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Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening

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Abstract

C9orf72 genetic mutation is the most common genetic cause of ALS/FTD accompanied by abnormal protein insufficiency. Induced pluripotent stem cell (iPSC)-derived two-dimensional (2D) and three-dimensional (3D) cultures are providing new approaches. Therefore, this study established neuronal cell types and generated spinal cord organoids (SCOs) derived from C9orf72 knockdown human iPSCs to model ALS disease and screen the unrevealed phenotype. Wild-type (WT) iPSC lines from three healthy donor fibroblasts were established, and pluripotency and differentiation ability were identified by RT-PCR, immunofluorescence and flow cytometry. After infection by the lentivirus with C9orf72-targeting shRNA, stable C9-knockdown iPSC colonies were selected and differentiated into astrocytes, motor neurons and SCOs. Finally, we analyzed the extracted RNA-seq data of human C9 mutant/knockout iPSC-derived motor neurons and astrocytes from the GEO database and the inflammatory regulation-related genes in function and pathways. The expression of inflammatory factors was measured by qRT-PCR. The results showed that both WT-iPSCs and edited C9-iPSCs maintained a similar ability to differentiate into the three germ layers, astrocytes and motor neurons, forming SCOs in a 3D culture system. The constructed C9-SCOs have features of spinal cord development and multiple neuronal cell types, including sensory neurons, motor neurons, and other neurons. Based on the bioinformatics analysis, proinflammatory factors were confirmed to be upregulated in C9-iPSC-derived 2D cells and 3D cultured SCOs. The above differentiated models exhibited low C9orf72 expression and the pathological characteristics of ALS, especially neuroinflammation.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

ALS :

Amyotrophic lateral sclerosis

C9orf72 :

Chromosome 9 open reading frame 72

WT :

Wild type;

HiPSCs :

Human induced pluripotent stem cells

SOX2 :

SRY-box transcription factor 2

OCT4 :

Octamer-binding transcription factor 4

SSEA4 :

Stage-specific embryonic antigen 4

NSCs :

Neural stem cells

GFAP :

Glial fibrillary acidic protein

CHAT :

Choline acetyltransferase

FOXA2 :

Forkhead box A2

IL :

Interleukin

TNFα :

Tumour necrosis factor alpha-like

TGFβ :

Transforming growth factor beta

VEGF :

Vascular endothelial growth factor

IFNγ :

Interferon γ

Cos :

Cerebral organoids

SCOs :

Spinal cord organoids

3D :

Three-dimensional

GDNF :

Glial-derived neurotrophic factor

BDNF :

Brain-derived neurotrophic factor

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Acknowledgements

This work was supported by Hebei Medical University, and funded by Natural Science Foundation of China (81801278), China Scholarship Council (201608130015), Natural Science Foundation of Hebei Province (H2019206637), Key Natural Science Foundation of Hebei Province (H2020206557), Overseas researcher Program in Hebei Provincial Department of human resources and social security (C20190509), Natural Science Foundation of Hebei Province (H2015206409), Natural Science Foundation of Hebei Province (H2023206266).

Funding

Funded by Natural Science Foundation of China (81801278), China Scholarship Council (201608130015), Natural Science Foundation of Hebei Province (H2019206637), Key Natural Science Foundation of Hebei Province (H2020206557), Overseas researcher Program in Hebei Provincial Department of human resources and social security (C20190509), Natural Science Foundation of Hebei Province (H2015206409), Natural Science Foundation of Hebei Province (H2023206266).

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Authors and Affiliations

  1. Hebei Medical University-University of Galway Stem Cell Research Center, Hebei Medical University, Shijiazhuang, 050017, Hebei Province, China

    Ruiyun Guo, Yimeng Chen, Jinyu Zhang, Zijing Zhou, Baofeng Feng, Xiaofeng Du, Xin Liu, Jun Ma & Huixian Cui

  2. Hebei Research Center for Stem Cell Medical Translational Engineering, Shijiazhuang, 050017, Hebei Province, China

    Ruiyun Guo, Yimeng Chen, Jinyu Zhang, Zijing Zhou, Baofeng Feng, Xiaofeng Du, Xin Liu, Jun Ma & Huixian Cui

  3. Hebei Technology Innovation Center for Stem Cell and Regenerative Medicine, Shijiazhuang, 050017, Hebei Province, China

    Ruiyun Guo, Yimeng Chen, Jinyu Zhang, Zijing Zhou, Baofeng Feng, Xiaofeng Du, Xin Liu, Jun Ma & Huixian Cui

  4. Human Anatomy Department, Hebei Medical University, Shijiazhuang, 050017, Hebei Province, China

    Baofeng Feng, Jun Ma & Huixian Cui

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  1. Ruiyun Guo
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Contributions

JM and HXC conceived and designed the study. RYG conducted the research and investigation process, including performed the experiments and YMC performed the GEO data collection and analysis. JYZ collected the references and extracted and cross-checked the data. RYG and JM wrote the first draft of the manuscript. JM, HXC and RYG revised and discussed the final edition. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Jun Ma or Huixian Cui.

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The human biopsy collection was approved by the Committee of Ethics on Experimentation of Hebei Medical University (assurance no. 20190505).

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The authors declare that they have no competing interests.

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a Hebei Medical University-National University of Ireland Galway Stem Cell Research Center, Hebei Medical University, Shijiazhuang, Hebei Province 050017, China.

b Hebei Research Center for Stem Cell Medical Translational Engineering, Shijiazhuang, Hebei Province 050017, China.

c Human Anatomy Department, Hebei Medical University, Shijiazhuang, Hebei Province 050017, China.

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Guo, R., Chen, Y., Zhang, J. et al. Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening. Mol Neurobiol (2023). https://doi.org/10.1007/s12035-023-03836-4

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