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Updated: Sep 30, 2025
Production and Characterization of Human Macrophages from Pluripotent Stem Cells
Published on: April 16, 2020
Efficient Method to Differentiate Mouse Embryonic Stem Cells into Macrophages in vitro
Qimin Hai1, Juying Han1, Sophia Wells1
1Department of Cardiovascular & Metabolic Sciences, Cleveland Clinic, Cleveland, OH 44195, USA.
Abstract:
Macrophages are key cells in the innate immune system and play a role in a variety of diseases. However, macrophages are terminally differentiated and difficult to manipulate genetically via transfection or through CRISPR-Cas9 gene editing. To overcome this limitation, we provide a simplified protocol for the generation of mouse embryonic stem cells-derived macrophages (ESDM). Thus, genetic manipulation can be performed using embryonic stem cells, selecting for the desired changes, and finally producing macrophages to study the effects of the previous genetic manipulation. These studies can contribute to many areas of research, including atherosclerosis and inflammation. Production of ESDM has been previously achieved using embryoid body (EB) intermediates. Here, we optimized the EB method using a simplified medium, reducing the number of recombinant proteins and medium recipes required. Our EB-based differentiation protocol consists of three stages: 1) floating EB formation; 2) adherence of EBs and release of floating macrophage progenitors; and, 3) terminal differentiation of harvested macrophage progenitors. The advantages of this protocol include achieving independent floating EBs in stage 1 by using a rocker within the tissue culture incubator, as well as the exclusion of small EBs and cell clusters when harvesting macrophage progenitors via cell filtration.
Insights
Generating genetically modified macrophages is challenging. This study presents a simplified protocol using mouse embryonic stem cells (ESCs) to create macrophages for disease research, such as inflammation.
Area of Science:
- Immunology
- Cell Biology
- Developmental Biology
Background:
- Macrophages are crucial innate immune cells involved in numerous diseases.
- Their terminally differentiated nature impedes genetic manipulation via transfection or CRISPR-Cas9.
- Existing methods for generating stem cell-derived macrophages often require complex protocols.
Purpose of the Study:
- To develop a simplified and optimized protocol for generating mouse embryonic stem cells-derived macrophages (ESDM).
- To enable genetic manipulation of macrophages for studying disease mechanisms.
- To facilitate research in areas like atherosclerosis and inflammation.
Main Methods:
- Optimization of the embryoid body (EB) method for ESDM production.
- Utilizing a simplified culture medium, reducing recombinant protein requirements.
- A three-stage protocol: floating EB formation, EB adherence and progenitor release, and terminal differentiation.
- Employing a rocker for independent floating EBs and cell filtration for progenitor harvesting.
Main Results:
- A simplified, three-stage protocol for ESDM generation was established.
- The optimized method reduces the complexity of culture media and recombinant protein usage.
- Independent floating EBs and purified macrophage progenitors were successfully obtained.
Conclusions:
- The simplified EB-based protocol facilitates genetic manipulation of macrophages.
- This method provides a valuable tool for studying macrophage function in various diseases.
- The optimized protocol streamlines ESDM production for immunological research.
