Poster Presentation ESA-SRB-ANZOS 2025 in conjunction with ENSA

Engineering placentoid cultures to model human placental villi complexity in vitro (128634)

Ashley Williams 1 , Georgia Kafer 1
  1. University of the Sunshine Coast, Moreton Bay, QLD, Australia

Aims

The maternal–fetal interface of the placenta consists of villi containing specialised trophoblast cells. Organoids offer a promising approach to model placental villi in vitro; however, current methods fail to replicate key aspects of villous structure and trophoblast cell characteristics1, 2, 3. This study aims to validate human stem cell-derived ‘placentoids’, grown on 3D-printed scaffolds, to better mimic villous architecture and support the differentiation of diverse trophoblast subtypes within a single system.

Methods

Polydimethylsiloxane (PDMS) villi scaffolds were fabricated using moulds printed with a 3D inkjet printer (Agilista, Keyence; 635×400 dpi, 15 μm resolution)4. PDMS (1:10) was poured into moulds, degassed under vacuum, and cured overnight at room temperature. Scaffolds were rendered wettable by ozone treatment prior to cell seeding. Human pluripotent stem cells (PSCs) were maintained in mTeSR1 before differentiation. PSCs were disaggregated into colonies (<250 μm) and seeded onto scaffolds. Trophoblast differentiation was induced using StemPro medium supplemented with BMP4 (10 ng/mL), SB431542 (20 µM), and SU5402 (20 µM). Cultures were maintained for up to 14 days and fixed with 4% paraformaldehyde for immunofluorescent staining and imaging using an Olympus FV3000 confocal microscope.

Results

Cells covered scaffold surfaces and lost pluripotency markers (Oct4) by day 3 (n = 3). Gradual expression of syncytiotrophoblast (STB) and extravillous trophoblast (EVT) markers was observed throughout the culture period. By day 12, cytotrophoblast cells (Ki67 and ITGB4 positive) were present alongside STBs (hCGβ and SDC1 positive) and EVTs (HLA-G positive). By day 14, HLA-G positive EVT bridges had formed between scaffold projections.

Conclusion

This placentoid culture system supports the differentiation of multiple trophoblast subtypes within a single culture system. Moreover, as placentoids more closely resemble placental villous structure than existing organoid models, this system provides a valuable tool for studying human placental villi development and dysfunction.

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  2. Haider S, Meinhardt G, Saleh L, Kunihs V, Gamperl M, Kaindl U, et al. Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta. Stem cell reports. 2018;11(2):537-51. https://dx.doi.org/10.1016/j.stemcr.2018.07.004.
  3. Sato M, Inohaya A, Yasuda E, Mogami H, Chigusa Y, Kawasaki K, et al. Three-dimensional human placenta-like bud synthesized from induced pluripotent stem cells. Scientific reports. 2021;11(1):14167-. https://dx.doi.org/10.1038/s41598-021-93766-9.
  4. Kamei K, Mashimo Y, Koyama Y, Fockenberg C, Nakashima M, Nakajima M, et al. 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients. Biomed Microdevices. 2015;17(2):36. https://dx.doi.org/10.1007/s10544-015-9928-y.