Establishment of an iPSCs-Derived Cerebral Cortex Organoid Model for Neurotoxicity Assessment
Jong Hyun Kim Prof.
Department of Biological Science, Hyupsung University, Hwasung-si 18330, Republic of Korea.
Purpose
Brain organoid studies using human pluripotent stem cells offer promising drug screening and neurotoxicity testing tools. However, establishing an optimal model for motor cortex-like organoids containing upper motor neurons, such as glutamatergic neurons and GABAergic neurons derived from human iPSCs, remains challenging. This study addresses these challenges.
Methods
Differentiating human-derived iPSCs into glutamatergic neurons and GABAergic neurons formed mixed 3D spheres. We characterized these cells through immunocytochemistry (ICC) and quantitative PCR (qPCR). Neurotoxicity was evaluated using WST-8 cell viability assays, comparing treated and untreated groups.
Methods
Differentiating human-derived iPSCs into glutamatergic neurons and GABAergic neurons formed mixed 3D spheres. We characterized these cells through immunocytochemistry (ICC) and quantitative PCR (qPCR). Neurotoxicity was evaluated using WST-8 cell viability assays, comparing treated and untreated groups.
Results
The 3D motor cortex-like organoids derived from iPSCs displayed gene expression profiles (vGlut1, vGlut2, ARPP21, and GABA) that closely paralleled those of human primary motor cortex tissue. Following organoid formation, cellular viability (Caspase 3) and safety (GAP43, ATG5) were evaluated within both 2D and 3D culture systems. The findings demonstrated that the 3D model not only provided superior cellular stability but also closely recapitulated the structural and functional hallmarks of primary motor cortex tissue, underscoring its potential as a high-fidelity model for neurobiological and neurotoxicological studies. Furthermore, toxicity assessments using various drugs demonstrated clear distinctions between non-toxic and toxic compounds, as well as dose-dependent effects, underscoring the model's sensitivity and relevance for neurotoxicity evaluations
Conclusion
This study suggests that combining 3D motor cortex-like organoids iPSC-derived models with neurotoxicity assays could provide a robust tool for drug toxicity assessment in neurodevelopmental research.
Keywords: Motor cortex-like organoids, Glutamatergic neurons, GABAergic neurons
Figure 1. Construction and characterization of 3D brain organoids using iPSC-derived glutamatergic and GABAergic cells.
(A) Scheme of 3D brain organoid generation from glutamatergic and GABAergic neurons. The iPSCs were differentiated the neuroepithelial cells. Neuroepithelial cells were induced into Glutamatergic progenitors and GABAergic progenitors by treating them with cyclopamine and puromorphine, respectively. (B and C) The iPSCs were differentiated to Glutamatergic neurons or GABAergic neurons for 30 days. At 30days, the 3D organoids were stained with specific antibodies such as anti- vGluta1, -Map2b, -GABA, -Tuj1 and DAPI. The organoids were observed under the confocal microscope.
Figure 2. Characterization of cell lines-indued organoid, iPSCs differentiation-induced organoid and brain tissues.
(A) The representative of images was indicated the SK-N-SH cells and iPSC induced 3D models, respectively. (B) qPCR analysis of gene expression levels for vGlut1, vGlut2, ARPP21, and GABA in various models in cell line induced organoid, human neuroblastoma cell line (3D model), 2D iPSC-derived model and human brain tissue. (C) mRNA expression analysis of neuronal markers such as GAP43, ATG5 and caspase 3 in the 2D and 3D iPSC-derived models compared to human brain tissue.
Establishment of an iPSCs-Derived Cerebral Cortex Organoid Model for Neurotoxicity Assessment
Jong Hyun Kim Prof.
Department of Biological Science, Hyupsung University, Hwasung-si 18330, Republic of Korea.
Purpose
Brain organoid studies using human pluripotent stem cells offer promising drug screening and neurotoxicity testing tools. However, establishing an optimal model for motor cortex-like organoids containing upper motor neurons, such as glutamatergic neurons and GABAergic neurons derived from human iPSCs, remains challenging. This study addresses these challenges.
Methods
Differentiating human-derived iPSCs into glutamatergic neurons and GABAergic neurons formed mixed 3D spheres. We characterized these cells through immunocytochemistry (ICC) and quantitative PCR (qPCR). Neurotoxicity was evaluated using WST-8 cell viability assays, comparing treated and untreated groups.
Methods
Differentiating human-derived iPSCs into glutamatergic neurons and GABAergic neurons formed mixed 3D spheres. We characterized these cells through immunocytochemistry (ICC) and quantitative PCR (qPCR). Neurotoxicity was evaluated using WST-8 cell viability assays, comparing treated and untreated groups.
Results
The 3D motor cortex-like organoids derived from iPSCs displayed gene expression profiles (vGlut1, vGlut2, ARPP21, and GABA) that closely paralleled those of human primary motor cortex tissue. Following organoid formation, cellular viability (Caspase 3) and safety (GAP43, ATG5) were evaluated within both 2D and 3D culture systems. The findings demonstrated that the 3D model not only provided superior cellular stability but also closely recapitulated the structural and functional hallmarks of primary motor cortex tissue, underscoring its potential as a high-fidelity model for neurobiological and neurotoxicological studies. Furthermore, toxicity assessments using various drugs demonstrated clear distinctions between non-toxic and toxic compounds, as well as dose-dependent effects, underscoring the model's sensitivity and relevance for neurotoxicity evaluations
Conclusion
This study suggests that combining 3D motor cortex-like organoids iPSC-derived models with neurotoxicity assays could provide a robust tool for drug toxicity assessment in neurodevelopmental research.
Keywords: Motor cortex-like organoids, Glutamatergic neurons, GABAergic neurons
Figure 1. Construction and characterization of 3D brain organoids using iPSC-derived glutamatergic and GABAergic cells.
(A) Scheme of 3D brain organoid generation from glutamatergic and GABAergic neurons. The iPSCs were differentiated the neuroepithelial cells. Neuroepithelial cells were induced into Glutamatergic progenitors and GABAergic progenitors by treating them with cyclopamine and puromorphine, respectively. (B and C) The iPSCs were differentiated to Glutamatergic neurons or GABAergic neurons for 30 days. At 30days, the 3D organoids were stained with specific antibodies such as anti- vGluta1, -Map2b, -GABA, -Tuj1 and DAPI. The organoids were observed under the confocal microscope.
Figure 2. Characterization of cell lines-indued organoid, iPSCs differentiation-induced organoid and brain tissues.
(A) The representative of images was indicated the SK-N-SH cells and iPSC induced 3D models, respectively. (B) qPCR analysis of gene expression levels for vGlut1, vGlut2, ARPP21, and GABA in various models in cell line induced organoid, human neuroblastoma cell line (3D model), 2D iPSC-derived model and human brain tissue. (C) mRNA expression analysis of neuronal markers such as GAP43, ATG5 and caspase 3 in the 2D and 3D iPSC-derived models compared to human brain tissue.