Culture and In Vitro Differentiation of Human iPSCs
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For in vitro differentiation, iPSC colonies were detached from the feeder layers en bloc using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV [], 1 mM CaCl2, and 20% KSR; day 0) and cultured in suspension in bacteriological dishes to form EBs in a humidified atmosphere of 3% CO2.
- From day 1 to 4 of EB formation, 3 μM dorsomorphin , 3 μM SB431542 , and 3 μM BIO ((2′Z, 3′E)-6-bromoindirubin-3′-oxime) were added.
- In addition, 1 μM retinoic acid and 1 μM purmorphamine were added on days 4 and 7, respectively, and maintained thereafter until day 16 .
- The medium was changed every 2 days.
- On day 16, the EBs were enzymatically dissociated into single cells using TrypLE Select , and the dissociated cells were cultured in suspension at a density of 1 × 105 cells/ml in proliferation medium consisting of medium'>serum-free medium (media hormone mix [MHM]; 2.
- The medium was changed every 4∼6 days for…
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For in vitro differentiation, iPSC colonies were detached from the feeder layers en bloc using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV [], 1 mM CaCl2, and 20% KSR; day 0) and cultured in suspension in bacteriological dishes to form EBs in a humidified atmosphere of 3% CO2.
- From day 1 to 4 of EB formation, 3 μM dorsomorphin , 3 μM SB431542 , and 3 μM BIO ((2′Z, 3′E)-6-bromoindirubin-3′-oxime) were added.
- In addition, 1 μM retinoic acid and 1 μM purmorphamine were added on days 4 and 7, respectively, and maintained thereafter until day 16 .
- The medium was changed every 2 days.
- On day 16, the EBs were enzymatically dissociated into single cells using TrypLE Select , and the dissociated cells were cultured in suspension at a density of 1 × 105 cells/ml in proliferation medium consisting of medium'>serum-free medium (media hormone mix [MHM]; 2.
- The medium was changed every 4∼6 days for approximately 15∼20 days to form the first neurospheres.
- To passage neurospheres, the first neurospheres were dissociated in the same manner as described above and cultured at a density of 1 × 105 cells/ml in proliferation medium without purmorphamine for approximately 15∼20 days.
- To assay neurosphere differentiation, undissociated 5–7 neurospheres were plated onto coverslips 10 mm in diameter coated with poly-L-ornithine and growth-factor-reduced Matrigel (50× dilution, thin coated), and cultured in differentiation medium that consisted of MHM supplemented with 2% B27 supplement, NEAA, 60 ng/ml T3, 10 ng/ml hLIF , and 25 ng/ml CNTF for 2–6 weeks in a humidified atmosphere of 5% CO2.
- Half of the medium was changed every 2 or 3 days.
- For the quantitative analysis of the differentiation efficiency into OL lineage cells, the numbers of neurosphere colonies containing more than 40 marker-positive cells (≥40 cells, oligodendrocyte [++]), those containing less than 40 marker-positive cells (1–39 cells, oligodendrocyte [+]), and those without marker-positive cells (oligodendrocyte [−]) were counted and are presented as the percentage of total neurosphere colonies.
- To examine the expression of ER-stress markers, O4+ differentiated cells were purified 4 weeks after the attachment of the neurospheres using MACS technology with an anti-O4 antibody.
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Astrocyte culturing and differentiation
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Astrocytes were differentiated from iPSCs as described.
- Briefly, NPCs were cultured in MEM/F12 supplemented with 1% N2 , 0.1% B27 , 1% nonessential amino acids , 1% penicillin/streptomycin , 1% GlutaMAX solution , 20 ng/mL LIF and 20 ng/mL EGF for 4–6 weeks.
- Spheres were then transferred to MEM/F12 supplemented with 1% N2, 0.1, % B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX, 20 ng/mL FGF-2 and 20 ng/mL EGF , and mechanically passaged every 2 weeks.
- To generate monolayer cultures, spheres were dissociated with papain and plated in wells coated with Matrigel ( 1:80).
- Monolayers were split with Accutase approximately every 3–4 d, or when confluent.
- ifferentiated astrocytes were created by culturing the cells in medium'>Neurobasal medium supplemented with 0.2% B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX solution, and 10 ng/mL CNTF for…
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Astrocytes were differentiated from iPSCs as described.
- Briefly, NPCs were cultured in MEM/F12 supplemented with 1% N2 , 0.1% B27 , 1% nonessential amino acids , 1% penicillin/streptomycin , 1% GlutaMAX solution , 20 ng/mL LIF and 20 ng/mL EGF for 4–6 weeks.
- Spheres were then transferred to MEM/F12 supplemented with 1% N2, 0.1, % B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX, 20 ng/mL FGF-2 and 20 ng/mL EGF , and mechanically passaged every 2 weeks.
- To generate monolayer cultures, spheres were dissociated with papain and plated in wells coated with Matrigel ( 1:80).
- Monolayers were split with Accutase approximately every 3–4 d, or when confluent.
- ifferentiated astrocytes were created by culturing the cells in medium'>Neurobasal medium supplemented with 0.2% B27, 1% nonessential amino acids, 1% penicillin/streptomycin, 1% GlutaMAX solution, and 10 ng/mL CNTF for 2 weeks.
- Before survival analysis, astrocytes were dissociated with Accutase and cultured at a density of 2×104 cells per well of a 96-well plate.
- 2–3 days later, differentiated astrocyte cultures were transfected with pGW1-mApple using Lipofectamine 2000, as described above.
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