Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.
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ABSTRACT:
SUBMITTER: Yin X
PROVIDER: S-EPMC8024766 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neuronal migration is necessary in the process of the formation of brain architecture. Recently, we demonstrated that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons exhibit directional migration in vitro. However, it remains unclear how the cell shape is involved in their migration. In this study, we performed live imaging analyses using human iPSC-derived dopaminergic neurons. Our automated method, which can automatically identify the cell body shape and the cell position at specific time points, revealed that healthy iPSC-derived dopaminergic neurons migrate according to their shape. This migration behavior was out of accord in neurons derived from iPSCs carrying an RELN deletion. Our findings provide a novel theory that cell body orientation is related to the stability of movement direction for human dopaminergic neurons, under the regulation of RELN.
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Project description:Unlabelled: The progressive death of dopamine producing neurons in the substantia nigra pars compacta is the principal cause of symptoms of Parkinson's disease (PD). Stem cells have potential therapeutic use in replacing these cells and restoring function. To facilitate development of this approach, we sought to establish a preclinical model based on a large nonhuman primate for testing the efficacy and safety of stem cell-based transplantation. To this end, we differentiated baboon fibroblast-derived induced pluripotent stem cells (biPSCs) into dopaminergic neurons with the application of specific morphogens and growth factors. We confirmed that biPSC-derived dopaminergic neurons resemble those found in the human midbrain based on cell type-specific expression of dopamine markers TH and GIRK2. Using the reverse transcriptase quantitative polymerase chain reaction, we also showed that biPSC-derived dopaminergic neurons express PAX6, FOXA2, LMX1A, NURR1, and TH genes characteristic of this cell type in vivo. We used perforated patch-clamp electrophysiology to demonstrate that biPSC-derived dopaminergic neurons fired spontaneous rhythmic action potentials and high-frequency action potentials with spike frequency adaption upon injection of depolarizing current. Finally, we showed that biPSC-derived neurons released catecholamines in response to electrical stimulation. These results demonstrate the utility of the baboon model for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches for the treatment of PD.SignificanceFunctional dopamine neurons were produced from baboon induced pluripotent stem cells, and their properties were compared to baboon midbrain cells in vivo. The baboon has advantages as a clinically relevant model in which to optimize the efficacy and safety of stem cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. Baboons possess crucial neuroanatomical and immunological similarities to humans, and baboon pluripotent stem cells can be differentiated into functional neurons that mimic those in the human brain, thus laying the foundation for the utility of the baboon model for evaluating stem cell therapies.
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