Biology subjects

Karakis, V.

Publications and source records attributed to Karakis, V..

3 recordsLinked to original sources

bioRxiv2024

Derivation of human trophoblast stem cells from placentas at birth

Human trophoblast stem cells (hTSCs) have emerged as a powerful tool for modeling the placental cytotrophoblast (CTB) in vitro. hTSCs were originally derived from CTBs of the first trimester placenta or blastocyst-stage embryos in trophoblast stem cell medium (TSCM) that contains epidermal growth factor (EGF), the glycogen synthase kinase-beta (GSK3{beta}) inhibitor CHIR99021, the transforming growth factor-beta (TGF{beta}) inhibitors A83-01 and SB431542, valproic acid (VPA), and the Rho-associated protein kinase (ROCK) inhibitor Y-27632. Here we show that hTSCs can be derived from CTBs isolated from the term placenta, using TSCM supplemented with a low concentration of mitochondrial pyruvate uptake inhibitor UK5099 and lipid-rich albumin (TUA medium). Notably, hTSCs could not be derived from term CTBs using TSCM alone, or in the absence of either UK5099 or lipid-rich albumin. Strikingly, hTSCs cultured in TUA medium for a few passages could be transitioned into TSCM and cultured thereafter in TSCM. hTSCs from term CTBs cultured in TUA medium as well as those transitioned into and cultured in TSCM thereafter could be differentiated to the extravillous trophoblast and syncytiotrophoblast lineages and exhibited high transcriptome similarity with hTSCs derived from first trimester CTBs. We anticipate that these results will enable facile derivation of hTSCs from normal and pathological placentas at birth with diverse genetic backgrounds and facilitate in vitro mechanistic studies in trophoblast biology. Significance statementHuman trophoblast stem cells (hTSCs) derived from early pregnancy have emerged as a powerful tool for modeling early human placental development in vitro. However, restrictions on derivation of cell lines from fetal tissues limit the availability and genetic diversity of hTSC lines. Also, the associated pregnancy outcome of hTSCs derived from early gestation is unknown. Here we describe the derivation and characterization of hTSCs from placentas obtained at birth. Our results will enable derivation of genetically diverse hTSCs associated with normal and pathological pregnancies, enabling research in placental biology.

developmental biology↗

bioRxiv2021

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

Human 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 PKC/{beta} 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.

developmental biology↗

bioRxiv2019

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

Trophoblasts are the principal cell type of the placenta. The use of human trophoblast stem cells (hTSCs) as a model for studies of early placental development is hampered by limited genetic diversity of existing hTSC lines, and constraints on using human fetal tissue or embryos needed to generate additional cell lines. Here we report the derivation of two distinct stem cells of the trophectoderm lineage from human pluripotent stem cells. The first is a CDX2- stem cell equivalent to primary hTSCs - they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ putative human trophectoderm stem cell (hTESC) with distinct cell culture requirements and differences in gene expression and differentiation relative to hTSCs. Derivation of hTSCs and hTESCs from pluripotent stem cells significantly enables construction of models for normal and pathological placental development.

developmental biology↗