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Method Article
Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.
Lucy Gardner
Centre for Trophoblast Research, University of Cambridge
Corresponding Author
Margherita Turco
Centre for Trophoblast Research, University of Cambridge
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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This protocol describes the generation of long-term, genetically-stable organoid cultures of extraembryonic fetal trophoblasts from human first trimester placentas. Trophoblast organoids are consistently established within one to two weeks. They can be expanded long term, cryopreserved and resurrected. Villous cytotrophoblasts (VCT) expressing EpCam, Ki67 and TP63 form an outer cell layer with deposition of basement membrane in contact with Matrigel. Differentiated, multinucleated syncytial cytotrophoblasts (SCT) form in the centre of the organoid, analogous to the intervillous space. They secrete placental specific peptides and hormones such as hCG, GDF15, PSGs and PAPPA. Characteristic features of SCT are confirmed by electron microscopy (EM). In addition, the organoids can be differentiated to invasive HLA-G+ extravillous trophoblasts (EVT) which digest and migrate out through the Matrigel. In summary, we describe the generation of trophoblast organoids from human extraembryonic tissues that grow as complex 3D structures with fusion of VCT to hCG-secreting SCT, anatomically and functionally closely resembling the villous placenta in vivo.
Keywords
human, placental, trophoblast, organoids, extraembryonic, 3-D culture, Matrigel
Lucy GARDNER
commented on 10 January, 2020
Medium formulations • Basic wash medium - HAMS/F12 medium (Biosera #SM-H0096) plus penicillin/ streptomycin • 0.2% trypsin-250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma #E9884)/PBS • Collagenase V solution 1.0mg/ml (Sigma #C9263) in Hams F12/10% FBS • Trophoblast Organoid Medium (TOM, Table 1). • Freeze medium – 20% DMSO in TOM. • Trophoblast Organoid Medium (TOM) - Advanced DMEM/F12, N2 supplement, B27 supplement minus vitamin A, Primocin 100μg/mL, N-Acetyl-L-cysteine 1.25 mM, L-glutamine 2mM, recombinant human EGF 50 ng/mL, CHIR99021 1.5µM, recombinant human Rspondin-1 80ng/mL, recombinant human FGF-2 100ng/mL, recombinant human HGF 50ng/mL, A83-01 500nM, prostaglandin E2 2.5µM, Y-27632 2µM. • EVT medium #1 – Advanced DMEM/F12, 2-mercaptoethanol 0.1mM, penicillin/streptomycin solution 0.5%, BSA 0.3%, ITS-X supplement 1%, NRG1 100ng/ml, A83-01 7.5µM, Knockout serum replacement 4%. • EVT medium #2 - Advanced DMEM/F12, 2-mercaptoethanol 0.1mM, penicillin/streptomycin solution 0.5%, BSA 0.3%, ITS-X supplement 1%, A83-01 7.5µM, Knockout serum replacement 4% ie: EVT medium #1 without NRG1.
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CURRENT STATUS: Posted
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Posted 30 Nov, 2018
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Biological techniques
Associated Publications
Trophoblast organoids as a model for maternal–fetal interactions during human placentation
by Margherita Y. Turco, Lucy Gardner, Richard G. Kay, +15
Nature (27 November, 2018)
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This is a preprint. It has not completed peer review.
Method Article
Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.
Lucy Gardner
Centre for Trophoblast Research, University of Cambridge
Corresponding Author
Margherita Turco
Centre for Trophoblast Research, University of Cambridge
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Nov, 2018
Published
On 09 Sep, 2020
Community comments: 1
Metrics
Comments: 1
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version here.
This protocol describes the generation of long-term, genetically-stable organoid cultures of extraembryonic fetal trophoblasts from human first trimester placentas. Trophoblast organoids are consistently established within one to two weeks. They can be expanded long term, cryopreserved and resurrected. Villous cytotrophoblasts (VCT) expressing EpCam, Ki67 and TP63 form an outer cell layer with deposition of basement membrane in contact with Matrigel. Differentiated, multinucleated syncytial cytotrophoblasts (SCT) form in the centre of the organoid, analogous to the intervillous space. They secrete placental specific peptides and hormones such as hCG, GDF15, PSGs and PAPPA. Characteristic features of SCT are confirmed by electron microscopy (EM). In addition, the organoids can be differentiated to invasive HLA-G+ extravillous trophoblasts (EVT) which digest and migrate out through the Matrigel. In summary, we describe the generation of trophoblast organoids from human extraembryonic tissues that grow as complex 3D structures with fusion of VCT to hCG-secreting SCT, anatomically and functionally closely resembling the villous placenta in vivo.
Lucy GARDNER
commented on 10 January, 2020
Medium formulations • Basic wash medium - HAMS/F12 medium (Biosera #SM-H0096) plus penicillin/ streptomycin • 0.2% trypsin-250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma #E9884)/PBS • Collagenase V solution 1.0mg/ml (Sigma #C9263) in Hams F12/10% FBS • Trophoblast Organoid Medium (TOM, Table 1). • Freeze medium – 20% DMSO in TOM. • Trophoblast Organoid Medium (TOM) - Advanced DMEM/F12, N2 supplement, B27 supplement minus vitamin A, Primocin 100μg/mL, N-Acetyl-L-cysteine 1.25 mM, L-glutamine 2mM, recombinant human EGF 50 ng/mL, CHIR99021 1.5µM, recombinant human Rspondin-1 80ng/mL, recombinant human FGF-2 100ng/mL, recombinant human HGF 50ng/mL, A83-01 500nM, prostaglandin E2 2.5µM, Y-27632 2µM. • EVT medium #1 – Advanced DMEM/F12, 2-mercaptoethanol 0.1mM, penicillin/streptomycin solution 0.5%, BSA 0.3%, ITS-X supplement 1%, NRG1 100ng/ml, A83-01 7.5µM, Knockout serum replacement 4%. • EVT medium #2 - Advanced DMEM/F12, 2-mercaptoethanol 0.1mM, penicillin/streptomycin solution 0.5%, BSA 0.3%, ITS-X supplement 1%, A83-01 7.5µM, Knockout serum replacement 4% ie: EVT medium #1 without NRG1.
1 comment flagged for moderation
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Associated Publications
Trophoblast organoids as a model for maternal–fetal interactions during human placentation
by Margherita Y. Turco, Lucy Gardner, Richard G. Kay, +15
Nature (27 November, 2018)