Constructing Human 3D Striatum-Substantia Nigra Organoids to Model Medium Spiny Neuron Projection Deficits in Huntington’s Disease

Background Introduction

Huntington’s Disease (HD) is a neurodegenerative disorder leading to significant deterioration of the motor system, primarily characterized by defects in medium spiny neurons (MSNs) that project from the striatum to the substantia nigra (SN). One of the challenges in studying the pathophysiology of HD is the lack of effective human models, especially in vitro models that can simulate these neuronal circuit defects. Although the cause of HD is known to be due to the abnormal expansion of CAG repeats in the huntingtin gene, effective therapies remain lacking, possibly in part due to the absence of analog models for pharmacological research.

In HD patients, dysfunction in the striatum-substantia nigra (STR-SN) circuit leads to a series of motor-related symptoms. Recently, human brain organoids derived from human pluripotent stem cells (hPSCs) have been considered promising experimental models for studying human brain development and the pathogenesis of brain diseases. However, despite some success with techniques using region-specific brain organoids, organoid assembloids that simulate STR-SN microcircuits have not yet been fully established.

Source and Author Information

This paper was co-authored by Shanshan Wu, Yuan Hong, Chu Chu, Yixia Gan, Xinrui Li, Mengdan Tao, Da Wang, Hao Hu, Zhilong Zheng, Qian Zhu, Xiao Han, Wanying Zhu, Min Xu, Yi Dong, Yan Liu, and Xing Guo. It was published on May 21, 2024, in the Proceedings of the National Academy of Sciences (PNAS, doi: 10.1073/pnas.2316176121). The article declares no conflicts of interest and is openly published on PNAS Direct Submission. The related article’s editor is Ted M. Dawson from Johns Hopkins University School of Medicine.

Research Methods

Research Process

1. Generation of Human Striatal Organoids: Initially, functional MSNs were differentiated from hPSCs based on previous reports. Researchers modified and optimized a differentiation protocol, treating neural epithelial cells with Sonic Hedgehog (SHH) early on to promote their differentiation into lateral ganglionic eminence (LGE) progenitor cells containing striatal projection neurons.

2. Construction of 3D STR-SN Organoid Assemblies: By fusing striatal organoids with SN organoids, researchers generated three-dimensional (3D) STR-SN organoid assembloids. Using AAV-HSyn-GFP-mediated viral tracing, extensive projections from MSNs in the striatal organoids to SN organoids were observed, forming synaptic connections with GABAergic neurons in the SN organoids.

3. Generation and Fusion of HD Patient-Derived iPSCs Organoids: Induced pluripotent stem cells (iPSCs) from HD patients were used to generate organoids, forming HD STR-SN organoid assembloids.

Detailed Experimental Steps

• Striatal Organoid Differentiation: Various SHH concentrations were tested to find optimal differentiation conditions, resulting in LGE precursor markers NKX2.1 and GABAergic neuron marker GAD67 expression at D30 and D60.

• Single-cell RNA Sequencing (scRNA-Seq): scRNA-Seq was performed on D30 and D60 striatal organoids to verify their regional specificity.

• Generation and Validation of Fused Organoids: GFP-labeled striatal organoids were fused with SN organoids to create STR-SN assembloids, and their synaptic connections were validated using immunofluorescence and electron microscopy techniques.

• Calcium Imaging and Electrophysiological Detection: Functional synaptic connections in the organoids were detected using calcium imaging and optogenetics.

Research Results

Striatal Organoid Generation

• At D30 and D60, these organoids exhibited gene expression patterns unique to striatal region differentiation and functional fibroblast-like currents (Figures 1C-E).

• RNA sequencing results showed that MSNs in the organoids presented transcriptomic characteristics similar to those of human fetal brain striatum. Electrophysiological recordings indicated typical fast, inactivating inflow and outflow currents, and spontaneous rhythmic discharges in these cells.

Construction and Functional Detection of 3D STR-SN Assemblies

• Optogenetics and whole-cell patch recordings confirmed that MSNs in the assemblies formed functional synaptic connections with GABAergic and dopaminergic neurons. The fused organoids exhibited significantly enhanced calcium activity, indicating higher functional maturity compared to standalone striatal organoids.

HD Patient-Derived Organoids and Assemblies

• Three HD patient-derived iPSC striatal organoids were generated, demonstrating significant mHTT aggregation features (Figures 2B-E), and a significant reduction in the number of MSNs. At D30, cell proliferation in HD organoids was markedly decreased, and apoptosis rates significantly increased.

• Calcium signaling in HD organoid assemblies was notably reduced, and optogenetic recordings and electrophysiological tests indicated significantly decreased neuronal activity, showing evident neuronal connectivity and functional deficits in HD patients (Figures 3A-F, 4A-C).

Drug Treatment Research

• The therapeutic effect of Brain-Derived Neurotrophic Factor (BDNF) on HD organoids was explored, resulting in significant improvements in the number and function of MSNs in HD organoids, with neuronal projections and calcium signaling recovering close to control levels (Figures 4G-K).

Research Conclusion and Significance

By constructing human STR-SN assemblies, this study provides a human-derived in vitro platform for studying STR-SN circuit deficits in HD. This platform not only offers reference value and guidance for other neurodegenerative diseases but also demonstrates its potential application in drug testing. Future research can continue to use this platform to explore the development and neural connectivity of various neuropsychiatric disorders, driving related therapeutic strategy studies.

Research Highlights

• Innovation: Established the first human-derived STR-SN assemblies, successfully simulating in vivo neuronal circuit deficits.

• Diversity: Included iPSCs from different sources, offering broad application potential for diverse pathologies.

• Drug Testing: Demonstrated the reliability and efficacy of this assembly in drug testing, especially in the application of BDNF treatment for HD.

Additional Valuable Information

This research was supported by multiple research funding projects in China, including the National Key Research and Development Program and the National Natural Science Foundation. All raw and processed single-cell RNA sequencing data have been stored in the Gene Expression Omnibus (GEO) database, ensuring transparency and ease of reference for other researchers. Through this study, the research team provides a powerful tool and platform for HD and other neurodegenerative diseases research, aiding in the further understanding of these diseases’ pathogenesis and promoting the development and implementation of new therapies.