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Matrix directs trophoblast differentiation in a bioprinted organoid model of early placental development

ABSTRACT: Trophoblast organoids offer a unique opportunity to study mechanisms orchestrating placental growth and development during pregnancy. However, many organoid cultures rely on extracellular matrix reagents that are highly variable and unable to be tuned to reflect in vivo tissues. Here we describe the first bioprinted placental organoid model, generated using first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol matrix. Bioprinted organoids were compared comprehensively to classic Matrigel-embedding using functional assays, immunofluorescence microscopy, transcriptomic and proteomic analyses. Organoids differentiated spontaneously from cytotrophoblasts into two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs), with bioprinted organoids driven towards EVT differentiation. Bioprinted organoids were exposed to inflammation and treated with aspirin or metformin to assess their effects on trophoblast organoid formation and viability. Further, we reversed the inside-out architecture of ACH-3P organoids by suspension culture. Organoid suspension caused STBs to form a syndecan-1+ outer layer on the periphery of organoids, reflecting placental tissue. Here, we present an alternative trophoblast organoid model with further tuning potential to reflect the placental microenvironment in physiological and pathological pregnancies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell Differentiation Involved In Embryonic Placenta Development, Cell Culture

SUBMITTER: Matthew O'Rourke

LAB HEAD: Lana McClements

PROVIDER: PXD056796 | Pride | 2025-07-28

REPOSITORIES: Pride

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			Action	DRS
	18102023_2D_Cells.raw	Raw
	18102023_2D_Cells_R02.raw	Raw
	18102023_2D_Cells_R03.raw	Raw
	18102023_BvM2_BO.raw	Raw
	18102023_BvM2_BO_R02.raw	Raw

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Matrix directs trophoblast differentiation in a bioprinted organoid model of early placental development

Project description:Trophoblast organoids offer a unique opportunity to investigate mechanisms orchestrating placental growth and development during pregnancy. However, many organoid cultures rely on extracellular matrix reagents that are highly variable and unable to be tuned to reflect different tissues. Here we describe a new bioprinted placental organoid model using immortalised first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol matrix. Bioprinted organoids were comprehensively compared to classical Matrigel-embedding using functional assays, immunofluorescence microscopy, transcriptomic and proteomic analysis. Organoids spontaneously differentiated from cytotrophoblasts into the two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs), with bioprinted organoids driven towards EVT differentiation. Bioprinted organoids were exposed to inflammation and treated with aspirin or metformin to assess their effects on trophoblast organoid formation and viability. Further, we reversed the inside-out architecture of ACH-3P organoids by transferring immature organoids to suspension culture. Organoid suspension caused STB to form a syndecan-1+ outer layer on the periphery of organoids, reflecting placental tissue. We present an alternative trophoblast organoid model with potential for further tuning to accurately reflect the placental microenvironment and provide more reliable insights into early placental development.

2025-07-28 | GSE279994 | GEO

Gene expression profiles of trophoblast cell lines

Project description:Invasive extravillous trophoblasts (EVTs) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular marker expression with their primary counterpart, it is unknown to what extent they recapture the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytrophoblasts and extravillous trophoblasts. To identify EVT-specific gene expression signatures in trophoblast cell lines, we calcuted differentially expressed genes between pre-defined groups based on the distinct origins of the five trophoblast cell lines under investigation. Comparison 1 comprised EVT, HTR-8/Svneo and SGHPL-5 vs choriocarcinoma cells (ACH-3P, BeWo, JEG-3). Comparison 2 comprised EVT, ACH-3P, BeWo, JEG-3 vs extravillous trophoblast cell lines (HTR-8/SVneo, SGHPL-5).

2010-09-23 | E-GEOD-20510 | biostudies-arrayexpress

Non-neuronal acetylcholine as an endogenous regulator of proliferation and differentiation of Lgr5-positive stem cells in mice

Project description:Acetylcholine (ACh) has been considered a neurotransmitter residing in central, parasympathetic and neuromuscular synapses of mammals. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids exhibit a noticeable capacity for ACh synthesis that is sensitive to a potent inhibitor of choline acetyltransferase (ChAT). Imaging mass spectrometry reveals distribution of endogenous ACh that is localized in intestinal epithelial layer in the cultured organoids as well as in mouse small intestinal epithelium in vivo, suggesting non-neural resources of ACh. Treatment of organoids with carbachol down-regulates growth of organoids and expression of marker gene for each epithelial cell. On the other hand, antagonists for mAChRs enhances growth and differentiation of organoids, indicating involvement of mAChRs in regulating proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice. Gene expression patters of gut, crypt, y-organoid and o-organoid, respectively

2013-08-15 | E-GEOD-49864 | biostudies-arrayexpress

Effect of PRR15 knockdown on human trophoblast cell gene expression

Project description:Proline rich 15 (PRR15) is a small nuclear protein required for normal conceptus development in the sheep. We used microarrays to assess the changes in the trophoblast transcriptome when PRR15 expression was diminished. The human first-trimester trophoblast cell line, ACH-3P, was infected with control lentivirus expressing no shRNA or lentivirus expressing shRNA to target PRR15 mRNA for degradation, with three biological replicates per treatment.

2012-10-11 | E-GEOD-41459 | biostudies-arrayexpress

Single cell trajectory modeling identifies a primitive trophoblast state defined by BCAM enrichment

Project description:The establishment and function of the human placenta is dependent on specialized cells called trophoblasts. Unfortunately, little is known about the cellular and molecular processes controlling human trophoblast stem cell maintenance and differentiation into mature trophoblast sub-populations/cell states. To address this, we here report transcriptomic data from n=7 first trimester human placental tissues, n=3 regenerative human trophoblast stem cell (hTSC) derived trophoblast organoids, and n=3 EVT-differentiated hTSC derived organoid cultures at single-cell resolution. This study sought to identify the molecular programs and cell states important in trophoblast progenitor establishment, renewal, and differentiation. Placental tissues were collected following elective termination at the British Columbia Women’s Hospital CARE clinic. hTSC cultures were established as described and sequenced on either day 0 of culture (for regenerative organoid colonies), or day 7 of culture (for EVT-differentiated organoid colonies).

2021-12-14 | GSE174481 | GEO

Stem cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens

Project description:Trophoblast organoids derived from placental villi provide a 3D model system of human placental development, but access to first-trimester tissues is limited. Here we report that trophoblast stem cells isolated from naïve human pluripotent stem cells (hPSCs) can efficiently self-organize into 3D stem cell-derived trophoblast organoids (SC-TOs) with a villous architecture similar to primary trophoblast organoids. Single cell transcriptome analysis reveals the presence of distinct cytotrophoblast and syncytiotrophoblast clusters and a small cluster of extravillous trophoblasts, which closely correspond to trophoblast identities in the post-implantation embryo. These organoid cultures display clonal X chromosome inactivation patterns previously described in the human placenta. We further demonstrate that SC-TOs exhibit selective vulnerability to emerging pathogens (SARS-CoV-2 and Zika virus), which correlates with expression levels of their respective entry factors. The generation of trophoblast organoids from naïve hPSCs provides an accessible 3D model system of the developing placenta and its susceptibility to emerging pathogens.

2022-04-29 | GSE172241 | GEO

Methylation profiling of human trophoblast organoids providing a model for maternal-fetal interactions in early pregnancy and placenta tissue for comparison

Project description:Generation of long-term, genetically-stable organoid cultures of extra-embryonic fetal trophoblast cells from human early placentas. Methylation profiling of the human trophoblast organoids and early placenta tissue.

2018-11-28 | E-MTAB-7204 | biostudies-arrayexpress

Transcriptomic profile of human placenta derived trophoblast organoid cultures with its tissue of origin, human placental villi.

Project description:The aim of this microarray experiment was to compare the overall transcriptomic profile of human placenta derived trophoblast organoid cultures with its tissue of origin, human placental villi. As the placental villi contains both trophoblast and stromal populations, we have included placenta derived stromal cultures in this comparison.

2018-11-28 | E-MTAB-6683 | biostudies-arrayexpress

Next generation trophoblast organoids with apical-out trophoblast polarity and central villous core-like cavity

Project description:Human trophoblast organoid serves as a revolutionary tool for studying trophoblast cell development and maternal-fetal interactions. However, currently reported trophoblast organoids exhibited opposite trophoblast polarity to in vivo or a solid cell spheroid lacking of central villous core-like cavity, both of which fail to model the physiological structure of in vivo placenta villi. Here, using human naïve embryonic stem cells, we constructed a trophoblast organoid with both peripheral apical-out bilaminar trophoblast cells and central villous core-like cavity, named ppTO. Besides, ppTO simulated the typical molecular and secretory features of in vivo trophoblast. Single-cell RNA sequencing (scRNA-seq) analysis demonstrated that the development of ppTO simulated in vivo trophoblast syncytialization process and pan-trophoblast transcriptome features. Using ppTO, we revealed that the syncytialization process relied on a suppressed HIPPO signaling for initiating syncytialization and followed by a reactivated HIPPO signaling for STB maturation. By comparative study with reported organoids, we revealed the important role of the expression and orientation of polarity molecule aPKC in the formation and spatial patterning of STB within trophoblast organoids. In summary, we develop a trophoblast organoid simulating the physiological structure and molecular features of placental villous trophoblast for studying the mechanisms underlying trophoblast development and function.

2025-07-09 | GSE272513 | GEO

Human cytotrophoblast organoids

Project description:Human cytotrophoblast organoid cultures were established from the villous trophoblast of first trimester placentas. We analyzed the global expression profile of the cytotrophoblast organoids (CTB-ORG) and compared to the profile of the tissue of origin i.e. villous cytotrophoblast (vCTB) as well as to differentiated syncytiotrophoblast (STB) and placental fibroblasts (FIB).

2018-08-02 | GSE109976 | GEO

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