Human macrophage biology research has been limited because it relies on collecting cells from donors. This protocol shows a method in which we can produce human macrophages from pluripotent stem cells in the laboratory. This is a robust protocol that can be scaled up to produce macrophages in large quantities for cell therapies or research.
Pluripotent stem cells can be genetically manipulated. So in this protocol, we can generate macrophages with a specific phenotype, fluorescent tags, and also macrophages to model disease states. Visual demonstration of this protocol is useful because the use of the easy passage tool won't be familiar to many researchers.
Also, the level of care needed when replenishing media and harvesting cells is hard to put into words. When the cultured cells have reached 80%confluency, replace the spent culture medium with 1.5 milliliters of fresh medium. Holding the culture vessel in one hand, roll a disposable cell passaging tool across the plate in one direction.
All blades in the roller must be touching the plate. Maintain uniform pressure while rolling. Repeat rolling in the same direction until the whole well has been covered.
Rotate the culture vessel 90 degrees and repeat rolling. With a sterile pipette, use media in the well to dislodge cut colonies. Aspirate CELLstart and replace media.
Transfer cells at a one-to-four ratio onto pre-coated wells prepared as described in the manuscript. To begin the differentiation process on day zero, add 2.25 milliliters of stage one media into two wells of an ultra low attachment six-well plate. Then for one 80%confluent well of iPSCs in a six-well plate, replace the maintenance media with 1.5 milliliters of stage one media.
Cut colonies using a new cell passaging tool. Using a pipette, transfer the cut colonies into the two wells of the ultra low attachment six-well plate. On day two, adjust the cytokine concentrations by adding 0.5 milliliters of hESC SFM media to the six-well plate containing the day two embryoid bodies.
On day four, coat four wells of a six-well tissue culture plate with 0.1%gelatin. After incubating for at least 10 minutes, remove the gelatin and add 2.5 milliliters of stage two media. Collect formed embryoid bodies from the two wells of a six-well plate and place them in a 50 milliliter centrifuge tube.
Allow them to settle at the bottom of the tube by gravity. Then carefully aspirate the media from the tube and resuspend the embryoid bodies in two milliliters of stage two media. Transfer 10 to 15 embryoid bodies to a gelatin-coated well containing 2.5 milliliters of stage two media and incubate at 37 degrees Celsius with 5%carbon dioxide.
For two to three weeks, change the media every three to four days. Plating of the EBs is crucial. Having fewer than 10 or more than 15 EBs or having them unevenly distributed can lead to low yields of macrophages.
After two to three weeks, the embryoid bodies start releasing nonadherent hematopoietic cells into suspension. Using a 40 micrometer strainer, collect these cells into a 50 milliliter tube and then replenish the media. Centrifuge the suspension of hematopoietic cells at 200 times g for three minutes.
Resuspend the hematopoietic cells in stage three media. Then plate the cells at a density of 0.2 times 10 to the sixth cells per milliliter. Change the stage three media every five days.
After nine to 11 days, the macrophages will be fully differentiated and fully functional. Add eight times 10 to the fourth iPSC-derived macrophages into 200 microliters of stage three media to each well of a 96-well plate. Add 100 microliters of bead solution to each well containing iPSC-derived macrophages.
Use a high-content imaging system to image the plate. Acquire three or more fields across each well to obtain a good representation. iPSC colonies, embryoid bodies, hematopoietic suspension cells, and mature macrophages were morphologically distinct.
iPSC-derived macrophages were large, had single small oval-shaped nuclei and had abundant cytoplasm containing many vesicles. Macrophage phenotype was assessed by flow cytometry. Mature iPSC-derived macrophages expressed the lineage marker CD45 and the macrophage maturation marker 25F9 and were negative for monocyte immature macrophage marker CD93.
iPSC-derived macrophages expressed several lineage myeloid markers and were positive for immune modulation marker CD86. A small proportion of the macrophages expressed chemokine receptors CX3CR1, CCR2, CCR5, and CCR8. iPSC-DM phagocytic ability was evaluated using bioparticles and a high-content imaging system.
Bioparticles were nonfluorescent when added to the macrophage cultures, but after phagocytosis fluoresced bright green in the intracellular acidic pH. The phagocytic fraction represents the proportion of macrophages that phagocytosed bioparticles and the phagocytic index is the measure of the number of bioparticles that each macrophage ingested. Macrophages can change phenotype in response to environmental cues.
RT-PCR was used to quantify upregulated mRNA expression of genes. Macrophages treated with LPS and interferon gamma or IL4 showed a significantly lower percentage of phagocytic cells when compared to naive macrophages. Macrophages treated with IL10 showed an increased percentage of phagocytic cells and an increased phagocytic index.
This protocol could be used to provide an off-the-shelf source of cells to treat diseases such as chronic liver disease where macrophages have been shown to be therapeutic in animal models. iPSC-derived macrophages can be used to study macrophages associated with several disease states. For example, we use these cells to study macrophages associated with tumors in breast cancer.
We have modified the phenotype of iPSC-derived macrophages by genetic programming using a single transcription factor called KLF1. With this, we produced macrophages that look like erythroid island macrophages and they have provided insights into the production and maturation of red blood cells.
