Differentiation of Induced Pluripotent Stem Cells (iPSCs) into Microglia
Table of Contents
Introduction
Welcome! If you're looking to generate microglia from iPSCs, you're in the right place. These cells are an incredible resource for investigating neurodegenerative diseases. This guide will walk you through a streamlined, weekend-free method to successfully differentiate your iPSCs into fully mature microglia. Let's get started!
Materials
- iPSCs
- Accutase
- Matrigel (0.5 mg/ml)
- 6-well culture plates
- E8-like medium
- ROCK inhibitor Y-27632 (HB2297)
- DMEM/F12 medium
- Sodium bicarbonate
- Sodium chloride
- L-Ascorbic acid (HB1238)
- Insulin-Transferrin-Selenium (ITS-G)
- BMP-4
- StemPro-34 SFM (1X)
- GlutaMax Supplement
- FGF2-G3
- SCF
- VEGF 165
- IL-3
- M-CSF
- Flt3L
- Thrombopoietin (TPO)
| Component | Final Concentration |
|---|---|
| DMEM/F12 medium | 1x |
| Sodium bicarbonate | 543 mg/l |
| Sodium chloride | 1000 mg/l |
| L-Ascorbic acid (HB1238) | 64 mg/l |
| Insulin-Transferrin-Selenium (ITS-G) | 1x |
| BMP-4 | 80 ng/ml |
| Component | Final Concentration |
|---|---|
| StemPro-34 SFM (1X) | 1x |
| GlutaMax Supplement | 2 mM |
| FGF2-G3 | 25 ng/ml |
| SCF | 100 ng/ml |
| VEGF 165 | 80 ng/ml |
| Component | Final Concentration |
|---|---|
| StemPro-34 SFM (1X) | 1x |
| GlutaMax Supplement | 2 mM |
| IL-3 | 50 ng/ml |
| SCF | 50 ng/ml |
| M-CSF | 50 ng/ml |
| Flt3L | 50 ng/ml |
| Thrombopoietin (TPO) | 5 ng/ml |
Protocol
- Day 0 (iPSC Plating): Detach the iPSCs utilizing Accutase and plate them at a density of 3.15x10^5 cells per well into a 6-well plate previously coated with Matrigel (0.5 mg/ml). Culture the cells in an E8-like medium supplemented with ROCK inhibitor Y-27632 (HB2297) at 10 µM.
- Days 1-4 (Mesoderm Induction): To begin differentiation into the mesodermal lineage, exchange the medium in each well every day with 2 ml of the prepared Mesoderm Induction medium.
- Days 5-6 (Hematopoietic Specification): Daily, replace the medium with 2 ml of fresh Hematopoietic Specification medium to encourage the transition of mesodermal cells into hemangioblasts. 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.
- Days 7-14 (Myeloid Progenitor Expansion): Refresh the medium each day with 2 ml of Myeloid Expansion medium to support differentiation into myeloid progenitors. From day 10 through day 14, harvest the supernatant daily and centrifuge it at 300 xg for 6 minutes to capture detached cells undergoing maturation. Carefully remove the supernatant, resuspend the resulting cell pellet in fresh medium, and return the cells back to their initial well.
References
- Woodburn SC, Bollinger JL and Wohleb ES. The semantics of microglia activation: neuroinflammation, homeostasis, and stress. J Neuroinflammation 18, 258 (2021). https://doi.org/10.1186/s12974-021-02309-6
- Abud EM, Ramirez RN, Martinez ES et al. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. (2017). doi: 10.1016/j.neuron.2017.03.042
- Muffat J, Li Y, Yuan B et al. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nat Med 22 (2016). https://doi.org/10.1038/nm.4189
- Chen SW, Hung YS, Fuh JL et al. Efficient conversion of human induced pluripotent stem cells into microglia by defined transcription factors. Stem Cell Reports. (2021). doi: 10.1016/j.stemcr.2021.03.010
- Douvaras P, Sun B, Wang M, et al. Directed Differentiation of Human Pluripotent Stem Cells to Microglia. Stem Cell Reports. (2017) doi: 10.1016/j.stemcr.2017.04.023