Journal Article (Video)

DZNE-2021-00261

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring.

Dhingra, A. (First author)DZNE* ; Täger, J. (First author)DZNE* ; Bressan, E. (First author)DZNE* ; Rodriguez-Nieto, S.DZNE* ; Bedi, M.-S.DZNE* ; Bröer, S.DZNE* ; Sadikoglou, E.DZNE* ; Alves Fernandes, N. R.DZNE* ; Castillo Lizardo, M. G.DZNE* ; Rizzu, P.DZNE* ; Heutink, P. (Last author)DZNE*

2020
JoVE Cambridge, MA

This record in other databases:    

Please use a persistent id in citations: doi:10.3791/61525

Abstract: 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.

Keyword(s): Automation (MeSH) ; Carbon Dioxide (MeSH) ; Cell Count (MeSH) ; Cell Culture Techniques: methods (MeSH) ; Cell Differentiation (MeSH) ; Cell Survival (MeSH) ; Cells, Cultured (MeSH) ; Cerebral Cortex: cytology (MeSH) ; Dopaminergic Neurons: cytology (MeSH) ; Humans (MeSH) ; Image Processing, Computer-Assisted (MeSH) ; Induced Pluripotent Stem Cells: cytology (MeSH) ; Mesencephalon: cytology (MeSH) ; Neural Stem Cells: cytology (MeSH) ; Neuronal Outgrowth (MeSH) ; User-Computer Interface (MeSH) ; Carbon Dioxide

Note: Section Biology

---

Contributing Institute(s):

1. Applied Genomics of Neurodegenerative Diseases (AG Rizzu)
2. Genome Biology of Neurodegenerative Diseases (AG Heutink 1)

Research Program(s):

1. 345 - Population Studies and Genetics (POF3-345) (POF3-345)

Appears in the scientific report 2020

---

Database coverage:
; ; ; BIOSIS Previews ; Biological Abstracts ; Clarivate Analytics Master Journal List ; Essential Science Indicators ; IF < 5 ; JCR ; PubMed Central ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

---