Loss of p57KIP2expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells

A complete hydatidiform mole (CHM) is androgenetic in origin and characterized by enhanced trophoblastic proliferation and the absence of fetal tissue. In 15 to 20% of cases, CHMs are followed by malignant gestational trophoblastic neoplasms including choriocarcinoma. Aberrant genomic imprinting may be responsible for trophoblast hypertrophy in CHMs, but the detailed mechanisms are still elusive, partly due to the lack of suitable animal or in vitro models. We recently developed a culture system of human trophoblast stem (TS) cells. In this study, we apply this system to CHMs for a better understanding of their molecular pathology. CHM-derived TS cells, designated as TSmolecells, are morphologically similar to biparental TS (TSbip) cells and express TS-specific markers such as GATA3, KRT7, and TFAP2C. Interestingly, TSmolecells have a growth advantage over TSbipcells only after they reach confluence. We found that p57KIP2, a maternally expressed gene encoding a cyclin-dependent kinase inhibitor, is strongly induced by increased cell density in TSbipcells, but not in TSmolecells. Knockout and overexpression studies suggest that loss of p57KIP2expression would be the major cause of the reduced sensitivity to contact inhibition in CHMs. Our findings shed light on the molecular mechanism underlying the pathogenesis of CHMs and could have broad implications in tumorigenesis beyond CHMs because silencing ofp57KIP2is frequently observed in a variety of human tumors.

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Laminin switches terminal differentiation fate of human trophoblast stem cells under chemically defined culture conditions

10.1101/2021.09.30.462667 ◽

2021 ◽

Author(s):

Victoria Karakis ◽

Thomas McDonald ◽

Abigail Cordiner ◽

Adam Mischler ◽

Adriana San Miguel ◽

...

Keyword(s):

Stem Cells ◽

Culture System ◽

Culture Conditions ◽

Extravillous Trophoblast ◽

Mechanistic Studies ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Defined Culture

AbstractHuman trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we do not utilize transforming growth factor-beta inhibitors or a passage step for EVT differentiation, or forskolin for STB formation. Strikingly, under these conditions, presence of a single additional extracellular cue – lam-inin-1 – switched the terminal differentiation of hTSCs from STB to the EVT lineage. Activation of the sphingosine-1 receptor 3 receptor (S1PR3) using a chemical agonist could drive EVT differentiation of hTSCs in the absence of exogenous laminin, albeit less efficiently. To illustrate the utility of a chemically defined culture system for mechanistic studies, we examined the role of protein kinase C (PKC) signaling during hTSC differentiation to the EVT lineage. Inhibition of PKCα/β signaling significantly reduced HLA-G expression and the formation of HLA-G+ mesen-chymal EVTs during hTSC differentiation mediated by laminin exposure; however, it did not prevent commitment to the EVT lineage or STB differentiation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation, and will enable mechanistic studies in vitro.SignificanceDespite its importance to a healthy pregnancy, early human placental development remains poorly understood. Mechanistic studies are impeded by restrictions on research with human embryos and fetal tissues, and significant differences in placentation between humans and commonly used animal models. In this context, human trophoblast stem cells (hTSCs) have emerged as attractive in vitro models for the epithelial cytotrophoblast of the early gestation human placenta. Here we describe chemically defined culture conditions for differentiation of hTSCs to the two major differentiated cell types – extravillous trophoblast and syncytiotrophoblast. These culture conditions enable in vitro studies to reveal molecular mechanisms regulating hTSC differentiation.

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Role of autocrine bone morphogenetic protein Signaling in trophoblast stem cells

Biology of Reproduction ◽

10.1093/biolre/ioab213 ◽

2021 ◽

Author(s):

Jennie Au ◽

Daniela F Requena ◽

Hannah Rishik ◽

Sampada Kallol ◽

Chandana Tekkatte ◽

...

Keyword(s):

Stem Cells ◽

Bone Morphogenetic Protein ◽

Bmp Signaling ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Morphogenetic Protein ◽

Bmp Pathway

Abstract The Bone Morphogenetic Protein (BMP) pathway is involved in numerous developmental processes, including cell growth, apoptosis, and differentiation. In mouse embryogenesis, BMP signaling is a well-known morphogen for both mesoderm induction and germ cell development. Recent evidence points to a potential role in development of the extra-embryonic compartment, including trophectoderm-derived tissues. In this study, we investigated the effect of BMP signaling in both mouse and human trophoblast stem cells (TSC) in vitro, evaluating the expression and activation of the BMP signaling response machinery, and the effect of BMP signaling manipulation during TSC maintenance and differentiation. Both mTSC and hTSC expressed various BMP ligands and the receptors BMPR1A and BMPR2, necessary for BMP response, and displayed maximal active BMP signaling when undifferentiated. We also observed a conserved modulatory role of BMP signaling during trophoblast differentiation, whereby maintenance of active BMP signaling blunted differentiation of TSC in both species. Conversely, the effect of BMP signaling on the undifferentiated state of TSC appeared to be species-specific, with SMAD-independent signaling important in maintenance of mTSC, and a more subtle role for both SMAD-dependent and -independent BMP signaling in hTSC. Altogether, these data establish an autocrine role for the BMP pathway in the trophoblast compartment. As specification and correct differentiation of the extra-embryonic compartment are fundamental for implantation and early placental development, insights on the role of the BMP signaling in early development might prove useful in the setting of in vitro fertilization as well as targeting trophoblast-associated placental dysfunction.

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Trophoblast stem cells - methods of isolation, histological and cellular characteristic, and their possible applications in human and animal models

Medical Journal of Cell Biology ◽

10.2478/acb-2020-0012 ◽

2020 ◽

Vol 8 (3) ◽

pp. 95-100

Author(s):

Rafał Sibiak ◽

Michał Jaworski ◽

Saoirse Barrett ◽

Rut Bryl ◽

Paweł Gutaj ◽

...

Keyword(s):

Stem Cells ◽

Marker Gene ◽

Experimental Studies ◽

Placental Tissue ◽

Trophoblast Cell ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Transcription Profiles ◽

Trophoblast Stem

AbstractThe placenta is a part of feto-maternal unit that develops from the maternal decidua basalis and fetal-derived trophoblast cells. The regulation of its early development is extremely intricate, albeit the elusive trophoblast stem cells (TSCs) are thought to give rise to the fetal part of the placenta. TSCs may be isolated in both animal and human models. In detail, TSCs can be efficiently obtained from the early conceptus tissues – blastocysts or early placental tissue. The isolation of murine TSCs pave the way for analyses of human trophoblast cell lineages. Both human and animal stem cells retain similar characteristic properties – the ability for unrestricted self-renewal and differentiation into all trophoblast cell lines. Nevertheless, there are some essential differences across the various species which are especially pronounced when pertaining to their distinct optimal cell culture requirements. Moreover, there are several crucial discrepancies in the stemness marker gene transcription profiles between human and murine TSCs models. In vitro TSC models can be adapted to the elucidation of the pathophysiology of various reproductive complications. For instance, their properties may illustrate the conditions observed during the implantation or simulate the state of abnormal placentation. Observations gained from the experimental studies could potentially explain the cause of some cases of infertility, preeclampsia, and fetal growth abnormalities.Running title: Update on the trophoblast stem cells

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Human trophoblast stem cells: Real or not real?

Placenta ◽

10.1016/j.placenta.2017.01.003 ◽

2017 ◽

Vol 60 ◽

pp. S57-S60 ◽

Cited By ~ 11

Author(s):

Ching-Wen Chang ◽

Mana M. Parast

Keyword(s):

Stem Cells ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17708690 ◽

2017 ◽

Vol 38 (5) ◽

pp. 888-903 ◽

Cited By ~ 10

Author(s):

Allison M Andrews ◽

Evan M Lutton ◽

Lee A Cannella ◽

Nancy Reichenbach ◽

Roshanak Razmpour ◽

...

Keyword(s):

Endothelial Cells ◽

Neurovascular Unit ◽

Fetal Brain ◽

Barrier Properties ◽

Endothelial Activation ◽

Fetal Tissue ◽

In Vitro Models ◽

Adult Brain ◽

Alternative Source

Endothelial cells (ECs) form the basis of the blood–brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs ( aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs ( fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

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Derivation of macaque trophoblast stem cells

10.1101/2020.03.17.995407 ◽

2020 ◽

Author(s):

Jenna Kropp Schmidt ◽

Michael G. Meyer ◽

Gregory J. Wiepz ◽

Lindsey N. Block ◽

Brittany M. Dusek ◽

...

Keyword(s):

Stem Cells ◽

Culture Media ◽

Syncytium Formation ◽

First Trimester ◽

Gonadotropin Secretion ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Mhc Class I Molecule

AbstractNonhuman 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 (TSC) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first trimester placental villous cytotrophoblasts followed by culture in TSC medium to “reprogram” the cells to a proliferative state. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was >4,000-fold higher in ST culture media compared to TSC media. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and macaque placental nonclassical MHC class I molecule, Mamu-AG. TSC differentiation to extravillous trophoblasts (EVTs) with or without the ALK-5 inhibitor A83-01 resulted in differing morphologies but similar expression of Mamu-AG and CD56 as assessed by flow cytometry, hence further refinement of relevant EVT markers is needed. Our preliminary characterization of macaque TSCs suggests that these cells represent a proliferative, self-renewing TSC population capable of differentiating to STs in vitro thereby establishing an experimental model of primate placentation.

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Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro

Embryonic Stem Cell Protocols ◽

10.1385/1-59745-037-5:35 ◽

2006 ◽

pp. 35-44 ◽

Cited By ~ 1

Author(s):

Satoshi Tanaka

Keyword(s):

Stem Cells ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Transplantation of human trophoblast stem cells to treat Parkinsonian rats and its mechanisms

Journal of Cell Science & Therapy ◽

10.4172/2157-7013.s1.15 ◽

2011 ◽

Vol 01 (S1) ◽

Author(s):

Jau- Nan Lee

Keyword(s):

Stem Cells ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Human trophoblast-derived exosomes attenuate doxorubicin-induced cardiac injury by regulating miR-200b and downstream Zeb1

Journal of Nanobiotechnology ◽

10.1186/s12951-020-00733-z ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Jie Ni ◽

Yihai Liu ◽

Lina Kang ◽

Lian Wang ◽

Zhonglin Han ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Cardiac Fibrosis ◽

Cardiac Injury ◽

Cardiomyocyte Apoptosis ◽

Luciferase Reporter ◽

Human Trophoblast ◽

Trophoblast Stem

AbstractHuman trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether trophoblast stem cell-derived exosomes (TSC-Exos) can protect cardiomyocytes from doxorubicin (Dox)-induced injury remains unclear. In the present study, TSC-Exos were isolated from the supernatants of human trophoblasts using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. In vitro, primary cardiomyocytes were subjected to Dox and treated with TSC-Exos, miR-200b mimic or miR-200b inhibitor. Cellular apoptosis was observed by flow cytometry and immunoblotting. In vivo, mice were intraperitoneally injected into Dox to establish a heart failure model. Then, different groups of mice were administered either PBS, adeno-associated virus (AAV)-vector, AAV-miR-200b-inhibitor or TSC-Exos via tail vein injection. Then, the cardiac function, cardiac fibrosis and cardiomyocyte apoptosis in each group were evaluated, and the downstream molecular mechanism was explored. TSC-Exos and miR-200b inhibitor both decreased primary cardiomyocyte apoptosis. Similarly, mice receiving TSC-Exos and AAV-miR-200b inhibitor exhibited improved cardiac function, accompanied by reduced apoptosis and inflammation. The bioinformatic prediction and luciferase reporter results confirmed that Zeb1 was a downstream target of miR-200b and had an antiapoptotic effect. TSC-Exos attenuated doxorubicin-induced cardiac injury by playing antiapoptotic and anti-inflammatory roles. The underlying mechanism could be an increase in Zeb1 expression by the inhibition of miR-200b expression. In summary, this study sheds new light on the application of TSC-Exos as a potential therapeutic tool for heart failure.

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