Neural Differentiation and spinal cord organoid generation from induced pluripotent stem cells (iPSCs) for ALS modelling and inflammatory screening.
Ruiyun GuoYimeng ChenJinyu ZhangZijing ZhouBaofeng FengXiaofeng DuXin LiuJun MaHuixian CuiPublished in: Molecular neurobiology (2023)
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.
Keyphrases
- induced pluripotent stem cells
- spinal cord
- single cell
- rna seq
- spinal cord injury
- neuropathic pain
- wild type
- stem cells
- flow cytometry
- poor prognosis
- oxidative stress
- amyotrophic lateral sclerosis
- induced apoptosis
- crispr cas
- emergency department
- genome wide
- endothelial cells
- cell death
- binding protein
- traumatic brain injury
- cerebral ischemia
- bioinformatics analysis
- high throughput
- wastewater treatment
- cell cycle arrest
- diabetic rats
- endoplasmic reticulum stress
- dna methylation
- cognitive impairment
- drug induced
- protein protein
- electronic health record
- deep learning
- drug delivery
- mesenchymal stem cells
- amino acid
- blood brain barrier
- long non coding rna
- subarachnoid hemorrhage