Deciphering the mitochondrial DNA reconfiguration in human pluripotent stem cells and differentiated neurons"

• Applicants: Professor Alessandro Prigione, Ph.D.; Andrea Rossi, Ph.D.
• Subject Area: Experimental Models for the Understanding of Nervous System Diseases ; Cell Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 579817425

Mitochondrial DNA (mtDNA) heteroplasmy, the coexistence of wild-type and mutant mtDNA molecules within the same cell, can have significant impact on overall cellular metabolism and health. High heteroplasmy of certain mtDNA variants is associated with severe inherited mitochondrial diseases that impair tissues that are highly dependent on mitochondrial energy, such as the central nervous systems. We and others have previously demonstrated that mtDNA heteroplasmy can undergo significant changes during the reprogramming of human fibroblasts into induced pluripotent stem cells (iPSCs). Monitoring the mtDNA state could thus represent an important quality control step for characterizing iPSCs. However, two obstacles currently limit the routine assessment of mtDNA in iPSCs: 1) the lack of understanding of the biological mechanisms underlying mtDNA changes during iPSC generation; 2) available analytical pipelines are cumbersome and lack standardization across laboratories. In this proposal, we aim to address these two aspects to ultimately improve the field of iPSCs and their translational implications. In our hypothesis-driven work, we will assess different chemical and genetic manipulations to modulate mtDNA heteroplasmy in established iPSC lines and during the reprogramming of fibroblasts into iPSCs. For mtDNA analysis, we will employ our newly developed Mitopore pipeline which enables cost-effective and streamlined mtDNA profiling. In parallel, we will generate induced neurons (iNeurons) from iPSCs derived from patients with mitochondrial diseases carrying pathogenic mtDNA variants. By coupling neuronal functionality with mtDNA changes at single-cell resolution in iNeurons, we aim to uncover the impact of mtDNA defects on human neuronal function. Taken together, our project might shed light on the mechanisms underlying mtDNA reconfiguration in iPSCs and the impact of heteroplasmy on neuronal functionality. These studies could have important implications for establishing quality control analyses of iPSCs and for driving the development of innovative human models of mtDNA disorders.

• DFG Programme: Research Grants