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Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

ABSTRACT: Calcium regulation is a critical process in neurons, and Ca²⁺ signaling is a major contributor to neurological disorders including Parkinson's disease (PD). Here, combining calcium imaging with whole-cell Ca²⁺ current recording, we provide a detailed protocol for measuring Ca²⁺ homeostasis in dopaminergic (DA) neurons derived from human induced pluripotent stem cells (hiPSCs). This approach can be applied to investigate the role of Ca²⁺ homeostasis in neuronal functionality as well as in disease processes. For complete details on the use and execution of this protocol, please refer to Kim et al. (2020).

SUBMITTER: Yin X

PROVIDER: S-EPMC8024766 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

Yin Xiling X Kim Jungwoo Wren JW Liu Shiyu S Dawson Ted M TM Dawson Valina L VL

STAR protocols 20210323 2

Calcium regulation is a critical process in neurons, and Ca2+ signaling is a major contributor to neurological disorders including Parkinson's disease (PD). Here, combining calcium imaging with whole-cell Ca2+ current recording, we provide a detailed protocol for measuring Ca2+ homeostasis in dopaminergic (DA) neurons derived from human induced pluripotent stem cells (hiPSCs). This approach can be applied to investigate the role of Ca2+ homeostasis in ...[more]

PMID: 33855307

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Project description:Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson's disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a "floor plate protocol" generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH- cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH- neuronal cell populations and that the "floor plate protocol" is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

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.

Dataset Information

Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

Publications

Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

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