Differentiation of Neurons (iNeurons) from iPSCs
Table of Contents
Introduction
Human induced pluripotent stem cells (iPSCs) are established by genetically reprogramming somatic cells to an embryonic-like, pluripotent state. These highly versatile cells can subsequently be differentiated into nearly any specialized cell type in the human body, providing an invaluable model for investigating developmental biology and disease pathways. Differentiation into neurons is possible in under 3-weeks, providing a valuable model for the study of basic neuronal biology alongside research into neurodegeneration and Alzheimer's.
Materials
- KOLF2 iPSCs
- E8 basal medium
- Vitronectin-coated 6-well plates
- EDTA (HB5135)
- Accutase
- ROCK inhibitor Y-27632 (HB2297)
- Doxycycline hyclate (HB4608)
- Geltrex
- DMEM/F12 medium
- N2 supplement
- GlutaMAX
- Non-essential amino acids
- 2-Mercaptoethanol
- Neurobasal Plus Medium
- B27 plus supplement
- Recombinant human NT-3 protein (HB8373)
- Recombinant human BDNF protein (HB3485)
- PDL hydrobromide
- 96-well plates
- 4% formaldehyde
- PBS buffer with Tween 20 (20x) (HB8088)
- Donkey serum
- Primary neuronal marker antibody (see our range of validated neuronal markers)
- Secondary antibodies (see our range of validated secondary antibodies)
- DAPI (HB0747)
Media A (iPSC Media)
| Component | Concentration |
|---|---|
| E8 medium | 1x |
|
ROCK inhibitor Y-27632 (HB2297) |
10 µM |
| Doxycycline hyclate (HB4608) | 1 µg/ml |
Media B (dox-NIM A Media)
| Component | Concentration |
|---|---|
| DMEM/F12 medium | 1x |
| N2 supplement | 1x |
| GlutaMAX | 1x |
| Non-essential amino acids | 1x |
| 2-Mercaptoethanol | 50 µM |
| Doxycycline hyclate (HB4608) | 1 µg/ml |
Protocol
- Maintenance of iPSC line: Transduce KOLF2 iPSCs with an inducible NGN2 transgene. Maintain the NGN2 KOLF2 iPSCs in E8 basal medium using 6-well plates coated with vitronectin. Use EDTA (HB5135) to passage the cells.
- Generation and Culture of NGN2 iNeurons (Day 0): Detach the NGN2 iPSCs utilizing accutase. Resuspend the cells to create a single cell suspension in Media A. Plate the cell suspension at a density of 25,000 cells/cm2 on a 6-well plate previously coated with Geltrex.
- Days 1 and 2: Completely exchange the culture media with freshly prepared Media B (dox-NIM A).
- Day 3: Passage cells using accutase and resuspend them in Media C (dox-NIM B). Seed the cells into a 96-well plate (pre-treated with 100 μg/ml PDL hydrobromide and coated with Geltrex) at a density of 10,000 cells / well.
- Days 4 to 7: Execute a 75% media exchange on days 4 - 6 using fresh Media C (dox-NIM B). Take extreme care to avoid mechanically disturbing the differentiating neurons during this process.
- Day 8 onwards: Replace 50% of the media volume with fresh Media C (dox-NIM B) every other day. This partial exchange method limits the neurons' exposure to air, which helps preserve cell attachment and culture quality.
- Immunocytochemistry (Day 18): Fix the mature iNeurons by incubating in 4% formaldehyde. Wash the cells thoroughly using PBS. Stain the cells for neuronal specific markers (e.g. βIII-Tubulin, NeuN or Neurofilament L) using our comprehensive immunocytochemistry (ICC) protocol.
References
- Takahashi, K. et al. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell PMID: 18035408
- Zhang, T.-Z. et al. 2022. Chimeric cerebral organoids reveal the essentials of neuronal and astrocytic APOE4 for Alzheimer's tau pathology. Signal Transduct. Target. Ther. PMID: 35691989
- Zhang, Y. et al. 2013. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron. PMID: 23764284