NOVEL FINDINGS OF EXOSOMES IN HUMAN PLACENTA ORGANOIDS

Abstract

Objective

Placenta is essential for coordinating the maternal response to pregnancy and releases molecules that affect maternal physiology. Placenta organoids offer a novel model for studying placental development and function, enabling researchers to explore interactions among the placenta's cell types. Exosomes, 30-150 nm extracellular vesicles formed via the endosomal pathway, transport signaling molecules and regulate cellular functions. Syncytiotrophoblast is the main source of placenta-derived extracellular vesicles. While exosome profiles of various tissue organoid models are documented, data on human placenta organoids (HPO) is lacking. This study aims to investigate if the exosome profile of HPOs can mimic that of in vivo human placenta.

Materials and Methods

In our study, with consent obtained from patients at the Obstetrics and Gynecology Clinic of Koç University Hospital, CTB isolation was performed from surgically obtained material of 6-9 weeks healthy pregnancy elective terminations (curettage), and HPO created by embedding the CTB cells in Matrigel. HPOs cultured at 37°C in a 5% CO2 environment with trophoblast organoid medium (TOM). HPOs were characterized using immunofluorescence with antibodies labeled with anti-Ki67, anti-CYT7, and anti-bHCG. TOM-conditioned medium was collected on days3 and 5 of the organoid culture. An explant culture (ExC) was established from the same villi used for HPO production and its conditioned media were collected at 24h and 48h. Exosomes were isolated using ultracentrifugation (100,000g for 2h). Exosome characterization involved size and concentration analysis using Nanoparticle Tracking Analysis (NTA) and detection of CD63 protein, an exosome-specific surface marker, using Western Blot.

Results

In HPOs, CTB cells located in the outer part of the organoids were specifically marked by the nuclear expression of the proliferation marker Ki67 and the cytoplasmic expression of CYT7. Additionally, STB cells in the inner part of the placenta organoid were specifically identified by bHCG labeling. The NTA results of HPOs and ExC revealed distinct particle concentrations over time. For HPO, the particle concentration (particles/ml) was 1.71 × 10¹⁰±9.32 × 10⁸ on day3 and 7.39 × 10¹⁰±1.37 × 10¹⁰ by day5. In comparison, explant cell culture showed a particle concentration (particles/ml) of 4.73 × 10¹⁰±5.11 × 10⁹ at 24h and 8.69 × 10⁹±2.55 × 10⁸ at 48h. CD63 protein expression confirmed the successful isolation of exosomes from HPO and ExC.

Conclusion

This research provides the first evidence of exosomes released from HPOs, showing that these organoids can functionally mimic the in vivo placenta. This finding is crucial for future studies, suggesting that HPOs could be used in drug trials and for comparing exosome profiles in diagnosing and treating pregnancy-related diseases like preeclampsia and IUGR.