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.