9429 human fibroblasts (Coriel Cell Repository, Camden, New Jersey) were reprogrammed to induced pluripotent stem cells and induced to neural progenitor cells (NPCs) in house according to the methods outlined by Brennand and colleagues (20 (link)). 5535 NPCs were obtained from the Brennand laboratory (21 ). hiPSC-derived NPCs were plated on Matrigel (Fisher, Waltham, Massachusetts) or Poly-Ornithine (Sigma, St. Louis, Missouri)/Laminin (Invitrogen, Waltham, Massachusetts) and passaged 1:6 every 4 days. NPCs were cultured in NPC medium (DMEM/F12 + Glutamax (Invitrogen), 1× N2 (Invitrogen), 1× B27-Vitamin A (Invitrogen), 1ug/ml Laminin (Invitrogen), 20 ng/ml FGF-2 (Peprotech, Rocky Hill, New Jersey) and dissociated in Accutase (Fisher) for 5 min at 37°C. hiPSC-derived neurons were cultured on Matrigel in Neuron Medium (DMEM/F12 + Glutamax, 1× N2, 1× B27 with Vitamin A (Invitrogen), 20 ng/ml BDNF (Shenandoah Biotechnology, Warwick, Pennsylvania), 20 ng/ml GDNF (Shenandoah Biotechnology), 1 mM dibutryl-cyclic AMP (Sigma), and 200 nM ascorbic acid (Stem Cell Technologies, Vancouver, Canada) for all experiments.
Protocol detail
Differentiation of hiPSC to NPC and Neurons
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Matrigel
Poly-Ornithine
Laminin
DMEM/F12 + Glutamax
B27-Vitamin A
FGF-2
Accutase
B27 with Vitamin A
BDNF
dibutryl-cyclic AMP
ascorbic acid
Accutase
Ascorbic acid
Cell
Cyclic amp
Fgf 2
Fibroblasts
Gdnf
Hipsc
Human
Induced pluripotent stem cells
Laminin
Matrigel
Neural progenitor cells
Neuron
Ornithine
Poly
Stem cell
Vitamin a
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independent variables
- Reprogramming of 9429 human fibroblasts to induced pluripotent stem cells
- Differentiation of induced pluripotent stem cells to neural progenitor cells (NPCs)
- Culture conditions for NPCs: Matrigel or Poly-Ornithine/Laminin
- Culture conditions for hiPSC-derived neurons: Matrigel, Neuron Medium
- Number of NPCs obtained (5535) from the Brennand laboratory
- Passage rate and duration for NPCs (1:6 every 4 days)
- Cell line: 9429 human fibroblasts (Coriel Cell Repository, Camden, New Jersey)
- NPC culture medium: DMEM/F12 + Glutamax, 1× N2, 1× B27-Vitamin A, 1ug/ml Laminin, 20 ng/ml FGF-2
- Neuron culture medium: DMEM/F12 + Glutamax, 1× N2, 1× B27 with Vitamin A, 20 ng/ml BDNF, 20 ng/ml GDNF, 1 mM dibutryl-cyclic AMP, 200 nM ascorbic acid
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