Abstract: DNA damage response (DDR) is a critical process involved in cancer, aging, and neurodegenerative diseases. Understanding how genetic variations influence individual responses to DNA damage is essential for developing personalized prevention and treatment strategies. Although, the use of induced pluripotent stem cells (iPSCs) for in vitro population genetics has gained popularity, traditional approaches, where iPSC lines are cultured in mono-culture, are time-consuming, expensive, and prone to technical variability. To address these challenges, the "village-in-a-dish" strategy has emerged. In this strategy, iPSCs from multiple donors representing the “normal” population, are cultured and stimulated together in a single dish. This platform increases throughput, reduces variability, and lowers costs, making it particularly useful for mimicking population studies in vitro, which require high donor numbers. Here, we established a pipeline to generate both small (5–10 iPSC lines) and large (up to 85 iPSC lines) villages, minimizing growth rate variability and enabling monitoring of cluster composition. For the first time, using cytoplasmic dyes, we demonstrated that iPSC clusters can form from both single and multiple donors. Additionally, we confirmed that the village platform does not affect the growth rates of the iPSC lines. We are now exploring how DDR, induced by X-ray and cisplatin, varies across different donors. After testing X-ray exposure on four iPSC lines (in mono-culture), we performed bulk RNA sequencing at multiple time points to capture variability and identify the optimal time point for scaling up towards the use of the village in a dish setup.
