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 neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFß) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFß signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFß inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFß inhibition prevented STB formation. 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.

Two distinct trophectoderm lineage stem cells from human pluripotent stem cells.

The trophectoderm layer of the blastocyst-stage embryo is the precursor for all trophoblast cells in the placenta. Human trophoblast stem (TS) cells have emerged as an attractive tool for studies on early trophoblast development. However, the use of TS cell models is constrained by the limited genetic diversity of existing TS cell lines and restrictions on using human fetal tissue or embryos needed to generate additional lines. Here we report the derivation of two distinct stem cell types of the trophectoderm lineage from human pluripotent stem cells. Analogous to villous cytotrophoblasts in vivo, the first is a CDX2- stem cell comparable with placenta-derived TS cells-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+ stem cell with distinct cell culture requirements, and differences in gene expression and differentiation, relative to CDX2- stem cells. Derivation of TS cells from pluripotent stem cells will significantly enable construction of in vitro models for normal and pathological placental development.

A quantitative image analysis platform for assessing trophoblast differentiation.

Immunofluorescence microscopy is extensively used in characterization of trophoblast differentiation in vitro. However, such data is primarily used to confirm the presence of protein markers or qualitatively compare levels of protein markers across experimental conditions. Imaging data, when processed and analyzed appropriately can provide quantitative and spatial information, and provide biological insight. Towards this end, here we present MATroph, an open-source MATLAB-based computational tool to process images generated by immunofluorescent microscopy. MATroph automatically executes a series of image processing operations, including the classification of red, blue, and green channels from images, background extraction, morphological operations, and image filtering. From the isolated blue channels corresponding to nuclear staining, this tool generates numerical values for cell number. Additionally, relative levels and spatial location of proteins are obtained by mapping red and green channel pixels to blue pixels by assigning minimum pixel distance between the blue and other color objects. Thus, this tool provides information about intracellular protein accumulation areas. Additionally, this tool can also classify cells as single cells or part of colonies, and extract information on protein levels for each; this is particularly useful for quantitative studies on extravillous trophoblast maturation. We provide a user-guide to analyze the relative levels of markers relevant to human trophoblast stem cell self-renewal and differentiation. Importantly, MATroph is composed of a simple MATLAB algorithm, and its implementation requires minimal expertise in programming.

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 (GSK3ß) inhibitor CHIR99021, the transforming growth factor-beta (TGFß) 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.