A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids

Area of Science:

• Cell Biology
• Vascular Biology
• Biotechnology

Background:

• 3D cell culture models are crucial for studying complex biological processes.
• Endothelial cells form blood vessels and are key in vascular development and disease.
• Modeling mosaic mutations in endothelial cells is challenging but important for understanding vascular malformations.

Purpose of the Study:

• To create a simple 3D endothelial spheroid model with mosaic gene expression.
• To utilize adeno-associated virus (AAV) transduction for gene delivery.
• To establish a platform for studying post-zygotic mutations in vascular malformations.

Main Methods:

• Two immortalized human endothelial cell lines were cultured in 96-well plates.
• Spontaneous aggregation and spheroid formation were monitored.
• Adeno-associated virus (AAV) transduction was used to achieve mosaic gene expression (eGFP).

Main Results:

• hCMEC/D3 cells spontaneously formed robust, stable 3D spheroids within 3 days.
• These spheroids developed a basement membrane and expressed CD31.
• AAV1 and AAV2^QUADYF transduction resulted in sustained, mosaic eGFP expression without toxicity.

Conclusions:

• A simple method for generating 3D endothelial spheroids with mosaic gene expression was established.
• This model facilitates the study of vascular malformations caused by mosaic mutations.
• The model is suitable for assessing 3D phenotypes and drug/radiation sensitivities.