TITLE: 3.5 Doxycycline-Induced Differentiation
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is part 3.3 of the "</span><a href="https://www.protocols.io/private/C65DB81838BC11EBA61A0A58A9FEAC2A/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Induced Neurons for the Study of Neurodegenerative  and Neurodevelopmental Disorders</span></a><span>" collection of protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> 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.</span></div></div>

[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 , add the diluted PLL solution to the cell culture plates and distribute equally so that the entire well is covered.
?. [3.5 Doxycycline-Induced Differentiation]
Seed the iPSCs at a density of 30,000–50,000 cells per cm2 in mTeSR™1 medium with ROCKi supplemented with .
[doxycycline]
?. [3.5 Doxycycline-Induced Differentiation]
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).
?. [3.5 Doxycycline-Induced Differentiation]
Incubate at .
37 °C
?. [3.5 Doxycycline-Induced Differentiation]
Dilute the laminin in  to a final concentration of  and add to the PLL-coated cell culture plates. Incubate at  for approximately . Prior to use, simply aspirate the coating solution and seed the cells without washing the plates.
[PBS with Ca2+ and Mg2+]
37 °C
?. [3.5 Doxycycline-Induced Differentiation]
On the next day, wash the cells with  and change the medium to mTeSR™1 w/o ROCKi supplemented with . Change the medium daily until day 4 (Fig. 4).
[PBS w/o Ca2+ and Mg2+]
[doxycycline]
?. [3.5 Doxycycline-Induced Differentiation]
Wash three times with ddH2O on the next day.
?. [3.5 Doxycycline-Induced Differentiation]
Change half of the medium on day 5 of differentiation to BrainPhys™ medium with supplements. Repeat changing half of the medium 2 days later.
?. [3.5 Doxycycline-Induced Differentiation]
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.