Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Extracellular matrix drives trophoblast differentiation in placental organoids (#228)

Claire Richards 1 2 , Hao Chen 3 , Matthew O'Rourke 3 , David Gallego Ortega 4 5 6 , Amy Bottomley 7 , Matthew Padula 1 , Philip Hansbro 3 , Louise Cole 8 , Lana McClements 1
  1. School of Life Sciences, University of Technology Sydney, Ultimo, NSW, Australia
  2. Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia
  3. Centre for Inflammation, Centenary Institute and School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia
  4. School of Biomedical Engineering, Faculty of Engineering and Information Technology, University of Technology Sydney, Ultimo, NSW, Australia
  5. Garvan Institute of Medical Research. The Kinghorn Cancer Centre. , Darlinghurst, NSW, Australia
  6. School of Clinical Medicine. Faculty of Medicine, University of New South Wales, Kensington, NSW, Australia
  7. Microbial Imaging Facility at the Australian Institute for Microbiology and Infection, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia
  8. Microbial Imaging Facility at the Australian Institute for Microbiology and Infection, Faculty of Science, University of Technology Sydney, Ultimo, NSW, Australia

Preeclampsia is a cardiovascular disorder of pregnancy with limited treatment options, in part due to the lack of reliable models of human pregnancy. Placental organoids, three-dimensional cultures derived from stem cells, offer a new experimental model of placental development. However, most organoid cultures rely on Matrigel which varies between batches and cannot be tuned for composition and stiffness. We aimed to assess the role of extracellular matrix on trophoblast organoids by comparing those generated by Matrigel-embedding and bioprinting within a synthetic matrix.

ACH-3P trophoblast cells were embedded in Matrigel or bioprinted in a polyethylene glycol (PEG)-based matrix using a RASTRUM platform (Inventia Life Science) and maintained in Ham’s F12 culture medium (10% FBS, 1% penicillin-streptomycin) for up to 12 days. Organoid formation and growth were captured by live cell imaging. Organoid metabolism and viability were analysed by Alamar Blue assay and fluorescent labelling, respectively. Organoids were harvested for analysis of trophoblast differentiation by confocal microscopy and differential expression analysed at the gene and protein levels by single cell RNA sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).

Cells encapsulated within the Matrigel and PEG matrix self-formed organoids within 2-3 days, demonstrating invasive properties within the matrix. The presence of key trophoblast subtypes was confirmed by immunofluorescence labelling for key trophoblast markers. Single cell RNA sequencing revealed a greater proportion of extravillous trophoblasts and comparatively fewer syncytiotrophoblasts within bioprinted organoids compared to Matrigel-derived. This was confirmed at the protein level, with bioprinted organoids displaying significantly increased levels of extravillous trophoblast markers human leukocyte antigen G (HLA-G) and integrin subunit alpha 5 (ITGA5).

Here, we present a novel approach to placental organoid generation using highly tuneable and reproducible synthetic hydrogels. This study highlights an increased capacity to study trophoblast responses to their environment and differentiation pathways.