Differentiation Of Trophoblast Stem Cells Research Articles

Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation.

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.

FBXL12 regulates T-cell differentiation in a cell-autonomous manner.

Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells including embryonic, adult tissue and cancer stem cells. The SCF(FBXL) (12) complex is an authentic ubiquitin ligase that targets ALDH3 for degradation. FBXL12 is essential for the differentiation of trophoblast stem cells into specific cell types in the placenta during mouse embryogenesis, but its physiological functions in adult tissues have remained unknown. We have now investigated the role of the FBXL12-ALDH3 axis in the thymus, in which FBXL12 was most abundant among adult mouse tissues examined. During T-cell differentiation, FBXL12 is most abundant in CD4(+) CD8(+) (DP) cells, with its expression declining as these cells differentiate into CD4(+) CD8(-) or CD4(-) CD8(+) (SP) cells. T cells of FBXL12-null mice manifested a differentiation block at the DP-SP transition that was associated with ALDH3 accumulation in DP cells. This differentiation block was also apparent in wild-type mouse recipients of FBXL12-null bone marrow transplants as well as in FBXL12-null fetal thymic organ culture, suggesting that it is a cell-autonomous phenomenon in the thymus rather than an indirect effect of altered systemic conditions. Our results thus indicate that, in addition to its role in placental development, the FBXL12-ALDH3 axis is required for maturation of undifferentiated thymocytes.

CITED2 modulation of trophoblast cell differentiation: insights from global transcriptome analysis.

Trophoblast stem (TS) cells possess the capacity to differentiate along a multi-lineage pathway yielding several specialized cell types. The regulatory network controlling trophoblast cell differentiation is poorly understood. Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) has been implicated in the regulation of placentation; however, we know little about how CITED2 acts to influence trophoblast cells. Rat Rcho-1 TS cells can be manipulated to proliferate or differentiate into specialized trophoblast lineages and are an excellent model for investigating trophoblast differentiation. CITED2 transcript and protein showed a robust induction during Rcho-1 TS cell differentiation. We used an shRNA knockdown approach to disrupt CITED2 expression in order to investigate its involvement in trophoblast cell differentiation. RNA-sequencing was used to examine the impact of CITED2 on trophoblast cell differentiation. CITED2 disruption affected the differentiating trophoblast cell transcriptome. CITED2 possessed a prominent role in the regulation of cell differentiation with links to several signal transduction pathways and to hypoxia-regulated and coagulation processes. In summary, our findings indicate that CITED2 contributes to the regulation of trophoblast cell differentiation.

Ggnbp2 Is Essential for Pregnancy Success via Regulation of Mouse Trophoblast Stem Cell Proliferation and Differentiation.

The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.

Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation

Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.

Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells.

Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson’s disease.

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

Rho GDIalpha Modulates Rabbit Trophoblast Stem Cell Survival and Migration.

Trophoblast stem cells differentiate into different trophoblast cell populations that are indispensable for successful pregnancy through interactions with the maternal uterine decidua. Rho GTPases play an important role in the regulation of trophoblast stem cell (TSC) self-renewal and differentiation; however, the role of Rho GDP-dissociation inhibitors (Rho GDIs) remains unclear. Here we report that overexpression of Rho GDIalpha resulted in rapid apoptosis of TSCs, while its knockdown promoted proliferation. Moreover, Rho GDIalpha knockdown also enhanced TSC invasion. Collectively, these results establish a potential mechanism whereby TSCs can balance growth and apoptosis, and thus ensure normal fetal development.

Activin Regulates Self-renewal and Differentiation of Trophoblast Stem Cells by Down-regulating the X Chromosome Gene Bcor

The development of a functional placenta is largely dependent upon proper proliferation and differentiation of trophoblast stem cells (TSCs). Activin signaling has long been regarded to play important roles during this process, but the exact mechanism is largely unknown. Here, we demonstrate that the X chromosome gene BCL-6 corepressor (Bcor) is a critical downstream effector of activin to fine-tune mouse TSC fate decision. Bcor was specifically down-regulated by activin A in TSCs in a dose-dependent manner, and immediately up-regulated upon TSC differentiation. Knockdown of Bcor partially compensated for the absence of activin A in maintaining the self-renewal of TSCs together with FGF4, while promoting syncytiotrophoblast differentiation in the absence of FGF4. Moreover, the impaired trophoblast giant cell and spongiotrophoblast differentiation upon Bcor knockdown also resembled the function of activin. Reporter analysis showed that BCOR inhibited the expression of the key trophoblast regulator genes Eomes and Cebpa by binding to their promoter regions. Our findings provide us with a better understanding of placental development and placenta-related diseases.

The placental sulfate transporter, Slc13a4, is critical for skeletal development in mice.

The placental sulfate transporter, Slc13a4, is critical for skeletal development in mice.

Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal–placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4) enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4–7days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

152 Stem Cell Coated Metallic Coronary Stents Show Accelerated Strut Coverage without Excessive Neointimal Proliferation in a Porcine Model

IntroductionThe success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these...

Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

The Gap Junction Channel encoding Genes Connexin31 (Gjb3) and Connexin31.1 (Gjb5) have Directly Opposed Effects on Trophoblast Stem Cell Differentiation during Mouse Placental Development

The Gap Junction Channel encoding Genes Connexin31 (Gjb3) and Connexin31.1 (Gjb5) have Directly Opposed Effects on Trophoblast Stem Cell Differentiation during Mouse Placental Development

Hypoxia and Trophoblast Differentiation: A Key Role for PPARγ

Tissue oxygen tension regulates differentiation of multiple types of stem cells. In the placenta, hypoxia has been associated with abnormal trophoblast differentiation and placental insufficiency syndromes of preeclampsia (PE) and intrauterine growth restriction (IUGR). Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor involved in many cellular processes, including differentiation. We have previously shown that PPARγ-null trophoblast stem (TS) cells show a defect in differentiation to labyrinthine trophoblast, instead differentiating preferentially to trophoblast giant cells (TGC). Since PPARγ is known to be regulated by hypoxia in adipose tissue, we hypothesized that there may be a link between oxygen tension, PPARγ expression, and trophoblast differentiation. We found that hypoxia reduced PPARγ expression by a mechanism independent of both hypoxia-inducible factor (HIF) and histone deacetylases (HDACs). In addition, PPARγ partially rescued hypoxia-induced inhibition of labyrinthine differentiation in wild-type TS cells but was not required for hypoxia-induced inhibition of TGC differentiation. Finally, we show that induction of labyrinthine trophoblast differentiation by HDAC inhibitor treatment is independent of both PPARγ and Gcm1. We propose a model with two pathways for labyrinthine trophoblast differentiation of TS cells, one of which is dependent on PPARγ and inhibited by hypoxia. Since hypoxia is associated with PE and IUGR, we propose that PPARγ may at least partially mediate hypoxia-induced placental insufficiency and as such may be a promising therapeutic target for these disorders.

Embryonic stem cell-derived trophoblast differentiation: a comparative review of the biology, function, and signaling mechanisms

The development of the placenta is imperative for successful pregnancy establishment, yet the earliest differentiation events of the blastocyst-derived trophectoderm that forms the placenta remain difficult to study in humans. Human embryonic stem cells (hESC) display a unique ability to form trophoblast cells when induced to differentiate either by the addition of exogenous BMP4 or by the formation of cellular aggregates called embryoid bodies. While mouse trophoblast stem cells (TSC) have been isolated from blastocyst outgrowths, mouse ESC do not spontaneously differentiate into trophoblast cells. In this review, we focus on addressing the similarities and differences between mouse TSC differentiation and hESC-derived trophoblast differentiation. We discuss the functional and mechanistic diversity that is found in different species models. Of central importance are the unique signaling events that trigger downstream gene expression that create specific cellular fate decisions. We support the idea that we must understand the nuances that hESC differentiation models display so that investigators can choose the appropriate model system to fit experimental needs.

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.

Differentiation-Driven Nucleolar Association of the Mouse Imprinted Kcnq1 Locus

The organization of the genome within the mammalian nucleus is nonrandom, with physiologic processes often concentrated in specific three-dimensional domains. This organization may be functionally related to gene regulation and, as such, may play a role in normal development and human disease processes. However, the mechanisms that participate in nuclear organization are poorly understood. Here, we present data characterizing localization of the imprinted Kcnq1 alleles. We show that nucleolar association of the paternal allele (1) is stimulated during the differentiation of trophoblast stem cells, (ii) is dependent upon the Kcnq1ot1 noncoding RNA, (3) does not require polycomb repressive complex 2, and (4) is not sufficient to preclude transcription of imprinted genes. Although nucleolar positioning has been proposed as a mechanism to related to gene silencing, we find that silencing and perinucleolar localization through the Kcnq1ot1 noncoding RNA are separable events.

The Maternally Expressed Gene Tssc3 Regulates the Expression of MASH2 Transcription Factor in Mouse Trophoblast Stem Cells through the AKT-Sp1 Signaling Pathway

Tssc3 is a maternally expressed/paternally silenced imprinted gene. Recent evidence suggests that the loss of TSSC3 results in placental overgrowth in mice. These findings showed that the TSSC3 gene functions as a negative regulator of placental growth. In this study, we describe the function of TSSC3 and its signaling pathway in mouse trophoblast stem (TS) cell differentiation. First of all, we tested Tssc3 expression levels in TS cells. TS cells expressed Tssc3, and its expression level was the highest from day 1 to 4 but was down-regulated at day 5 after the induction of differentiation. Overexpression of TSSC3 in TS cells up-regulated Gcm1 and Mash2, which are marker genes of mouse trophoblast differentiation. Down-regulation of TSSC3 by siRNA enhanced Pl1 and Tpbpa expression in TS cells cultured under stem cell conditions, suggesting the contribution of TSSC3 to the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts. TSSC3 activated the PI3K/AKT pathway through binding with phosphatidylinositol phosphate lipids and enhanced the activity of a promoter containing an E-box structure, which is the binding sequence of the Mash2 downstream target gene promoter. PI3K inhibitor suppressed the promoter activity induced by TSSC3. TSSC3 induced Sp1 translocation from cytoplasm to nucleus through the PI3K/AKT pathway. Nuclear Sp1 activated the Mash2 transcription by Sp1 binding with a consensus Sp1-binding motif. This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway.

Adrenomedullin Promotes the Differentiation of Rat Trophoblast Stem Cells.

Adrenomedullin Promotes the Differentiation of Rat Trophoblast Stem Cells.