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 2

Generation and characterization of striatal MSN subtypes from hESCs by 3D-default XFSC.

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Generation and characterization of striatal MSN subtypes from hESCs by 3...
(A and B) Immunostaining of CTIP2/MEIS2/GABA/HO and CTIP2/MAP2/DARPP32/HO in neurons generated by 3D-default XFSC with or without SAG 2 weeks after neuron maturation. Scale bar: 50 μm. (C–E) Quantification of cells positive for GABA, CTIP2, MEIS2, MAP2, DARPP32, or marker combinations (CTIP2/MEIS2, CTIP2/MEIS2/GABA, DARPP32/CTIP2) among total cells with or without SAG treatment. (F and G) Representative images (F) and quantification (G) of GABA+/DARPP32+/CTIP2+ cells among total cells after neuron maturation for 2 and 10 weeks. Scale bar: 50 μm. (H) Growth curve showing long-term expansion capacity of 3D-default XFSC–derived LGE-like progenitors. (I) Schematic diagram illustrating timings of neural differentiation and maturation with 3D-default XFSC. (J) Quantification of cells positive for GABA, CTIP2, MEIS2, DARPP32, MAP2, or marker combinations (CTIP2/MEIS2/GABA, DARPP32/CTIP2) among total neurons across different patterning time points (T1–T5) with 3D-default XFSC. (K) Immunofluorescence image showing a GABA/MEIS2 double-positive neuron with typical spiny morphology. Scale bar: 50 μm. (L) Postsynaptic density protein 95 (PSD95), MAP2, and SYN immunostaining showing prominent co-localization of PSD95 and SYN. Scale bar: 10 μm. (M and N) Typical trace of sIPSCs (M) and post-recording immunostaining images (N) for biocytin/DARPP32/CTIP2. Scale bar: 50 μm. (O and P) RNAscope staining (O) and quantification (P) of GAD2 and VGLUT1. Scale bar: 50 μm. (R) Relative mRNA expression levels of canonical genes for D1- and D2-MSNs at different stages during 3D-default XFSC. (Q, S, T, and V) Representative immunofluorescence images (S and T) and quantification (Q and V) of FOXP2 and SP for D1-MSN, and SIX3 and ENK for D2-MSN. Scale bar: 50 μm. (U and W) RNAscope staining (U) and quantification (W) of DRD1 and DRD2. Scale bar: 50 μm. Data were analyzed by Student’s t test (for 2-group comparisons) or 1-way ANOVA (for multiple groups) and presented as the mean ± SEM; n = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.