Abstract
Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, representing a unique tool for in vitro modeling of Parkinson’s disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. For this reason, neurons differentiated in three dimensional (3D) in vitro cultures might better mimic the in vivo cellular environment for functional studies. Metabolic changes represent fundamental hallmarks in directing cell fate in the nervous system. In particular, PGC-1a is a key regulator of mitochondrial biogenesis, known to play an essential role in neuronal metabolism. Several studies have also highlighted that differentiation of PSCs induces a variety of mitochondrial morphological and functional changes. In this work, we used a 3D cell culture method based on the microencapsulation of hiPSCs in small alginate/fibronectin (Alg/Fn) beads and observed a faster metabolic shift during differentiation to DA neurons as compared to the conventional 2D protocol. The expression level of PGC1-a was significantly increased in the 3D cultures indicating an earlier and more pronounced shift to a post-mitotic neuronal identity. Furthermore, we observed reduced levels of mitochondrial superoxide production and higher levels of mitochondrial membrane potential at days 35 and 50 in the 3D neurons. Accordingly, the mitochondrial morphology appeared more elongated for the 3D cultures at both time intervals. Finally, a transcriptional profile of the 2D and 3D neuronal cultures support a stronger maturation into post-mitotic neurons in the 3D system. Collectively, these data suggest the occurrence of an earlier metabolic switch in the Alg/Fn 3D scaffold compared to 2D cultures, indicating that it might offer an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons for PD modeling and therapy development.