here, we have utilized single cell RNA sequencing to profile two major hematopoietic organs in the mouse. The aorta-gonad-mesonephros (AGM) region was analyzed on embryonic day (E)10.5, just prior to the first emergence of HSCs, and the placenta on E11.5, prior to its peak of HSC potential. In addition to capturing endothelial and nascent hematopoietic stem cells, we also included cells of the surrounding embryonic tissue, allowing us to computationally predict cell-cell interactions based on ligand and receptor expression. This approach validated previously described regulators of HSC emergence, i.e. the recently published surface antigen CD44, while also predicting additional signaling pathways, which have not been implicated in HSC development so far. Using a morpholino based knockdown screening approach in zebrafish, we tested the effect of the predicted target ligand and receptor interactions on the presence of runx1+/cmyb+ cells in the aorta. Following this approach, we were able to identify novel modulators of HSC emergence in the AGM. Strikingly, direct comparison between the AGM and placental dataset revealed a population of cells within the placenta with a transcriptional signature highly similar to AGM hemogenic endothelium. Functional validation of these cells will test their ability to acquire hemogenic potential after in vitro maturation in the presence of a supportive stromal layer. Implementation of the transcriptional and cell-cell communication findings will aid enhancement of hiPSC-derived hematopoietic differentiation protocols toward the production of functionally competent HSCs.

Recently, direct cell fate conversion attempts between the embryonic and extra-embryonic lineage gained new momentum. Two concomitant publications were published, describing the successful generation of transgene-independent, self-renewing trophoblast stem cells (TSCs) from murine fibroblasts. Cells were faithfully converted, displaying high similarity to blastocyst or extraembryonic ectoderm derived TSCs. Here, we summarize and compare published attempts aiming at the direct induction of trophoblast-fate from either mouse embryonic stem cells or fibroblasts.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Researchers at the University of Cambridge, UK have succeeded in reconstructing mouse embryos by combining pluripotent embryonic and multipotent trophoblast stem cells in a 3D scaffold; the study from the laboratory of Professor Zernicka-Goetz, recently published in Science, provides a break-through tool to probe early mammalian development outside the uterus. Achieving a similar feat with human cells might necessitate reconsideration of the 14-day rule as a limitation of such research.

Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of B-cell Non-Hodgkin Lymphoma (NHL), representing a third of all new cases. DLBCL is further sub-divided into various molecular sub-types based on gene expression and co-occurring genetic alterations. Gene expression based- subtypes include the germinal center B-cell (GCB) and the activated B-cell type (ABC) subtypes, with the ABC sub-type having a poorer prognosis than the GCB sub-type. Interestingly, 30-40% of all DLBCL patients harbor mutations in key epigenetic regulators, EZH2, KMT2D, CREBBP, EP300, and mutations in histone proteins themselves. Through an unbiased cell-based phenotypic screen, we discovered that inhibition of lysine demethylases, specifically KDM4C and KDM4A, represents a vulnerability across 15 different DLBCL cell lines including germinal center B-cell (GCB) and activated B-cell (ABC) type lines, with a GI50 value between 75nM to ~200nM, while sparing leukemia lines. Consistently, treatment of xenograft-based animal models of DLBCL with a low dose of KDM4A/KDM4C inhibitor delivered intra-peritoneally three times a week, results in a drastic reduction of tumor burden. Both KDM4A and KDM4C catalyze the removal of histone H3 K9 di- and tri-methylation (H3K9me2/3) and H3K36 di- and tri-methylation (H3K36me2/3). H3K9me2/3 is associated with promoter and enhancer repression, while H3K36me2/3 is present in gene bodies during transcription but also functions as a chromatin repressor Consistently, we have identified key enhancers, including those associated with IKZF1, that are "decommissioned" after inhibition of KDM4A/KDM4C. Repressed enhancer activity, through loss of H3K27 acetylation and H3K4me1, and gain of H3K9me2/3, results in a rapid transcriptional downregulation of IKZF1 and its partners including IKZF3. Given the role of IKZF1 in the transcriptional regulation of key B Cell Receptor (BCR) signaling components, we show that KDM4A/KDM4C inhibition leads to a downregulation of SYK, a proximal BCR-signaling component, which likely precedes DLBCL cell apoptosis. In addition, we observed an activation of extra-lineage transcription factors such as CEBPA and CEBPB, which are normally repressed by IKZF1 in the lymphoid lineage. A concomitant downregulation of the B-cell gene expression program and an upregulation of the myeloid (CD14+ monocytic) gene expression program is also observed, implying a "trans-differentiation" of DLBCL cells into the monocyte lineage. This lineage-switch correlates with an increased population of CD14+ expressing cells. Finally, using DLBCL patient data sets, we can show that over-expression of either KDM4A or KDM4C is associated with poor prognosis in DLBCL patients. In summary, we have discovered that KDM4A/KDM4C inhibition results in an increase of repressive histone modifications at several intra-genic "enhancers" of genes that are responsible for the survival and proliferation of DLBCL cells. The elucidation of this unique epigenetic mechanism provides a strong rationale for the development of novel targeted therapies against both multiple subtypes of DLBCL. Figure. Figure. Disclosures Shipp: Bayer: Research Funding; AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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 extra-embryonic 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.

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 extra-embryonic 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.

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.

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.

SummaryTrophoblast 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.