Organoid generation from trophoblast stem cells highlights distinct roles for cytotrophoblasts and stem cells in organoid formation and expansion.

Background: Organoids are stem-cell derived, self-organised, three-dimensional cultures that improve in vitro recapitulation of tissue structure. The generation of trophoblast organoids using primary placental villous digests (containing cytotrophoblasts and trophoblast stem cells (TSC)) improved high-throughput assessment of early trophoblast differentiation. However, the relative contributions of cytotrophoblasts and TSCs to trophoblast organoid growth and differentiation remain unclear, with implications for model interpretation. Here we sought to generate organoids from side-population trophoblasts (SpTSCs) to better understand the contribution of TSC to trophoblast organoid formation.

Methods: Methods were adapted from Haider et al., 2018 to generate organoids from Okae TSCs (OkTSCs) or SpTSCs. Organoid growth was compared with primary villous trophoblast organoids and cellular composition interrogated by immunohistochemistry.

Results: Organoids can be derived from first-trimester SpTSCs that exhibit similar architecture to those from primary villous trophoblast. However, organoids established from pure TSC populations (OkTSC or SpTSC) have different growth dynamics to primary placental villous digest-derived organoids - with OkTSCs developing faster and spontaneously generating migratory cells, whilst SpTSC organoids grow more slowly. Importantly, depletion of SpTSC from first-trimester villous digests ablates organoid formation. Finally, the capacity of the side-population technique to isolate late-gestation TSC enabled the generation of trophoblast organoids from term placentae, although these were significantly smaller than their first-trimester SpTSC counterparts.

Discussion: Together, this work highlights the requirement of TSC for organoid formation, and the functional distinction between TSC and cytotrophoblasts. Proof-of-principle data demonstrating organoid generation from late gestation TSC isolated directly from the placenta lays the groundwork for future disease models.