3D cultured human medium spiny neurons functionally integrate and rescue motor deficits in Huntington’s disease mice
Yuting Mei, Yuan Xu, Xinyue Zhang, Ban Feng, Yingying Zhou, Qian Cheng, Yuan Li, Xingsheng Peng, Mengnan Wu, Lianshun Xie, Lei Xiao, Wenhao Zhou, Yuejun Chen, Man Xiong
Yuting Mei, Yuan Xu, Xinyue Zhang, Ban Feng, Yingying Zhou, Qian Cheng, Yuan Li, Xingsheng Peng, Mengnan Wu, Lianshun Xie, Lei Xiao, Wenhao Zhou, Yuejun Chen, Man Xiong
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Research Article Cell biology

3D cultured human medium spiny neurons functionally integrate and rescue motor deficits in Huntington’s disease mice

Abstract

Dysfunction of striatal medium spiny neurons (MSNs) is implicated in several neurological disorders, including Huntington’s disease (HD). Despite progress in characterizing MSN pathology in HD, mechanisms underlying MSN susceptibility remain unknown, driving the need for MSNs derived from human pluripotent stem cells (hPSCs), especially subtypes in research and therapy. Here, we established a scalable 3D-default culture system to produce striatal MSNs efficiently from hPSCs by activation of the endogenous sonic hedgehog (SHH) pathway. These cells expressed canonical markers of striatal progenitors and dopamine D1 (D1) and dopamine D2 (D2) MSNs and presented dynamic specification and transcriptional signatures that closely resemble endogenous MSNs at single-cell resolution, both in vitro and post-transplantation in HD mice with quinolinic acid (QA) lesions. Grafted human cells survived and matured into D1-/D2-like MSNs and projected axons to endogenous targets including globus pallidus externus, globus pallidus internus, and substantia nigra pars reticulata to reconstruct the basal ganglia pathways. Functionally, they displayed spontaneous synaptic currents, received regulation from host cortex and thalamus, and were modulated by dopamine to either enhance or reduce neuronal excitability, similar to the endogenous D1-/D2-MSNs, subsequently improving behavior in QA-lesioned HD mice. Our study presents a method for generating authentic MSNs, providing a reliable cell source for HD cell therapy, mechanistic studies, and drug screening.

Authors

Yuting Mei, Yuan Xu, Xinyue Zhang, Ban Feng, Yingying Zhou, Qian Cheng, Yuan Li, Xingsheng Peng, Mengnan Wu, Lianshun Xie, Lei Xiao, Wenhao Zhou, Yuejun Chen, Man Xiong

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Figure 4

Neuron maturation and neural circuit reconstruction of grafted striatal neurons in HD mice.

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Neuron maturation and neural circuit reconstruction of grafted striatal ...
(A) The strategy of experimental design for cell transplantation (transpl.) in HD model mice. (B and C) Immunostaining (B) of GFP/Hoechst and quantification (C) of ipsilateral brain area loss in HD model mice with or without cell transplantation. Scale bar: 1 mm. (D) Representative images showing GFP (nuclear localization sequence [nls])/GABA double-labeling in grafts 2MPT. Scale bar: 500 μm. White dashed box is magnified in right panel (a). Scale bar: 100 μm. White box is magnified in lower panel (b). Scale bar: 10 μm. (E and F) Representative immunofluorescence images of GABA/mCherry/CTIP2 and DARPP32/hN/CTIP2 for grafts 5MPT. Scale bar: 50 μm. (G) Representative immunofluorescence images of GABA/GFP/CTIP2 and DARPP32/GFP/CTIP2 for grafts 5MPT. Scale bar: 25 μm. (H) Representative images of DARPP32/CTIP2/GABA/hN using tyramide signal amplification–based (TSA-based) immunofluorescence in grafts at 5 months after transplantation (5MPT). (J–L) Quantification of marker-positive cells among grafted cells. (I and O) Representative immunofluorescence images (I) and quantification (O) of SP+ and ENK+ cells in grafts 5MPT. Scale bar: 50 μm. (M and N) RNAscope labeling and quantification of DRD1 and DRD2 in the grafts 3MPT. (P) Quantification of hNCAM+ fibers in target areas (GPe, GPi, SN) and other brain regions. (Q and R) Immunohistochemical staining of hNCAM 5MPT. Boxed regions in (Q) show magnified views in (R). The black dashed box in R is further magnified in the lower panels. Scale bars: 100 μm. (S) Quantification of hNCAM+ fiber density in different brain regions. (T) Representative immunofluorescence images of presynaptic hSYN colocalized with host GABAergic neurons in the striatum, GPe, GPi, or with host TH+ dopaminergic neurons in the SN 5MPT. Hippo., hippocampus; Hypothala., hypothalamus; MRN, median raphe nucleus; SC, superior colliculus; Thala., thalamus. Scale bars: 10 μm. n = 3–7 mice. Data were analyzed with Student’s t test and presented as the mean ± SEM. **P < 0.01, ***P < 0.001.