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Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta

Abstract

The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability to understand pregnancy disorders. Generating an in vitro model that recapitulates the unique features of the human placenta has been challenging. The first in vitro model system of human trophoblast that could be cultured long term and differentiated to syncytiotrophoblast (SCT) and extravillous trophoblast (EVT) was a two-dimensional (2D) culture system of human trophoblast stem cells. Here, we describe a protocol to isolate trophoblast from first-trimester human placentas that can be grown long term in a three-dimensional (3D) organoid culture system. Trophoblast organoids can be established within 2−3 weeks, passaged every 7–10 d, and cultured for over a year. The structural organization of these human trophoblast organoids closely resembles the villous placenta with a layer of cytotrophoblast (VCT) that differentiates into superimposed SCT. Altering the composition of the medium leads to differentiation of the trophoblast organoids into HLA-G+ EVT cells which rapidly migrate and invade through the Matrigel droplet in which they are cultured. Our previous research confirmed that there is similarity between the trophoblast organoids and in vivo placentas in their transcriptomes and ability to produce placental hormones. This organoid culture system provides an experimental model to investigate human placental development and function as well as interactions of trophoblast cells with the local and systemic maternal environment.

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Fig. 1: Summary of derivation, validation and quality control of trophoblast organoids.
Fig. 2: Derivation of trophoblast organoids.
Fig. 3: Troubleshooting: identifying glandular contamination and timing of passaging trophoblast organoid cultures.
Fig. 4: EVT differentiation and characterization.
Fig. 5: Controls and gating strategy for flow cytometric analysis.
Fig. 6: Clonal isolation, propagation and differentiation of trophoblast organoids.

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Acknowledgements

The authors are grateful to patients for donating tissue for research. We thank D. Moore and staff at Addenbrookes Hospital, Cambridge. This work was initiated and funded by the Centre for Trophoblast Research, University of Cambridge. G. J. Burton was funded by the Medical Research Council (MR/L020041/1). A. Moffett holds a Wellcome Trust joint investigator award with Dr F. Colucci (200841/Z/16/Z). M.Y. Turco received funding from the E.U. 7th Framework Programme for research, technological development and demonstration (PIEF-GA-2013-629785) and from the European Union’s Horizon 2020 research and innovation programme (Grant agreement no. [853546]). M.Y. Turco holds a Royal Society Dorothy Hodgkin Fellowship (DH160216) and a Royal Society Research Grant (RGF\R1\180028). We thank all members of the Moffett Laboratory for support.

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M.Y.T. and L.G. developed the organoid culture system for trophoblast in ref. 8. All the experiments undertaken while developing this protocol were carried out by M.A.S., R.C.F., L.G., and M.S.H. The manuscript was written by M.Y.T., M.A.S., L.G. and A.M. with help from R.C.F. and G.J.B. The figures were prepared by M.A.S., R.C.F., and M.S.H. All authors commented on the manuscript.

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Correspondence to Margherita Y. Turco.

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Turco, M. Y. et al. Nature 564, 263–267 (2018): https://doi.org/10.1038/s41586-018-0753-3

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Sheridan, M.A., Fernando, R.C., Gardner, L. et al. Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta. Nat Protoc 15, 3441–3463 (2020). https://doi.org/10.1038/s41596-020-0381-x

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