Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring
| Autor: | Eldem Sadikoglou, Joachim Täger, Manmeet-Sakshi Bedi, Noemia Fernandes, Stefanie Bröer, Peter Heutink, Salvador Rodriguez-Nieto, Ashutosh Dhingra, Patrizia Rizzu, Elisangela Bressan, Melissa Castillo-Lizardo |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Cell type
Neurite Cell Survival General Chemical Engineering Induced Pluripotent Stem Cells Neuronal Outgrowth Cell Cell Culture Techniques Cell Count Biology General Biochemistry Genetics and Molecular Biology cytology [Cerebral Cortex] Automation User-Computer Interface Neural Stem Cells Mesencephalon ddc:570 Precursor cell methods [Cell Culture Techniques] Image Processing Computer-Assisted medicine Humans CRISPR cytology [Neural Stem Cells] Induced pluripotent stem cell Cells Cultured cytology [Dopaminergic Neurons] cytology [Mesencephalon] Cerebral Cortex General Immunology and Microbiology Dopaminergic Neurons General Neuroscience Dopaminergic Cell Differentiation cytology [Induced Pluripotent Stem Cells] Carbon Dioxide medicine.anatomical_structure Cell culture Neuroscience |
| Zdroj: | JoVE journal Biology(162), 61525 (2020). doi:10.3791/61525 |
| ISSN: | 1940-087X |
| Popis: | Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6‒8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|