Generation and Functional Characterization of Monocytes and Macrophages Derived from Human Induced Pluripotent Stem Cells

Abstract

Monocytes and macrophages are essential for immune defense and tissue hemostasis. They are also the underlying trigger of many diseases. The availability of robust and short protocols to induce monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) will benefit many applications of immune cells in biomedical research. Here, we describe a protocol to derive and functionally characterize these cells. Large numbers of hiPSC‐derived monocytes (hiPSC‐mono) could be generated in just 15 days. These monocytes were fully functional after cryopreservation and could be polarized to M1 and M2 macrophage subtypes. hiPSC‐derived macrophages (iPSDMs) showed high phagocytotic uptake of bacteria, apoptotic cells, and tumor cells. The protocol was effective across multiple hiPSC lines. In summary, we developed a robust protocol to generate hiPSC‐mono and iPSDMs which showed phenotypic features of macrophages and functional maturity in different bioassays. © 2020 The Authors. Basic Protocol 1: Differentiation of hiPSCs toward monocytes Support Protocol 1: Isolation and cryopreservation of monocytes Support Protocol 2: Characterization of monocytes Basic Protocol 2: Differentiation of different subtypes of macrophages Support Protocol 3: Characterization of hiPSC‐derived macrophages (iPSDMs) Support Protocol 4: Functional characterization of different subtypes of macrophages

Optimised generation of iPSC-derived macrophages and dendritic cells that are functionally and transcriptionally similar to their primary counterparts

“…This is an increase in yield of 4.3x per cm 2 culture surface and has significant cost savings (1000USD per run) of expensive growth factors and cytokines. The new protocol produces 2.5-4 x10 6 CD14+ monocytes per harvest and up to 3.5 x10 7 cells over the course of a single differentiation run which compares very well to other recently published methods [21,22].…”

Large-Scale Production of Human iPSC-Derived Macrophages for Drug Screening

“…Cryopreservation of intermediate stages, in this case macrophage progenitors, would be highly desirable. While others reported progress in this area [ 49 ], we have not managed to cryopreserve cells with good recovery rates, which will be a point to further address and optimize in the near future. However, by introducing and characterizing the prolonged cultivation of macrophage progenitors in suspension culture, we were able to overcome this limitation by various means.…”

“…We managed a robust culture of myeloid factories in culture vessels with up to 1000 cm 2 , resulting in 20–60 million macrophage progenitors per harvest. Recently published work of others also aimed at increasing the yield or at decreasing the differentiation time of similar protocols [ 19 , 49 ]. It was shown that the myeloid factory state can be transferred to stirring cultures, which could be run in industry compatible bioreactors to generate sufficient cells for cell therapy approaches [ 19 ].…”

“…While stirring cultures have clear advantages in scalability, harvests from such cultures by sedimentation, filtration, and centrifugation are more laborious. A different study was successful in shortening the differentiation to 15 days [ 49 ] and in generating about 40 macrophage progenitors per iPSC in a single harvest differentiation. However, in order to obtain the macrophage progenitors, the study had to use a CD14 immunoferromagnetic purification step, which is not required for the method described here.…”

“…Moreover, they are a powerful tool to dissect which properties are intrinsic to all macrophages and which are due to ontogenetic differences or tissue residency [ 8 ]. One example for such difference is the efferocytotic capacity, which has been shown to be different between iPSC- and PBMC-derived macrophages [ 49 ]. This is in concordance with our findings reported here.…”