Feb 23, 2026
hiPSC-iAstro SOX9 NFIB Induction
- Nadine Schrode1
- 1Icahn School of Medicine at Mount Sinai
- Brennand Laboratory
Protocol Citation: Nadine Schrode 2026. hiPSC-iAstro SOX9 NFIB Induction. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bodml3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2026
Last Modified: February 23, 2026
Protocol Integer ID: 243770
Keywords: hiPSCs, NPCs, neuron, MPRA, CRISPR, FACS, morphology, iastro sox9 nfib induction direct induction of astrocyte, iastro sox9 nfib induction, astrocyte, hipsc
Funders Acknowledgements:
Kristen Brennand
Grant ID: R56MH101454
Abstract
Direct induction of astrocytes from hiPSCs using transcription factor induction
Guidelines
1. Doxycycline: 25 mg/mL dissolved in water (10000x stock). Sterilize with a 0.22 μm filter and store at -20°C. Protect from light.
2. CNTF: Reconstitute at 10 μg/ml in sterile 10 mM sodium phosphate containing 0.1% BSA. Aliquot and store at -20°C.
3. bFGF: Reconstitute at 1 mg/ml in sterile 5mM Tris, pH 7.6 containing 0.1% BSA. Aliquot and store at -20°C.
4. BMP4: Reconstitute at 10 μg/ml in sterile 4 mM HCl containing 0.1% BSA. Aliquot and store at -20°C.
5. dbcAMP: Reconstitute at 100 mg/ml in sterile water. Aliquot and store at -20°C. Protect from light.
6. N-acetyl-cysteine: Reconstitute at 50 mg/ml in sterile water. Aliquot and store at -20°C.
7. Heparin-binding EGF-like growth factor: Reconstitute at 50 μg/ml in sterile PBS containing 0.1% BSA. Aliquot and store at -20°C.
Materials
- DMEM/F-12 (ThermoFisher Scientific, cat. no. 31330038)
- Neurobasal (ThermoFisher Scientific, cat. no. 21103049)
- FBS (ThermoFisher Scientific, cat. no. 10082147)
- N2 supplement (ThermoFisher Scientific, cat. no. 17502001)
- B27 supplement (ThermoFisher Scientific, cat. no. 17504001)
- NEAA (ThermoFisher Scientific, cat. no. 11140035)
- Glutamax (ThermoFisher Scientific, cat. no. 35050061)
- Sodium Pyruvate (ThermoFisher Scientific, cat. no. 11360070)
- bFGF (Peprotech, cat. no. 100-18B)
- CNTF (Peprotech, cat. no. 450-13)
- BMP4 (Peprotech, cat. no. 120-05ET)
- dbcAMP (Sigma-Aldrich, cat. no. D0627-25MG)
- N-acetyl-cysteine (Sigma-Aldrich, cat. no. A8199-10G)
- Heparin-binding EGF-like growth factor (Sigma-Aldrich, cat. no. E4643-50UG)
- Polybrene (Sigma-Aldrich, cat. no. TR-1003-G)
- Doxycycline (Sigma-Aldrich, cat. no. D9891-100UG)
- Accutase (ThermoFisher Scientific, cat. no. A1110501)
- Matrigel (Corning, cat. no. 354230)
- Rock inhibitor (StemCell Technologies, cat. no. Y-27632)
Troubleshooting
Procedure
Day -2: Reagent setup
Expansion Medium:
DMEM/F-12
10% FBS
1% N2 supplement 1% Glutamax
FGF Medium:
Neurobasal
2% B27 supplement 1% NEAA
1% Glutamax
1% FBS
8 ng/ml FGF
5 ng/ml CNTF 10 ng/ml BMP4
Maturation Medium:
1:1 DMEM/F-12 and Neurobasal
1% N2
1% Sodium Pyruvate
1% Glutamax
5 μg/ml N-acetyl-cysteine
5 ng/ml heparin-binding EGF-like growth factor 10 ng/ml CNTF
10 ng/ml BMP4
500 μg/ml dbcAMP
Day -1
Dissociate hiPSC with Accutase and incubate 5x10^5^ cells with virus for 1 h.
Seed in Matrigel-coated 6-well plates with StemFlex containing 10 μM Rock inhibitor
Spinfect with Sox9 and NfiB virus for 1h at 1000g
Day 0
Aspirate medium and add 2 ml of fresh StemFlex containing 2.5 μg/ml of Doxycycline per well.
Days 1 and 2
Aspirate medium and add 2 ml of Expansion Medium containing 2.5 μg/ml of Doxycycline and pertinent antibiotics (for whatever viruses you used).
Day 3
Aspirate medium and add 2 ml of 3/4 of Expansion Medium and 1/4 of FGF Medium containing 2.5 μg/ml of Doxycycline per well.
Day 4
Aspirate medium and add 2 ml of 1/2 of Expansion Medium and FGF Medium containing 2.5 μg/ml of Doxycycline per well.
Day 5
Aspirate medium and add 2 ml of 1/4 of Expansion Medium and 3/4 of FGF Medium containing 2.5 μg/ml of Doxycycline per well.
Day 6
Aspirate medium and add 2 ml of FGF Medium containing 2.5 μg/ml of Doxycycline.
Day 7
Dissociate cells with Accutase and pellet at 400g for 5 min.
Replate cells in Matrigel-coated coverslips, petri dishes or wells according to desired output.
Day 8
Use the necessary amount of FGF Medium containing 2.5 μg/ml of Doxycycline.
Aspirate medium and add 1/4 FGF Medium and 3/4 Maturation Medium containing 2.5 μg/ml of Doxycycline.
Day 10
Aspirate half of the medium and add the same amount of Maturation Medium containing 2.5 μg/ml of Doxycycline.
From here, change half of the medium every 2-3 days.