The ability to generate functional macrophages from hiPSCs offers a unique opportunity to study macrophages' biology, model diseases, and develop cell-based therapies. Here, Creative Biolabs offers a detailed protocol for differentiating hiPSCs into functional macrophages.

Materials

• Human induced pluripotent stem cells (hiPSCs)
• Appropriate culture medium for hiPSC maintenance
• Matrigel-coated culture dishes
• Appropriate medium for hematopoietic differentiation
• Cell dissociation reagent
• Various other supplements

Methods

1. Maintenance and Expansion of hiPSCs
   • First, culture hiPSCs on Matrigel-coated dishes using a suitable culture medium.
   • Passage hiPSCs every 4-7 days using an appropriate cell dissociation reagent.
   • Maintain hiPSCs in an undifferentiated state by regularly changing the medium and supplementing with ROCK inhibitor if necessary.
2. Formation of Embryoid Bodies
   • Harvest undifferentiated hiPSCs using suitable cell dissociation reagent.
   • Gently triturate the hiPSCs into small clumps and transfer them into ultra-low attachment dishes.
   • Cultivate the hiPSCs in a suitable medium supplemented with rhBMP4 and rhVEGF for 4-7 days to form EBs. Change the medium every 2-3 days.
3. Hematopoietic Differentiation
   • After EB formation, collect the EBs and transfer them into a new dish.
   • Culture the EBs in a suitable medium supplemented with rhSCF, rhIL-3, and rhIL-6 for 5-7 days.
   • Monitor the emergence of hematopoietic progenitor cells under a microscope.
4. Macrophage Differentiation
   • After hematopoietic differentiation, transfer the emerging hematopoietic progenitor cells to RPMI medium supplemented with rhGM-CSF for 5-7 days.
   • Collect the differentiated macrophages for further characterization or functional assays.

Notes

• The quality of hiPSCs is crucial for successful differentiation. Regularly assess the pluripotency and karyotype stability of hiPSCs during maintenance.
• Optimize the timing and concentration of cytokines for hematopoietic and macrophage differentiation based on the specific requirements of the experiment.

Our protocol provides a comprehensive guide for the step-by-step differentiation of hiPSCs into macrophages. Creative Biolabs is dedicated to providing comprehensive services to researchers interested in utilizing macrophages for a variety of applications in biomedical research.

You can get more services by contacting us directly.

Reference

1. Pandya H, et al. Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nature neuroscience, 2017, 20(5): 753-759.