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Differentiation of induced pluripotent stem cells (iPSCs) into astrocytes

 

Hello Bio Protocol Header

 

Differentiating induced pluripotent stem cells (iPSCs) into astrocytes is a fantastic way to generate highly relevant glial cell models for drug discovery, neurotoxicity testing, and studying neurological diseases in vitro. By carefully directing the developmental pathways, you can efficiently derive functional astrocytes. This protocol provides a straightforward, step-by-step guide to help you achieve robust astrocyte differentiation in your lab!

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TCB-32 (Small Molecule FGF2 Replacement)

Synthetic, thermostable FGF2 replacement. Suitable for cell culture.

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TCB-541 (Small Molecule FGF2 Replacement)

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TCB-621 (Small Molecule FGF2 Replacement)

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Neuronal Induction

SB 431542

Potent, selective TGF-βRI ALK5, ALK4, ALK7 inhibitor

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Materials

  • Neurobasal Medium
  • N2 Supplement
  • B27 Supplement
  • EGF
  • Noggin
  • Anti-GFAP antibody ValidAbTM (HB8267)
  • DAPI (HB0747)
  • CNTF (HB8968)
  • GlutaMax

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Media Recipes

Aseptically combine components and filter the media before use.

Neural Embroid Body Forming Media

Component Concentration
DMEM/F12 1x
N2 Supplement 1x
B27 Supplement 1x
GlutaMax 1x
Noggin 500ng/ml
ROCK inhibitor Y-27632 (HB2297) 10 μM
SB 431542 (HB3555) 20 μM
One of: FGF2-G3 4 ng/ml
TCB-32 (HB12632) 400nM
TCB-541 (HB11050) 200nM
TCB-621 (HB17550) 100nM

FGF2 replacement with TCB compounds enables weekend-free feeding

Neural Induction Media

Component Concentration
DMEM/F12 1x
N2 Supplement 1x
B27 Supplement 1x
GlutaMax 1x
Noggin 500ng/ml
SB 431542 (HB3555) 20 μM
One of: FGF2-G3 10 ng/ml
TCB-32 (HB12632) 2µM
TCB-541 (HB11050) 1µM
TCB-621 (HB17550) 500nM

Daily media changes are critical to avoid aggregate disruption

Radial Glia Formation Media

Component Concentration
Neurobasal medium 1x
N2 Supplement 1x
EGF 10 ng/ml
One of: FGF2-G3 10 ng/ml
TCB-32 (HB12632) 2µM
TCB-541 (HB11050) 1µM
TCB-621 (HB17550) 500nM

Transitions to Neurobasal to promote precursor specification.

Please note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs. TCB compounds have not been optimised yet for this assay therefore concentrations may need adjusting. TCB concentrations are based upon the original 5x lower FGF2 concentration used by Lancaster et al.

 

Protocol

1
Day 0: Dissociate iPSCs (70-80% confluence) and plate at 5x105 cells/ml in neural embroid body forming medium. Transfer to low-attachment plates and centrifuge at 800 x g for 3 minutes to promote EB formation.
2
Days 1-5: Perform daily media changes using Neural Induction Media (these changes may be able to be reduced when using TCB compounds instead of FGF2). 
3
Day 6: Dissociate EBs using Accutase and replate at 5x105 cells/ml onto 0.5 μg/ml Matrigel-coated plates. Feed cells with Radial Glia Formation Media.
4
Days 7-8: Perform daily media changes with Radial Glia Formation Media (these changes may be able to be reduced when using TCB compounds instead of FGF2).
5
Days 9-11: Perform daily media changes with Radial Glia Formation Media (containing 20ng/ml CNTF (HB8968)) to initiate lineage specification.
6
Days 12-14: Perform daily media changes with Radial Glia Formation Media (containing 20ng/ml CNTF (HB8968) and without EGF).
7
Day 15: Transition to Radial Glia Formation Media (containing 20ng/ml CNTF (HB8968) and without EGF or FGF2 / TCB compounds) to promote maturation.
8
Days 16 - 31: Change media on Days 15, 16, 17, 19, 21, 23, 25, 27, and 29.
9
Days 32-34: Fix and permeabilize cells then carry out immunocytochemical anaylsis using astrocyte specific cell markers (e.g. GFAP, EAAT1, EAAT2, or ALDH1L1) to confirm an astrocytic phenotype. Please see our Immunocytochemistry (ICC) Protocol for full guide to staining and imaging of fixed cells.

References

1
Tcw et al., 2017 An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.Stem Cell Reports. PMID: 28757165
2
Emdad et al., 2012. Efficient differentiation of human embryonic and induced pluripotent stem cells into functional astrocytes.Stem Cells Dev. PMID: 21631388
3
Jovanovic et al., 2023 A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells.Stem Cell Reports. PMID: 37451260