scholarly journals Growing human trophoblasts in vitro: a review of the media commonly used in trophoblast cell culture

Reproduction ◽  
2020 ◽  
Vol 160 (6) ◽  
pp. R119-R128 ◽  
Author(s):  
Yohanes N S Nursalim ◽  
Cherie Blenkiron ◽  
Katie M Groom ◽  
Lawrence W Chamley
Keyword(s):  
Trophoblast Cell ◽  
Human Trophoblast ◽  
Vast Array ◽  
Trophoblast Stem ◽  
Gradual Development ◽  
The Media ◽  
Media Supplements ◽  
In The Mists ◽  
Trophoblast Culture

Trophoblasts are unique epithelial cells found only in the placenta. It has been possible to isolate and maintain human trophoblasts in in vitro culture for many decades. During this period there have been a vast array of media and supplements reported for trophoblast culture and often the reasons for using the media and specific supplements employed in any given laboratory have been lost in the ‘mists of time’. After a gradual development over many years this field has recently changed, with the publication of several reports of the isolation, growth and differentiation of human trophoblast stem or stem-like cells. This advance was made largely because of a greater understanding of the molecular pathways that control human trophoblasts and availability of media supplements that can be used to manipulate those pathways. We have searched the literature and here summarise many of the different media and supplements and describe how and why they were developed and are used to culture human trophoblasts.

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

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

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

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.

154. EXTRACELLULAR CALRETICULIN ALTERS ENDOTHELIAL CELL AND TROPHOBLAST CELL FUNCTIONS IN VITRO CONSISTENT WITH PRE-ECLAMPSIA

2010 ◽  
Vol 22 (9) ◽  
pp. 72
Author(s):  
N. M. Gude ◽  
K. E. Crawford ◽  
J. L. Stevenson ◽  
S. P. Brennecke
Keyword(s):  
Endothelial Cell ◽  
In Vitro Culture ◽  
Cell Number ◽  
Trophoblast Cell ◽  
Human Trophoblast ◽  
Migratory Activity ◽  
Cell Numbers ◽  
Cell Functions ◽  
Normotensive Pregnancy

Pre-eclampsia is a multisystem disorder of human pregnancy that involves abnormal placentation via insufficient trophoblast cell invasion of the maternal spiral arteries and widespread maternal endothelial cell dysfunction. Factors in plasma of pre-eclamptic women affect both trophoblast and endothelial cell functions during in vitro culture (1). The calcium-binding protein calreticulin is elevated in peripheral blood with pre-eclampsia compared to normotensive pregnancy (2). The aim of this study was to determine the effects of exogenous calreticulin at concentrations relevant to normotensive pregnancy (2 µg/mL) and to pre-eclampsia (5 µg/mL) on human trophoblast cell (HTR8) and microvascular endothelial cell (myometrial) numbers and migratory activity. Cell migration was measured by scratch assay; changes in cell number were measured by MTS assay (Promega). The results showed that calreticulin at 5µg/mL did not affect HTR8 cell number (control 68044+24542 cells, with calreticulin 72810 + 30673 cells, n = 3, P > 0.05) after 48 hours, but significantly inhibited migration of the cells by 48+11% compared to the control at 26 hours (n = 4, P < 0.02). Calreticulin at 5 µg/mL and under conditions that did not change cell number significantly increased migration of the myometrial endothelial cells by 39+7% (n = 4, P < 0.01) at 20 hours. Calreticulin at 5 µg/mL, however, significantly reduced endothelial cell numbers after 3–5 days (control 6213 + 1937 cells, with calreticulin 1937+728 cells, n = 6, P < 0.05). There was no significant change to the functions of either cell type with 2 µg/mL of calreticulin. In conclusion, exogenous calreticulin at a concentration consistent with that found in maternal blood with pre-eclampsia was shown to alter trophoblast and endothelial cell migratory activity and reduce endothelial cell numbers during in vitro culture. These results indicate that elevated circulating calreticulin may contribute to the cellular mechanisms that underlie the development of pre-eclampsia. (1) Harris et al, Reprod Sci, 2009, 16: 1082–90.(2) Gu et al, Molec Human Repro, 2008, 14: 309–15.

scholarly journals Investigation of human trophoblast invasion in vitro

2020 ◽  
Vol 26 (4) ◽  
pp. 501-513 ◽  
Author(s):  
Yassen Abbas ◽  
Margherita Y Turco ◽  
Graham J Burton ◽  
Ashley Moffett
Keyword(s):  
Cell Lines ◽  
First Trimester ◽  
Trophoblast Cell ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Human Trophoblast ◽  
Uterine Environment ◽  
Microfluidic Assays ◽  
Invasion Assays

Abstract BACKGROUND In humans, inadequate trophoblast invasion into the decidua is associated with the ‘great obstetrical syndromes’ which include pre-eclampsia, foetal growth restriction (FGR) and stillbirth. The mechanisms regulating invasion remain poorly understood, although interactions with the uterine environment are clearly of central importance. Extravillous trophoblast (EVT) cells invade the uterus and transform the spiral arteries. Progress in understanding how they invade has been limited due to the lack of good in vitro models. Firstly, there are no non-malignant cell lines that have an EVT phenotype. Secondly, the invasion assays used are of limited use for the small numbers of primary EVT available from first-trimester placentas. We discuss recent progress in this field with the generation of new EVT lines and invasion assays using microfluidic technology. OBJECTIVE AND RATIONALE Our aim is to describe the established models used to study human trophoblast invasion in vivo and in vitro. The difficulties of obtaining primary cells and cell lines that recapitulate the phenotype of EVT are discussed together with the advantages and pitfalls of the different invasion assays. We compare these traditional end point assays to microfluidic assays where the dynamics of migration can be measured. SEARCH METHODS Relevant studies were identified by PubMed search, last updated on February 2020. A search was conducted to determine the number of journal articles published using the cell lines JEG-3, BeWo, JAR, HTR-8/Svneo, Swan-71 and primary human extravillous trophoblast in the last 5 years. OUTCOMES Deep trophoblast invasion into the maternal decidua is a particular feature of human pregnancy. This invasion needs to be finely regulated to allocate resources between mother and baby. A reliable source of EVT is needed to study in vitro how the uterine environment regulates this process. First, we critically discuss the issues with the trophoblast cell lines currently used; for example, most of them lack expression of the defining marker of EVT, HLA-G. Recently, advances in human stem cell and organoid technology have been applied to extraembryonic tissues to develop trophoblast cell lines that can grow in two (2D) and three dimensions (3D) and differentiate to EVT. This means that the ‘trophoblast’ cell lines currently in use should rapidly become obsolete. Second, we critically discuss the problems with assays to study trophoblast invasion. These lack physiological relevance and have simplified migration dynamics. Microfluidic assays are a powerful tool to study cell invasion because they require only a few cells, which are embedded in 3D in an extracellular matrix. Their major advantage is real-time monitoring of cell movement, enabling detailed analysis of the dynamics of trophoblast migration. WIDER IMPLICATIONS Trophoblast invasion in the first trimester of pregnancy remains poorly understood despite the importance of this process in the pathogenesis of pre-eclampsia, FGR, stillbirth and recurrent miscarriage. The new technologies described here will allow investigation into this critical process.

scholarly journals Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

2003 ◽  
Vol 23 (10) ◽  
pp. 3566-3574 ◽  
Author(s):  
Jean-Louis Frendo ◽  
Delphine Olivier ◽  
Valérie Cheynet ◽  
Jean-Luc Blond ◽  
Olivier Bouton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Fusion ◽  
Primary Cultures ◽  
Placental Tissue ◽  
Trophoblast Cell ◽  
Direct Role ◽  
Human Trophoblast ◽  
Type D ◽  
Env Protein

ABSTRACT We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation.

PPARγ controls pregnancy outcome through activation of EG-VEGF: new insights into the mechanism of placental development

2015 ◽  
Vol 309 (4) ◽  
pp. E357-E369 ◽  
Author(s):  
Vanessa Garnier ◽  
Wael Traboulsi ◽  
Aude Salomon ◽  
Sophie Brouillet ◽  
Thierry Fournier ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Trophoblast Cell ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Migration And Invasion ◽  
Placental Development ◽  
Human Trophoblast ◽  
Pparγ Agonist

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants ( n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ+/− and PPARγ−/− mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ−/− mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion.

scholarly journals PENGARUH POLIFENOL TEH HIJAU TERHADAP PRODUKSI TNF-A (TUMOUR NECROSIS FACTOR-A) PADA KULTUR SEL TROFOBLAS MANUSIA YANG DIPAPAR GLUKOSA TINGGI 33 Mm

2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Siti Aisah ◽  
Sasmito Djati ◽  
Husnul Khotimah
Keyword(s):  
Green Tea ◽  
Placental Tissue ◽  
Trophoblast Cell ◽  
Tea Polyphenol ◽  
Human Trophoblast ◽  
Green Tea Polyphenol ◽  
Normal Glucose ◽  
Trophoblast Culture ◽  
Necrosis Factor ◽  
Number Of Cells

The purpose of this research was to study the effect of green tea polyphenol to TNF-á production on human trophoblast cell culture exposed by 33 mM glucose. Trophoblast culture isolated from human fetal placental tissue by sectio caessaria. Monolayer trophoblast cells that had been incubated for 3 days at 5% CO2; 37°C were divided into 2 groups: (1) normal glucose (5 mM) and (2) glucose 33 mM exposure, both divided into 2 sub groups: (a) without green tea polyphenol treatment, and (b) green tea polyphenol treatment 0,1; 0,2; and 0;4 mg/ml. Cells incubated for 3 days at 5% CO2; 37°C then analyzed cytotrophoblast cells characteristic. TNF-á level was measured by ELISA and analyzed with oneway ANOVA. Immunocytochemistry showed the number of cells that expressed TNF-á then analyzed descriptively. The results of this study showed that TNF-á level at 0,1; 0,2; and 0,4 mg/ml polyphenol were 2804,333 ñg/mL; 2513,222 ñg/ml; and 2739,889 ñg/ml respectively compared with 2739,889 ñg/mL normal glucose without polyphenol, and 2721,000 ñg/mL; 2612,111 ñg/mL; and 2566,555 ñg/mL compared with 2621,000 ñg/mL glucose 33 mM exposure without polyphenol. Number of cells that expressed TNF-á with 0,1; 0,2; and 0,4 mg/ml polyphenol treatment were 0%; 2%; and 5,5% compared with 0% normal glucose without polyphenol, and 82%; 0%; and 0% compared with 3% glucose 33 mM exposure without polyphenol. Green tea polyphenol exposure for 3 days at 0,1; 0,2; and 0,4 mg/ml didn’t significantly affect the decreasing of TNF-á production. Keywords: GDM, green tea, polyphenol, TNF-á, trophoblast

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

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.

scholarly journals Effect of steroids on transcription and secretion of Gal-1 by the human trophoblast cell line in vitro

2013 ◽  
Vol 65 (4) ◽  
pp. 1331-1337 ◽  
Author(s):  
Danica Cujic ◽  
Zanka Bojic-Trbojevic ◽  
Natasa Tosic ◽  
Sonja Pavlovic ◽  
Ljiljana Vicovac
Keyword(s):  
Cell Line ◽  
Culture Media ◽  
Mrna Level ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Human Trophoblast ◽  
Present Evidence ◽  
Trophoblast Cell Line ◽  
Synthetic Glucocorticoid

Galectin-1 (Gal-1) is a lectin with recently documented pro-invasive function in trophoblasts in vitro, whose regulation is currently insufficiently known. The potential involvement of steroid hormones, synthetic glucocorticoid dexamethasone (DEX), the sex steroid progesterone (PRG) and mifepristone (RU486) in the regulation of Gal-1 in the trophoblast-derived cell line HTR-8/SVneo was investigated. Gal-1 mRNA levels were assessed by real-time PCR. The effect on secretion of Gal-1 into the culture media was followed using the SELDI-TOF protein chip array. We present evidence that DEX and RU486 significantly reduced Gal-1 in the HTR-8/SVneo cell line at the mRNA level. In addition, trophoblast-derived HTR-8/SVneo cells were shown to secrete detectable Gal-1 protein, which was only slightly increased by PRG. The potential clinical relevance of these findings remains to be determined.

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

2019 ◽  
Vol 116 (52) ◽  
pp. 26606-26613
Author(s):  
Sota Takahashi ◽  
Hiroaki Okae ◽  
Norio Kobayashi ◽  
Akane Kitamura ◽  
Kanako Kumada ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Hydatidiform Mole ◽  
Fetal Tissue ◽  
In Vitro Models ◽  
Contact Inhibition ◽  
Gene Encoding ◽  
Human Trophoblast ◽  
Trophoblast Stem Cells ◽  
Trophoblast Stem

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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