Transcription Factor-Mediated Generation of Dopaminergic Neurons from Human iPSCs-A Comparison of Methods

• Published in: Cells (Basel, Switzerland) Vol. 13; no. 12; p. 1016
• Main Authors: McDonald, Kirstin O, Lyons, Nikita M A, Gray, Luca K C, Xu, Janet B, Schoderboeck, Lucia, Hughes, Stephanie M, Basak, Indranil
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.06.2024; MDPI
• Subjects: Amino acids; Antibodies; ASCL1; Brain-derived neurotrophic factor; Cell culture; Cell Differentiation; Cloning; CRISPR; Culture media; Disease; DNA binding proteins; Dopamine; Dopamine receptors; dopaminergic neurons; Dopaminergic Neurons - cytology; Dopaminergic Neurons - metabolism; Ethylenediaminetetraacetic acid; human-induced pluripotent stem cells; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Inhibitory postsynaptic potentials; International economic relations; Lentivirus - genetics; Lentivirus - metabolism; LMX1A; Medical research; Methods; Neurodegenerative diseases; Neurogenesis; Neurons; NURR1; Parkinson's disease; Penicillin; Pharmaceutical industry; Pluripotency; Population studies; Protocol; Skin diseases; Stem cells; Tetracycline; Tetracyclines; Transcription factors; Transcription Factors - metabolism; New Zealand; Australia; United States; Melbourne Victoria Australia; United States > US; directed differentiation; LMX1A; human-induced pluripotent stem cells; transcription factors; CRISPR-Cas9; dopaminergic neurons; NURR1; lentivirus transduction; ASCL1
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells13121016

Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.