Trophoblast Stem Cell Markers Research Articles

Molecular and histopathological characterization of seminoma patients with highly elevated human chorionic gonadotropin levels in the serum

Approximately 30% of seminoma (SEM) patients present with moderately elevated human chorionic gonadotropin (hCG) levels at first diagnosis. In case of high hCG serum levels, the presence of a non-SEM component, i.e. choriocarcinoma (CC), may be assumed. To characterize cases described as pure seminoma with high serum hCG levels, tissue samples and DNA were analyzed. Patient files from an international registry were screened for patients with SEM and extraordinarily high hCG serum levels. IHC and qRT-PCR analysis was performed for markers of SEM, embryonal carcinoma (EC) and CC/trophoblast cells. The cell lines TCam-2 (SEM), 2102EP, NCCIT, NT2/D1 (EC) and JAR, JEG3 and BeWo (CC) were included for comparison. Of 1031 SEM patients screened, 39 patients (3.7%) showed hCG serum levels > 1000 U/l. Of these, tumor material for IHC and RNA for qRT-PCR was available from n = 7 patients and n = 3 patients, respectively. Median pre-orchiectomy serum hCG level was 5356 U/l (range: 1224–40909 U/L). Histopathologically, all investigated samples were classified as SEM with syncytiotrophoblast sub-populations. SEM cells were SALL4+ / OCT3/4+ / D2-40+, while syncytiotrophoblast cells were hCG+ / GATA3+ / p63+ and SOX2−/CDX2−. qRT-PCR analysis detected trophoblast stem cell markers CDX2, EOMES and TFAP2C as well as the trophectoderm-specifier TEAD4, but not GATA3. Additionally, SOX17 and PRAME, but not SOX2, were detected, confirming the pure SEM-like gene expression signature of the analyzed samples. In conclusion, excessively increased hCG serum levels can appear in patients with pure SEM. To explain detectable hCG serum levels, it is important to diagnose the subtype of a SEM with syncytiotrophoblasts.

ESRRB Facilitates the Conversion of Trophoblast-Like Stem Cells From Induced Pluripotent Stem Cells by Directly Regulating CDX2.

Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.

Abstract 422: Placental Cdx2 Cells Regenerate Injured Myocardium

The limited regeneration of adult mammalian heart has prompted the need to recognize novel strategies that can restore contractile function in heart disease. However, in cell-based therapies the lack of an appropriate cell-type that can differentiate to cardiomyocytes in vivo persists as an ultimate unmet need. Our prior study demonstrates that experimental myocardial injury in pregnant mice triggers the flux of fetal cells via the maternal circulation into the injured heart where they undergo differentiation into diverse cardiac cell fates. Among those fetal cells, the expression of Caudal type homeobox2 (Cdx2); a trophoblast stem cell marker was unique. To understand the intriguing role of placental Cdx2 cells in cardiomyogenesis, we utilized a lineage-tracing strategy to label fetal-derived Cdx2 cells with enhanced green fluorescent protein (Cdx2-eGFP). Cdx2-eGFP cells were characterized and assayed for cardiac differentiation in vitro and in vivo using a mouse model of myocardial infarction. Cdx2-eGFP cells clonally proliferated and differentiated into spontaneously beating cardiomyocytes and vascular cells in vitro , signifying a multipotent nature compared to the Cdx2 negative cell population. When administered via tail vein to infarcted wild-type male mice, Cdx2-eGFP cells selectively and robustly homed to the injured heart and differentiated to cardiomyocytes and blood vessels, significantly improving the contractility noted by magnetic resonance imaging. Proteomics and immune transcriptomics studies of Cdx2-eGFP cells compared to embryonic stem (ES) cells reveal that they appear to retain ‘stem’-related functions of ES cells, but exhibit unique signatures for homing and survival in addition to being immunologically naive. Blocking CXCR4, during the migration of Cdx2-eGFP cells to SDF1α suggested a possible role for SDF1-CXCR4 signaling in the mechanistic basis of homing. Advancing towards a translational role, we demonstrate that CDX2 expressing cells can be isolated from the chorionic region of human term placenta. Our results herein may represent a paradigmatic shift in the way we approach early embryonic lineages and cell fate choices and will establish the translational potential of placental Cdx2 cells for cardiac repair.

Human Trophoblast Progenitor Cells Express and Release Angiogenic Factors.

Trophoblast stem cells develop from polar trophectoderm and give rise to the cells that generate the placenta. Trophoblast cells are responsible for the uterine invasion and vascular remodeling during the implantation of the embryo. However this knowledge is not yet to be confirmed for trophoblast progenitor cells (TPCs). In this study, we aimed to demonstrate that human TPCs (hTPCs) express and release angiogenic factors for the first time. TPCs were isolated from the term placenta. Then immunophenotyping was performed by FACS method by analyzing caudal type homeobox 2 (CDX2) and eomesodermin (EOMES). Immunofluorescence staining of CDX2 and EOMES was performed on these cells. Lastly, angiogenesis-related proteins were detected by western blot and ELISA assays. The isolated cells were positive for trophoblast stem cell markers CDX2 and EOMES in 92.5% and 92.7% of cells, respectively showing the characteristics of TPCs. The investigation of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2) at protein and mRNA level in comparison with human umbilical vein endothelial cells (HUVECs), revealed that human TPCs (hTPCs) have higher levels of VEGF and VEGFR1 transcripts. Additionally soluble forms of VEGF and VEGFR1 were detected in supernatants of hTPCs. With this information, TPCs seem to be promising for regenerative cell therapies by increasing angiogenesis.

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

A pregnancy case report of alagille syndrome; expression of NOTCH signaling in the placenta

Gestational protein restriction affects trophoblast differentiation.

Whether and how gestational protein restriction (PR) affects placental development and function remain unknown. To test the hypothesis that PR can affect trophoblast differentiation in mid-and late pregnancy, rats were fed a 20% or an isocaloric 6% protein diet from Day 1 to 14 or 18 of pregnancy and effects of PR on trophoblast differentiation were determined by changes in expressions of marker gene(s) for trophoblast lineages. At Day 18 of pregnancy, PR increased expressions of Esrrb, Id1 andId2 (trophoblast stem cell markers), decreased expressions of Ascl2 (spongiotrophblast cell marker) and Prl2c1 (trophoblast giant cell marker), but did not alter expressions of Gjb3 and Pcdh12(glycogen cell markers) in the junctional zone (JZ). In the labyrinth zone (LZ), PR did not change expressions of Prl2b1 (trophoblast giant cell marker), Gcm1 and Syna (syncytiotrophoblast cell markers), but decrease expression of Ctsq (sinusoidal trophoblast giant cell marker). These results indicate that PR impairs the differentiation of trophoblast stem cell into spongiotrophoblast and trophoblast giant cells in JZ, and formation of sinusoidal trophoblast giant cells in LZ.

Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos

Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.

Immunolocalization of stemness markers in tissue sections of the human placenta

OBJECTIVE: The trophoblast lineage is the first to differentiate during the early stages of embryonic development. Trophoblast stem (TS) cells have been identified in the mouse but have yet to be described in humans. Currently there are no reported markers of human placental stem/progenitor cells that distinguish them from their mature progeny. Our objective was to characterize expression of human embryonic stem cell (hESC) and TS cell markers in the human placenta across a wide spectrum of gestational ages using immunolocalization techniques.

Impaired placental trophoblast lineage differentiation in Alkbh1−/− mice

E. coli AlkB has been intensively studied since 1983, but the in vivo roles of its mammalian homologue Alkbh1 are unknown. We, therefore, created null mice for Alkbh1. Alkbh1 mRNA is expressed at highest levels in the trophoblast lineages of the developing placenta. Alkbh1(-/-) placentas have decreased expression of differentiated trophoblast markers including Tpbp, Gcm1, and Pl-1, and increased expression of the trophoblast stem cell marker Eomes. Alkbh1 localizes to nuclear euchromatin, and interacts strongly with Mrj, an essential placental gene that mediates gene repression by recruitment of class II histone deacetylases (HDACs). Competition experiments show Alkbh1 and HDAC4 binding to Mrj are mutually exclusive, which causes decreased HDAC activity and increased target gene expression. Our study demonstrates Alkbh1 performs important functions in placental trophoblast lineage differentiation and participates in mechanisms of transcriptional regulation.

Gradient oxygen differentially regulates trophoblast giant cell differentiation

Preeclampsia is a condition associated with approximately 7–10% of all pregnancies and is known to induce maternal hypertension, increasing the risk for IUGR and premature birth. An important feature of preeclampsia is altered differentiation of invading placental trophoblast cells. While early trophoblast development takes place in severe hypoxia, trophoblast cells encounter an increasing oxygen gradient as they migrate and invade the maternal tissues. In this study, we examined the functional, morphological, and molecular differentiation of Rcho-1 trophoblast cells under a range of oxygen concentrations. Severe hypoxia maintained the expression of trophoblast stem cell marker Id2 and inhibited the induction of palladin and CSH1 protein, and formation of stress fibers. Moderate hypoxia, however, was unable to sustain Id2 expression and low levels of palladin protein induction and stress fiber formation was observed. HIF-1alpha, a potential transcriptional mediator in low oxygen conditions exhibited constitutive mRNA expression under all oxygen conditions. In contrast, HIF-1alpha protein levels and reporter activity decreased with increasing oxygen levels. These results suggest that an oxygen gradient differentially modulates aspects of trophoblast giant cell differentiation in a stepwise fashion and that HIF-1alpha may play a key role in oxygen’s inhibition of trophoblast differentiation. This work was supported by NICHD, National Institute of Health Grant (HD045750, T.L.B).

Hypoxia Inhibits Differentiation of Lineage-Specific Rcho-1 Trophoblast Giant Cells1

Defects in placental development lead to pregnancies at risk for miscarriage and intrauterine growth retardation and are associated with preeclampsia, a leading cause of maternal death and premature birth. In preeclampsia, impaired placental formation has been associated with alterations in a specific trophoblast lineage, the invasive trophoblast cells. In this study, an RT-PCR Trophoblast Gene Expression Profile previously developed by our laboratory was utilized to examine the lineage-specific gene expression of the rat Rcho-1 trophoblast cell line. Our results demonstrated that Rcho-1 cells represent an isolated, trophoblast population committed to the giant cell lineage. RT-PCR analysis revealed that undifferentiated Rcho-1 cells expressed trophoblast stem cell marker, Id2, and trophoblast giant cell markers. On differentiation, Rcho-1 cells downregulated Id2 and upregulated Csh1, a marker of the trophoblast giant cell lineage. Neither undifferentiated nor differentiated Rcho-1 cells expressed spongiotrophoblast marker Tpbpa or labyrinthine markers Esx1 and Tec. Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers; however, hypoxia did inhibit the downregulation of the trophoblast stem cell marker Id2. Differentiation in hypoxia also blocked the induction of CSH1 protein. In addition, hypoxia inhibited stress fiber formation and abolished the induction of palladin, a protein associated with stress fiber formation and focal adhesions. Thus, Rcho-1 cells can be maintained as a proliferative, lineage-specific cell line that is committed to the trophoblast giant cell lineage on differentiation in both normoxic and hypoxic conditions; however, hypoxia does inhibit aspects of trophoblast giant cell differentiation at the molecular, morphological, and functional levels.

168 ESTABLISHMENT OF TROPHOBLAST STEM CELLS FROM MOUSE ANDROGENETIC AND PARTHENOGENETIC EMBRYOS

Mouse androgenetic and parthenogenetic embryos, which have two sets of paternal and maternal genomes, respectively, are lethal until Day 9.5 of pregnancy, because the growth of the extraembryonic tissues including placenta is inadequate. These results suggest that both parental genomes are involved in placental development. However, the reason for placental deficiency in androgenetic and parthenogenetic embryos has been unclear. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage, have been used to elucidate the mechanism of differentiation and gene function in the development of the placenta. In this study, to characterize trophoblast lineage in androgenetic and parthenogenetic embryos, we examined TS cells from these embryos, and assessed their ability to differentiate into trophoblast lineage. The ovulated MII oocytes from B6D2F1 (C57BL/6 � DBA2) female mice were used for the uniparental embryo production. In order to produce parthenogenetic embryos, the oocytes were activated in SrCl2 and cytochalasin B. The androgenetic embryos were produced by in vitro fertilization using enucleated oocytes. These uniparental embryos were cultured for 3.5–4.5 days to develop into blastocysts. TS-like cell lines were established from these blastocysts, as described previously (Tanaka et al. 1998 Science 282, 2072–2075). TS-like cells were cultured without feeder cells in conditioned medium containing FGF4 and heparin to maintain the stem cell conditions. The expression of 5 TS cell maker genes (Eomes, CdX2, Fgfr2, Ap2a, and Errb) in TS-like cells was analyzed by RT-PCR and northern blotting. In addition, the localization of CDX2 was detected using immunostaining. To cause their differentiation into giant cells, TS-like cells were cultured for 6 days in TS medium without FGF4 and heparin. The giant cells were detected by the expression of 2 giant cell marker genes, Hand1 and Pl-1. TS cells from fertilized embryos between B6D2F1 male and female mice were also established as controls. Five TS cell marker genes were expressed both in androgenetic TS-like cells (ATS) and in parthenogenetic ones (PTS). The CDX2 gene was localized in the nucleus in both ATS and PTS; however, the number of CDX2-positive cells decreased in PTS. After 6 days of differentiation, Hand1 and Pl-1 were expressed and giant cells were detected in differentiated derivatives in ATS. On the other hand, giant cells were not detected in differentiated derivatives in PTS. These results suggest that parental genomes may regulate the gene expression independently to differentiate into trophoblast lineage. In conclusion, TS-like cells from uniparental embryos are useful tools to aid the understanding of the function of the parental genomes during placental development.

Transforming Growth Factor-βInduces Differentiation of the Labyrinthine Trophoblast Stem Cell Line SM10

The mammalian placenta consists of different trophoblast cell types that assist in the variety of functions required for the maintenance of pregnancy. In rodents, labyrinthine trophoblasts of the placenta are especially important, because they are capable of differentiating into fused labyrinthine cells, which form the feto-maternal exchange surface. Even though the molecular signals triggering labyrinthine trophoblast differentiation are poorly understood, transforming growth factor-beta (TGF-beta) has been shown to be present in the placental environment and alter trophoblast development. In this study, we investigated the effects of TGF-beta on the differentiation of the labyrinthine trophoblast stem cell lines SM10 and HRP-1. RT-PCR analyses demonstrated that while the molecular expression of labyrinthine-specific lineage markers (Esx1, Tfeb, and Tec) was maintained in TGF-beta-treated SM10 and HRP-1 cells, TGF-beta induced the down-regulation of trophoblast stem cell markers Id2 and Cdx2. In contrast, TGF-beta induced the expression of a marker of differentiated labyrinthine trophoblasts, Gcm1, only in the SM10 cell line. Furthermore, we demonstrated an increased glucose uptake in the TGF-beta-treated SM10 cells, indicative of functional differentiation. Finally, cell fusion in TGF-beta-treated SM10 and HRP-1 cells was investigated by western blotting analysis of placental alkaline phosphatase and cadherin-11 and by microscopic analyses of cell morphology using green fluorescent protein (GFP) and rhodamine phalloidin staining. The western blotting and morphological analyses indicate TGF-beta-induced cell fusion and morphological differentiation in the SM10 cell line. The SM10 cell line will provide a new and unique model for detailed analysis of TGF-beta-induced molecular events associated with labyrinthine trophoblast differentiation and function.

Identification of Committed Placental Stem Cell Lines for Studies of Differentiation

Trophoblasts provide a model to investigate fundamental mechanisms of stem cell differentiation, but the availability of trophoblast stem cell lines is limited. Here we report the development of an RT-PCR-based lineage-specific profile as a method to identify the lineages of placental trophoblast cells routinely and specifically. This profiling method was used to analyze the mouse SM10 and rat HRP-1 cell lines, isolated from a region of the placental labyrinth, but of previously unidentified lineage. Using this profile, the expression of trophoblast stem cell markers was detected in the SM10 and HRP-1 cells. In contrast, no expression of a marker of differentiated labyrinthine trophoblast was detected. Additionally, both cell lines expressed labyrinthine trophoblast-specific genes and did not express lineage-specific markers of spongiotrophoblasts or trophoblast giant cells. Our results suggest that SM10 and HRP-1 cell lines are trophoblast stem cell-like cell lines that can be maintained in undifferentiated but committed state in cell culture. These cell lines express labyrinthine-specific genes and are committed to differentiate solely into functional labyrinthine trophoblasts. Our profiling method provides a new technique to identify stem cells and their lineage-specific differentiation. This method additionally indicates that SM10 and HRP-1 cell lines provide new systems for future studies of stem cell differentiation, allowing investigation of basic mechanisms of differentiation, which may provide insights into the biophysics of development of a specialized system. This method should also prove to be useful for identification of other stem cell lines and examination of lineage-specific commitment.