Molecular and functional characterization of hiPSCs derived dopaminergic neurons
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Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and by intracellular inclusions (Lewy bodies) in surviving neurons. The focal loss of neurons makes the disease particularly interesting for the stem cell field as it makes it an attractive target for patient derived cell substitution therapy. Therefore, in the recent years iPSCs have become a very important resource for the in vitro generation of a cell model of PD. Current standard protocols for defining maturity of iPSC derived DA neurons are illustrating expression of Tyrosine Hydroxylase (TH) along with varying midbrain markers like Lmx1a/b, FOXA2 and OTX2, paired with firing of action potentials of TH positive neurons. However, despite showing primary function of neurons as in the transduction of a chemoelectrical signal, positive electrophysiological identification of these cells are limited to single action potentials, lacking the display of synaptic function. We aim to address the electrophysiological development of iPSC derived DA neurons, using an established differentiation protocol (Kriks et al.). Under these conditions, we observed expression of molecular and functional characteristics of DA identity including expression of FOXA2, LMX1A, TH and PITX3. We also observe co-staining of TH with GIRK2 channel, a feature of A9 DA neurons, the subtype most susceptible to degeneration in PD. Additionally, we will supplement this protocol to accelerate this neuronal development, with particular attention to synapse function. We tracked the markers expression over time to understand the timing of midbrain DA neuron cell faith definition. The maturation process of cells was monitored throughout the differentiation by staining for markers of mature DA neurons (NURR1, MAPT/Tau) and synapse formation (PSD95, Synaptophysin) by Western Blot and immunocytochemical assays as well as qRT-PCR for monitoring transcriptional activation. To assess neuronal properties, whole-cell patch clamp recordings are obtained from neurons, measuring spontaneous and evoked action potentials, Na+-, K+-and Ca2+ mediated currents and miniature excitatory post-synaptic currents. Finally, we assess expression of Dopamine Transporter (DAT), Aromatic L-amino acid decarboxylase (AADC) and TH by immunocytochemistry over time and monitor stimulated DA release by MS-HPLC analysis of the culture media. The data presents a thorough characterization of an in vitro derived neuronal model that could allow a better understanding of maturation process of iPSC derived DA neurons.