3.5 Doxycycline-Induced Differentiation
Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp
Human induced pluripotent stem cells
Nucleofection
PiggyBac transposon
Lentiviral transduction
CRISPR/Cas9
Transcription factor-mediated neuronal differentiation
Astrocyte coculture
Abstract
This is part 3.3 of the "Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders" collection of protocols.
Collection Abstract: Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.
Before start
NB Introduction, Notes, and References are in the Collection Guidelines tab
Attachments
Steps
3.5 Doxycycline-Induced Differentiation
Neurons can be grown on Matrigel-coated cell culture dishes, however, especially for long-term neuronal differentiation, it is recommended to grow the neurons on cell culture plates coated with poly-L-lysine (PLL) and laminin. Dilute the PLL in ddH2O to a final concentration of 40μg/mL, add the diluted PLL solution to the cell culture plates and distribute equally so that the entire well is covered.
Incubate at 37°C .
Wash three times with ddH2O on the next day.
Dilute the laminin in 1x to a final concentration of 20μg/mL and add to the PLL-coated cell culture plates. Incubate at 37°C for approximately 4h 0m 0s. Prior to use, simply aspirate the coating solution and seed the cells without washing the plates.
Seed the iPSCs at a density of 30,000–50,000 cells per cm2 in mTeSR™1 medium with ROCKi supplemented with 0.5μg/mL.
On the next day, wash the cells with 1x and change the medium to mTeSR™1 w/o ROCKi supplemented with 0.5μg/mL. Change the medium daily until day 4 (Fig. 4).
![Fig. 4 Representative images of neuronal differentiation of human iPSCs expressing the neurogenic TFs Neurogenin-1 and Neurogenin-2 (iNGN cells) under the control of a doxycycline-inducible promoter [4]. Scale bar represents 100 μm](https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.bqhgmt3w/ea8g7vcw2.jpg "Fig. 4 Representative images of neuronal differentiation of human iPSCs expressing the neurogenic TFs Neurogenin-1 and Neurogenin-2 (iNGN cells) under the control of a doxycycline-inducible promoter [4]. Scale bar represents 100 μm")
When culturing the neurons for longer time periods, it is recommended to change the stem cell medium (mTeSR™1) to maturation medium (BrainPhys™ with supplements).
Change half of the medium on day 5 of differentiation to BrainPhys™ medium with supplements. Repeat changing half of the medium 2 days later.
After those two adaptation medium changes, it is sufficient to change half of the medium once per week. Volume loss due to evaporation should be compensated with ddH2O.
