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Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming.

ABSTRACT: Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was?>?4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.

SUBMITTER: Schmidt JK

PROVIDER: S-EPMC7644694 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming.

Schmidt Jenna Kropp JK Keding Logan T LT Block Lindsey N LN Wiepz Gregory J GJ Koenig Michelle R MR Meyer Michael G MG Dusek Brittany M BM Kroner Kamryn M KM Bertogliat Mario J MJ Kallio Avery R AR Mean Katherine D KD Golos Thaddeus G TG

Scientific reports 20201105 1

Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew ...[more]

PMID: 33154556

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Project description:Significance During pregnancy, extravillous trophoblasts (EVTs) detach from anchoring sites of the placenta, differentiate, and invade the maternal uterus to remodel its vasculature. Failures in this program contribute to placentation defects observed in gestational disorders such as spontaneous pregnancy loss, early-onset preeclampsia, and fetal growth restriction. However, critical regulators and signaling pathways controlling EVT development have been poorly elucidated. In the present study, we demonstrate that transforming growth factor-β (TGF-β) signaling plays a pivotal role in the differentiation program of EVTs. In vitro, loss of Wingless signaling was sufficient for EVT formation in self-renewing trophoblast models, whereas subsequent activation of TGF-β signaling accomplished differentiation into trophoblasts that display some features of in vivo EVTs. Abnormal placentation has been noticed in a variety of pregnancy complications such as miscarriage, early-onset preeclampsia, and fetal growth restriction. Defects in the developmental program of extravillous trophoblasts (EVTs), migrating from placental anchoring villi into the maternal decidua and its vessels, is thought to be an underlying cause. Yet, key regulatory mechanisms controlling commitment and differentiation of the invasive trophoblast lineage remain largely elusive. Herein, comparative gene expression analyses of HLA-G–purified EVTs, isolated from donor-matched placenta, decidua, and trophoblast organoids (TB-ORGs), revealed biological processes and signaling pathways governing EVT development. In particular, bioinformatics analyses and manipulations in different versatile trophoblast cell models unraveled transforming growth factor-β (TGF-β) signaling as a crucial pathway driving differentiation of placental EVTs into decidual EVTs, the latter showing enrichment of a secretory gene signature. Removal of Wingless signaling and subsequent activation of the TGF-β pathway were required for the formation of human leukocyte antigen-G+ (HLA-G+) EVTs in TB-ORGs that resemble in situ EVTs at the level of global gene expression. Accordingly, TGF-β–treated EVTs secreted enzymes, such as DAO and PAPPA2, which were predominantly expressed by decidual EVTs. Their genes were controlled by EVT-specific induction and genomic binding of the TGF-β downstream effector SMAD3. In summary, TGF-β signaling plays a key role in human placental development governing the differentiation program of EVTs.

Project description:BackgroundSERPINE2, one of the potent serpins belonging to the plasminogen activator (PA) system, is involved in the tissue remodeling. We previously demonstrated the expression patterns of Serpine2 in the mouse placenta and uterus, indicating that Serpine2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. In this study, we further investigated the expression pattern of SERPINE2 in the human placenta and explored possible functional roles of SERPINE2 in regulating trophoblast activity.MethodsPlacental tissues from various trimesters were collected for real-time reverse-transcription polymerase chain reaction quantification. Immunohistochemical staining was performed in placental tissues to assure localization of SERPINE2. SERPINE2 small interfering (si) RNA was applied to suppress its expression in villous explants and extravillous trophoblast-like 3A cells. Subsequent experiments to evaluate SERPINE2 levels, villous outgrowth, trophoblast invasion, and tube formation were performed.ResultsSERPINE2 messenger RNA was detected in the human placenta during pregnancy with the highest levels in the third trimester. The SERPINE2 protein was present in villous syncytiotrophoblasts and trophoblasts of chorionic villi for anti-SERPINE2 immunostaining. Extravillous trophoblasts in the chorionic plate and basal plate confronting the invasive face of anchoring villi were also positive. In most decidual cells, SERPINE2 was observed in the cytoplasm. In addition, fibrinoid deposit was weakly immunoreactive. Introduction of SERPINE2 siRNA into villous explants and trophoblast cells led to significantly reduced villous outgrowth, and trophoblastic migration and invasion. Moreover, capillary-like network formation of 3A cells in Matrigel was greatly attenuated by SERPINE2 siRNA and SERPINE2 antiserum.ConclusionsThese data identify the temporal and spatial SERPINE2 distribution in the human placenta and suggest its possible role in modulating tissue remodeling of extravillous trophoblasts in the placenta during pregnancy.

Project description:Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.

Dataset Information

Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming.

Publications

Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming.

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