De Santis, Rosa (2025) Reprogramming-to-organoids: how to cost-efficiently leverage hIPSCs to model human diseases. [Tesi di dottorato]

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Abstract

Human-induced pluripotent stem cells (hiPSCs) can differentiate into any cell type in the human body, making them a valuable resource in personalized and regenerative medicine. The use of hiPSC-derived organoids holds great promise for disease modeling and opens up exciting possibilities for evaluating drug responses at the organ level rather than the cell level. High costs and intensive workloads often hamper differentiation. In addition, expanding hiPSCs is also necessary to allow their use, which may cause cytogenetic instability and mutation accumulation. To address this, we designed a highly efficient protocol that enables reprogramming to organoids in just 14 days. This protocol avoids hiPSC expansion by using freshly reprogrammed hiPSCs (‘nascent’). Nascent hiPSCs are directly self-assembled in 3D epiblast-like cysts and differentiated into all three germ layers. This novel continuous three-dimensional process offers a fast-track approach to cost-effectively generating functional organoids, avoiding expansion passages. Here, we conducted a multi-omics analysis by profiling scRNA and scATAC on pooled hiPSCs at different days of reprogramming to characterize gene expression patterns and chromatin accessibility. This approach enabled us to construct a pluripotency gene regulatory network (GRN), confirming that nascent hiPSCs possess pluripotent capabilities for successful organoid differentiation. We complemented our analysis with scRNA and bulk RNA, collecting samples throughout the differentiation process. Using this reprogramming-to-organoid approach, we guided neuroectodermal cysts toward cortical brain maturation. We also generated functional organoids from the other germ layers, such as the heart, muscle, and liver. This system protects the genomic integrity of hiPSCs and enables the creation of an innovative, cost-effective method for producing patient-specific avatars.

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