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, 12 (2), 191-200

Human Pluripotent Stem Cell-Derived Striatal Interneurons: Differentiation and Maturation In Vitro and in the Rat Brain

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Human Pluripotent Stem Cell-Derived Striatal Interneurons: Differentiation and Maturation In Vitro and in the Rat Brain

Zoe Noakes et al. Stem Cell Reports.

Abstract

Striatal interneurons are born in the medial and caudal ganglionic eminences (MGE and CGE) and play an important role in human striatal function and dysfunction in Huntington's disease and dystonia. MGE/CGE-like neural progenitors have been generated from human pluripotent stem cells (hPSCs) for studying cortical interneuron development and cell therapy for epilepsy and other neurodevelopmental disorders. Here, we report the capacity of hPSC-derived MGE/CGE-like progenitors to differentiate into functional striatal interneurons. In vitro, these hPSC neuronal derivatives expressed cortical and striatal interneuron markers at the mRNA and protein level and displayed maturing electrophysiological properties. Following transplantation into neonatal rat striatum, progenitors differentiated into striatal interneuron subtypes and were consistently found in the nearby septum and hippocampus. These findings highlight the potential for hPSC-derived striatal interneurons as an invaluable tool in modeling striatal development and function in vitro or as a source of cells for regenerative medicine.

Keywords: Huntington's disease; caudal ganglionic eminence; cell replacement therapy; differentiation; human pluripotent stem cells; medial ganglionic eminence; striatal development; striatal interneurons.

Figures

Figure 1
Figure 1
Differentiation of hESCs into Striatal and Cortical Interneurons In Vitro (A–C) qPCR data presented as gene expression fold-change of SHH-treated samples relative to untreated controls on days 20 (A) and 45 (B) on a logarithmic y axis. Day 45 samples were also analyzed for their expression of cortical and striatal interneuron-specific markers (C). Data are presented as mean fold-change ± SEM from three independent experiments performed in H7 cells. p < 0.05, two-sample t test with equal variance not assumed. (D–F) Representative immunocytochemistry images of H7 control and SHH-treated cultures at days 20 (D) and 60 (E and F). Scale bars, 100 μm. (G–I) Images were counted for MGE and CGE progenitor markers (G and I) and interneuron subtype markers (H and I). Data presented are mean ± SEM from independent experiments performed in H7 (n = 3), H7-tauGFP (n = 2), H9 (n = 1), and iCas9 (n = 1) for day 20 and H7 (n = 3) and H7-tauGFP (n = 2) for day 60. p < 0.05, ∗∗∗p < 0.001, one-way ANOVA (COUP-TFII, SST, CR), Kruskal-Wallis test (FOXG1, ASCL1, OLIG2, PV), and two-way ANOVA with post hoc Bonferroni (NKX2.1).
Figure 2
Figure 2
hESC-Derived MGE/CGE-like Progenitors Become Functional GABAergic Neurons (A) Schematic of whole-cell patch clamp of H7-tauGFP+ neurons co-cultured with primary mouse astrocytes. (B) Basic membrane properties at days 45 (n = 25) and 60 (n = 16–19). RMP, resting membrane potential. (C) Representative traces of spontaneous activity (left). Post-synaptic potentials and action potentials were counted over 2 min (right: D45, n = 14; D60, n = 14). (D) Representative traces of evoked activity from current injection steps (left). The maximum number of evoked spikes was quantified in each cell (right: D45, n = 23; D60, n = 19). (E) Overlaid averaged traces of all D45 (blue, n = 15) and D60 (red, n = 16) first evoked spikes. (F) Amplitude and half-width of first evoked spikes. (G) Schematic illustrating second pipette for focal application of glutamate or GABA onto patched cell. (H) Representative traces showing glutamate- (Glut, 100 μM) and GABA (100 μM)-evoked currents (left) and their quantification (right: Glut, n = 5; GABA, n = 14). (I) Post hoc immunocytochemistry of AF555-filled neurons to confirm GAD67 (white) expression. Scale bars, 15 μm. (J) AF555-filled neurons were imaged and traced post hoc in Neurolucida. (K) Quantification of Sholl analysis intersections compared by two-way ANOVA with post hoc Bonferroni correction. (L) Total neurite length was divided into primary path length (white) and branch length (gray) (D45, n = 15; D60, n = 18). n represents number of cells from three independent experiments and all data plots show mean ± SEM of the cells recorded. All statistical analyses except for (K) were done by two-sample t test. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 3
Figure 3
Transplanted MGE/CGE-like Progenitors Adopt Region-Specific Morphologies (A) Representative immunohistochemistry images of H7-tauGFP+ (green) hESC-derived progenitors and neurons stained for HuNu (red) and counterstained with DAPI (blue) at 6, 12, and 20 weeks post-transplantation, in the striatum, septum, and hippocampus of rats. Visible GFP+ cells were traced using Neurolucida and representative examples are shown on the right. Scale bar, 50 μm. (B) Sholl profiles comparing cells in each brain region at 6, 12, and 20 weeks. Data presented are mean number of intersections per shell ± SEM of n = 9–26 cells. (C) The number of primary neurites, branch points, and terminations were compared by two-way ANOVA with post hoc Bonferroni. Horizontal bars show significant differences across time points color-coded to their respective brain regions (blue, striatum; red, septum; green, hippocampus) and black vertical bars show significant differences between brain regions at 20 weeks. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 4
Figure 4
Transplanted MGE/CGE-like Progenitors Differentiate into Striatal Interneurons (A–C) Representative immunohistochemistry images of brain sections from 6 (A), 12 (B), and 20 (C) weeks post-transplantation, with GFP+ (green) transplanted cells and DAPI (blue). (D) NKX2.1 (blue: 6 weeks, n = 6; 12 weeks, n = 5; 20 weeks, n = 3) and NeuN (orange: 6 weeks, n = 5; 12 weeks, n = 3; 20 weeks, n = 3) were counted as a percentage of HuNu+ and GFP+ cells, respectively. Two-way ANOVA reported no significant differences. Str, striatum; SVZ, subventricular zone; hip, hippocampus; sep, septum. Scale bars, 100 μm (A–C, top two panels), 250 μm (C, bottom three panels).

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