Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase

Abstract

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.

Multi-omics analysis identifies LBX1 and NHLH1 as central regulators of human midbrain dopaminergic neuron differentiation

“…Generation of paired transcriptomic and chromatin accessibility profiles of mDAN differentiation mDANs were enriched for transcriptomic and epigenomic profiling based on the expression of P2A-mCherry reporter, stably expressed under the control of the endogenous promoter of the TH gene, coding for tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis (Figure 1A, (19)).…”

“…mDANs were enriched for transcriptomic and epigenomic profiling based on the expression of P2A-mCherry reporter, stably expressed under the control of the endogenous promoter of the TH gene, coding for tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis (Figure 1A, (19)). The presence of the mCherry reporter allowed the purification of mDANs from heterogeneous iPSC-derived neuronal cultures by FACS at multiple time points of differentiation (Figure 1A-B).…”

“…The human iPSC line GM17602 (Coriell) was used in this study as a control and for the generation of a tyrosine hydroxylase (TH) reporter cell line. This iPSC line was previously characterized in (24) (called HFF) and used in (19) for the generation of the reporter line. Briefly, in the reporter line, the T2A coding sequence was fused with the mCherry open reading frame, and it was biallelically inserted in place of the stop codon in the endogenous TH locus using CRISPR/Cas9 editing (Figure 1A).…”

“…Only a few studies have considered the epigenetic landscape of mDANs during development (16)(17)(18). Furthermore, cellular heterogeneity present in the iPSC-derived cultures obscure physiological or biological insights from the cell type of interest (19,20).…”

Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons

“…Modulating the combination, concentration, and exposure time of crucial signaling molecules has yielded protocols for generating neurons and glia from iPSCs [ 23 ]. Thus far, researchers have developed protocols by which several neural and glial cell types could be generated, including glutamatergic neurons [ 24 ], GABAergic neurons [ 25 , 26 ], dopaminergic neurons [ 27 ], serotonergic neurons [ 28 ], MN [ 29 , 30 , 31 , 32 ], astrocytes [ 33 ], and microglia [ 34 , 35 ]. The improvement techniques and updated protocols in neural differentiation and maturation greatly facilitate the generation of iPSC-derived cells.…”