PD patient derived human induced pluripotent stem cells (iPSC) expressing SNCAA53T and the matched isogenic corrected iPSC line were generously provided by Dr. R. Jaenisch (Whitehead Institute MIT) and were previously characterized extensively [63 (link)]. iPSCs were maintained in mTeSR1 media (Stemcell, 85850) media or mTeSR plus (Stemcell, 100–0276) on Matrigel (Corning, 354234)-coated plates and differentiated into DA neurons using an established protocol [113 (link)]. Between day 25–30 after differentiation, neurons were seeded on coated plates with poly-D-lysine (Merck, P1149) and LAM/laminin (Merck, 11243217001) at a cell number of 3 × 105 per well (24-well plate) on 12-mm cover glasses for immunofluorescence or at 4 × 105 for western blot. Neurons were maintained in Neurobasal media (Thermo Fisher Scientific, 21103049) containing NeuroCult SM1 Neuronal Supplement (Stemcell, 05711) and 1% PenStrep until ~ day 90, when neurons were treated with 10 µg/mL rHsCTSD, replenished every 4 days with media replacement for 21–25 days. Detailed describtion for iPSn culturing during the microelectrode array (MEA) analysis is mentioned in the section ”Microelectrode array (MEA) analysis”.
Protocol detail
Differentiation and Characterization of SNCAA53T iPSC-Derived Dopaminergic Neurons
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Cells line
Glasses
Human induced pluripotent stem cells
Immunofluorescence
Ipscs
Laminin
Lysine
Matrigel
Microelectrode
Neurons
Patient
Poly
Stem
Stemcell
Supplement
Western blot
Corresponding Organization : Universitätsklinikum Erlangen
Other organizations : Kiel University, Universidade Nova de Lisboa, University of Würzburg, Northwestern University
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Variable analysis
independent variables
- Expression of SNCAA53T in iPSCs
- Survival and function of DA neurons
- Matched isogenic corrected iPSC line
- Culture conditions (mTeSR1 media, Matrigel-coated plates, Neurobasal media with NeuroCult SM1 Neuronal Supplement and 1% PenStrep)
- Differentiation protocol (established protocol [113])
- Neuron seeding density and culture duration (between day 25-30 and ~day 90)
- Positive control: Matched isogenic corrected iPSC line
- Negative control: Not explicitly mentioned
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