Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation

Abstract

Background: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction. Methods: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer. Results: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. Conclusions: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.