Differentiation of monocytes and polarized M1/M2 macrophages from human induced pluripotent stem cells

• Published in: STAR protocols 2024-03, Vol.5 (1), p.102827-102827, Article 102827
• Main Authors: Park, Tea Soon, Hirday, Rishabh, Quinn, Russell, Jacob, Sheela Panicker, Feldman, Ricardo A., Bose, Devika, Sharma, Ruchi, Bharti, Kapil
• Format: Article
• Language: English
• Subjects: Cell Biology; Cell Differentiation; Embryoid Bodies; Humans; Induced Pluripotent Stem Cells; Macrophages; Monocytes; Protocol; Stem Cells
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102827

Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. “monocyte factory.” We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. For complete details on the use and execution of this protocol, please refer to Karlson et al.1 and Panicker et al.2 [Display omitted] •Stepwise protocol of iPSC differentiation to produce monocytes•Details on monocytes cryopreservation and thawing processes•Monocyte differentiation to polarized M1/M2 macrophages•Characterization by flow cytometry, Ac-LDL uptake, and phagocytosis assay Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. “monocyte factory.” We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells.