Steering human testicular organoid architecture by avoiding EMT in Sertoli cells

Samenvatting

Background/aim: Human testicular organoids (TOs) derived from transgender testicular tissue show
promise for reprotoxicology studies. However, initial histological findings indicate that current TOs are
unable to replicate a seminiferous epithelial-like structure surrounded by interstitial cells, crucial for
mimicking the protective environment supporting germ cell differentiation. Instead, Sertoli cells in
TOs appear to undergo an epithelial-to-mesenchymal transition (EMT), contributing to the observed
architecture. High expression of pro-inflammatory cytokines IL6 and IL1β in TOs was detected and may
be the driver of Sertoli cell EMT. The immunostimulatory Toll-like receptors TLR2 and TLR4 have the
ability to activate these cytokines and were also found to be upregulated in TOs. Additionally, the lack
of physical support in the current set up, as well as a lack of specific Sertoli cell factors in the medium,
may also contribute. The hypothesis is that by either modulating inflammation to avoid EMT and
focusing on maintaining Sertoli cell phenotype, the desired architecture can be achieved.

Material & methods: Transgender testistissue was digested, and cells were seeded in a 3D Petri Dish®.
In the first experiment, CLI-095 and TL2-C29 were added in TO medium to inhibit TLR4 and TLR2,
respectively. In the second experiment, mesenchymal stem cells (MSCs) were included during TO
formation for their immunomodulatory and physical support abilities. In the third experiment, TOs
were seeded in a defined Sertoli cell-specific medium. Histological assessment of TOs was done
through H/PAS staining. Immunofluorescent staining for testis-specific markers was performed to
examine the reorganization of testicular cells within TOs. Expression levels of IL6 and IL1β were
determined by qRT-PCR.

Results: TL2-C29 and CLI-095 did not significantly reduce IL6 and IL1β expression, and no differences were
observed between control and treated TO architecture. Depending on their density, MSCs overgrew
testicular cells and filled the TOs with extracellular matrix, or changed very little their architecture.
The Sertoli cell-specific medium hindered organoid formation.

Conclusion: Recovery of the desired architecture was not achieved with TLR2/TLR4 inhibitors, MSCs, or the Sertoli
cell-specific medium.

Datum prijs	24 mei 2024
Originele taal	English
Begeleider	Samuel Madureira Silva (Advisor), Ellen Goossens (Promotor) & Yoni Baert (Promotor)