TITLE: Differentiation of iPSC into dopaminergic neurons
AUTHORS: Elisangela Bressan, Melanie Cobb, on behalf of the Foundational Data Initiative for Parkinson's Disease (FOUNDIN-PD)

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Induced pluripotent stem cell (iPSC)-derived dopaminergic neurons is a promising tool to model Parkinson's disease and of great interest for studying disease mechanisms. Our aim is to describe the protocol for differentiation of iPSC into dopaminergic (DA) neurons currently used in our laboratory. The protocol described here is based on a previously published dual-SMAD inhibition protocol (Kriks et al. Nature 2011). Minor modifications were required for the implementation of the protocol in our automated cell culture system (Dhingra et al. JoVE 2020). The differentiation takes 65 days and produces substantial amounts of MAP2 (neuron) and TH (DA neuron) positive cells.</div></div>

[STEPS]
?. [iPSC preparation for differentiation]
Grow iPSC on matrigel-coated plates with Essential E8 Flex medium until they cover 70-80% of the well area.
?. [iPSC preparation for differentiation]
Check if iPSC appear pluripotent and undifferentiated using a bright-field microscope. The iPSC should show a typical morphology with high nuclear-to-cytoplasm ratio, prominent nucleolus and densely packed colonies (Figure 1).
?. [iPSC preparation for differentiation]
Dissociate iPSC with Accutase into a single cell suspension.
?. [iPSC preparation for differentiation]
Dissociate iPSC into single cells with accutase (aprox. 30 min at 37 °C) and replate at 200,000 per cm2 on matrigel-coated plates and Essential 8 Flex medium (200 µL/cm2) supplemented with 10 µM Y-27632 (until day 0 of differentiation). Matrigel coating: Use the same matrigel concentration applied for iPSC growth advised in the certificate of analyis of the product. Increase the coating time to 12 hours at 37 °C for differentiation. Use plates immediately after coating.
?. [iPSC preparation for differentiation]
Once iPSC cover 100% of the well area, usually 24 to 48 after single cell replating, start the differentiation into dopaminergic neurons.
?. [Dopaminergic neuron differentiation]
A schematic representation of the differentiation protocol is shown in Figure 2.Start day 0 of differentiation by changing the culture medium to differentiation medium (KSR medium; 200 µL/cm2) supplemented with small molecules as described below:Day 0 - 1: 100 nM LDN193189, 10 µM SB431542Day 1 - 3: 100 nM LDN193189, 10 µM SB431542, 1 mM SHH, 2 µM Purmorphamine, 100 ng/mL FGF-8bDay 3 - 5: 100 nM LDN193189, 10 µM SB431542, 1 mM SHH, 2 µM Purmorphamine, 100 ng/mL FGF-8b, 3 µM CHIR99021Day 5 - 7: 100 nM LDN193189, 1 mM SHH, 2 µM Purmorphamine, 100 ng/mL FGF-8b, 3 µM CHIR99021Day 7 - 9: 100 nM LDN193189, 1 mM SHH, 3 µM CHIR99021Day 9 - 11: 100 nM LDN193189, 1 mM SHH, 3 µM CHIR99021KSR (knockout serum replacement) medium composition and storage: Mix 409.5 mL of knockout DMEM/F-12 medium with 75 mL knockout serum replacement (15%), 5 mL GlutaMAX (2 mM), 5 mL MEM Non-Essential Amino Acids (1%), 0.5 mL 2-mercaptoethanol (55 µM) and 5 mL Penicillin-Streptomycin (1%). Store media without small molecules and growth factors at 4 °C for up to one week or at -20 °C for up to four weeks.
?. [Dopaminergic neuron differentiation]
From day 5, combine KSR with N2 medium and perform media changes (200 µL/cm2) as described below:Day 0 - 5: 100 % KSRDay 5 - 7: 75% KSR/25% N2Day 7 - 9: 50% KSR/50% N2Day 9 - 11: 25% KSR/75% N2N2 medium composition and storage: Mix 475 mL Neurobasal medium with 5 mL N2 supplement (1%), 10 mL B27 supplement (2%), 5 mL GlutaMAX (2 mM) and 5 mL Penicillin-Streptomycin (1%). Store media without small molecules and growth factors at 4 °C for up to one week or at -20 °C for up to four weeks.
?. [Dopaminergic neuron differentiation]
From day 11, replace KSR/N2 by NB/B27 medium supplemented with the following small molecules and growth factors:3 µM CHIR99021 (until day 13)0.2 mM Ascorbic acid20 ng/mL BDNF 10 µM DAPT1 mM db-cAMP20 ng/mL GDNF1 ng/mL TGFβ3NB/B27 medium composition and storage: Mix 485 mL Neurobasal (NB) medium with 10 mL B27 supplement (2%) and 5 mL Penicillin-Streptomycin (1%). Store media without small molecules and growth factors at 4 °C for up to one week or at -20 °C for up to four weeks.
?. [Dopaminergic neuron differentiation]
On day 25, dissociate dopaminergic precursors into single cells with accutase (aprox. 40 min at 37 °C) and replate at 400,000 per cm2 on plates pre-coated with poly-l-ornithine (0.1 mg/mL), laminin (10 µg/mL) and fibronectin (2 μg/mL). Cultivate cells in differentiation medium (200 µL/cm2). Differentiation medium: NB/B27 medium supplemented with 0.2 mM Ascorbic acid, 20 ng/mL BDNF, 10 µM DAPT, 1 mM db-cAMP, 20 ng/mL GDNF , 1 ng/mL TGFβ3 and 10 µM Y-27632 (until day 26). NOTE 1: It is highly recommended to avoid the storage of differentiation medium containing small molecules and growth factors. NOTE 2: Dopaminergic precursors can be frozen on day 25 of differentiaion.NOTE 3: An additional replating at lower cell densities (100,000 per cm2) can be performed on day 32 of differentiation for single-cell imaging.
?. [Dopaminergic neuron differentiation]
On day 26, perform media change (200 µL/cm2) to remove dead cells and debris. Add freshly prepared differentiation medium supplemented with small molecules and growth factors, as described in step 8 (without Y-27632).
?. [Dopaminergic neuron differentiation]
From day 29, perform media changes every 3-4 days as described in step 9 .
?. [Dopaminergic neuron differentiation]
On day 65 of differentiation, process differentiated cells for assays as desired.