TITLE: A Simple Multistep Protocol for Differentiating Human Induced Pluripotent Stem Cells into Functional Macrophages
AUTHORS: Chandrayana Mukherjee, Christine Hale, Subhankar Mukhopadhyay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Macrophages differentiated from human induced pluripotent stem cells (hiPSCs) provide an alternative new tool overcoming some of the limitations of existing models for human macrophages, such as human macrophage-like cell lines and primary monocyte-derived macrophages. A combination of different cytokines and growth factors can differentiate hiPSCs toward myeloid lineage. Here we describe a simple multistep protocol for differentiating hiPSCs into functional macrophages. This includes derivation of three germ-line containing embryoid bodies (EBs) from iPSCs, generation of myeloid precursors from EBs, and finally maturation of myeloid precursors into functional macrophages. Technical procedure and specific culture conditions associated with each of these steps are discussed in detail.</div></div>

[STEPS]
?. [General Considerations]
Human iPSCs are delicate and more difficult to maintain in culture compared to most other conventional human cell lines. They show a natural tendency to spontaneously differentiate into fibroblast like cells. Cytokines and growth factors present in the medium allow them to be kept in pluripotent state; hence, daily change of media is essential.
?. [General Considerations]
iPSCs grow in individual colonies. During handling, it is critical that iPSCs remain as small clumps and not broken down into single cells. Hence, iPSC colonies should be always treated gently; do not pipette too harshly or too many times as this will result in single-cell colonies. Use wide-bore stripettes, and centrifuge slowly and with shorter duration where possible.
?. [General Considerations]
It is important to ensure that iPSC colonies remain separate in culture and do not fuse to each other during expansion to prevent spontaneous differentiation. However, low levels of spontaneous differentiation are sometimes unavoidable especially on feeder-dependent culture. Culture can be cleaned up by carefully selecting only pluripotent colonies during passage steps—this will eventually clean up the culture after few passages
?. [hiPSC Culture in Feeder-Free System]
Prepare vitronectin-coated maintenance plate at least 2 h before plating the hiPSCs. Thaw  at  and dilute it in . Add  into each well of 6-well tissue culture plates or add whole  into 100 mm tissue culture dish.
[aliquot of vitronectin]
4 Room temperature
[D-PBS]
[diluted vitronectin]
6 mL
?. [hiPSC Culture in Feeder-Free System]
Make sure that the vitronectin solution covers the whole surface of the plastic vessel and incubate at  for at least  or at  tightly sealed with Parafilm. If necessary, sealed plates can be stored at  for up to 5 days without removing the vitronectin solution (see Note 7).
4 Room temperature
4 °C
4 °C
?. [hiPSC Culture in Feeder-Free System]
Prepare a 15 mL centrifuge tube with  supplemented with  for each vial of iPSC that will be defrosted.
[E8 media]
[Rock inhibitor]
?. [hiPSC Culture in Feeder-Free System]
Centrifuge the tube at .
Centrifuge: 290 34, 3 min, 20 10
?. [hiPSC Culture in Feeder-Free System]
Place the plate into a humidified  incubator with . Once inside the incubator gently rock the plate in different directions to make sure that colonies are evenly distributed throughout the surface of the dish. Keep the plate undisturbed for at least  to allow colonies to attach to the vitronectin-coated surface.
37 °C
[CO2]
?. [hiPSC Culture in Feeder-Free System]
After  the majority of the colonies will attach to the surface. Remove all media from the dish to discard any unattached colonies and dead cells, and add – for each well of a 6-well plate or – for each 100 mm dish (see Note 10).
2 mL
[fresh E8 media without Rock inhibitor]
10 mL
12 mL
?. [hiPSC Culture in Feeder-Free System]
Change medium every day and replace with fresh E8 medium until cells are ~70–80% confluent and ready for passaging.
?. [hiPSC Culture in Feeder-Free System]
For passaging, start by preparing vitronectin-coated plates as before.
?. [hiPSC Culture in Feeder-Free System]
Once cells are sufficiently confluent, remove the spent media and gently wash the plate twice with an appropriate volume (~) of D-PBS.
5 mL
?. [hiPSC Culture in Feeder-Free System]
Remove the D-PBS and add required volume of  to cover the entire surface of the dish ( for each well of a 6-well plate, and  for 100 mm dish).
[PBS-EDTA solution]
1 mL
8 mL
?. [hiPSC Culture in Feeder-Free System]
Look under the microscope every 2 min to confirm that colonies are detaching and have changed their morphologies but do not wait too long such that the colonies are fully detached or broken up into single cells.
?. [hiPSC Culture in Feeder-Free System]
Carefully remove PBS-EDTA solution without disturbing iPSC colonies and add same volume of fresh E8 media. Gently pipette up and down using a 10 mL stripette to dislodge the colonies from the surface. If necessary repeat this step two more times to recover as many iPSC colonies as possible.
?. [hiPSC Culture in Feeder-Free System]
Collect the media and detached iPSC colonies into a new 15 mL centrifuge tube and centrifuge at .
Centrifuge: 290 34, 3 min, 20 10
?. [hiPSC Culture in Feeder-Free System]
Transfer colonies in a desired splitting ratio into a fresh vitronectin-coated dish with appropriate volume of E8 media supplemented with .
[Rock inhibitor]
Generally we recommend a splitting ratio of 1:10; but this depends on growth rates of individual iPSC lines and how frequently cells are passaged. Thus, the splitting ratio should be adjusted between 1:5 and 1:10 according to the requirement of individual user.
?. [hiPSC Culture in Feeder-Free System]
After remove medium and add fresh E8 medium (without Rock inhibitor); change medium every day until next passage.
?. [hiPSC Culture in Feeder-Free System]
To freeze iPS cells, freshly prepare the freezing mixture— in KSR ( + ) at . After lifting off iPSC colonies as described in Subheading "hiPSC Culture in Feeder-Free System", resuspend the cell pellet into desired volume of freezing mixture and quickly transfer  into each cryovial. We recommend one 100 mm dish (70–80% confluent) to divide into ten cryovials and each well of a 6-well plate into two cryovials for freezing. Immediately transfer the cryovials into a Mr. Frosty or CoolCell container and place them into  freezer . After  transfer the cryovials into liquid nitrogen for long-term storage.
[DMSO]
[KSR]
[DMSO]
0 Room temperature
[cell suspension]
-80 °C
?. [Culturing hiPSCs in Feeder-Dependent System]
At least 24 h before transferring the iPSCs from feeder-free culture, prepare feeder plates using inactivated mouse embryonic fibroblasts (MEFs). First coat the tissue culture plates by adding  into each well of 6-well plates or  for 100 mm dishes; ensure that whole surface of the dish is covered and then incubate at  for at least .
[0.1% gelatine solution]
8 mL
80 Room temperature
?. [Culturing hiPSCs in Feeder-Dependent System]
Thaw one vial (2 × 106 cell) of irradiated mouse embryonic fibroblasts into  in a 15 mL centrifuge tube and spin at .
[of human iPSC basal medium]
Centrifuge: 290 34, 20 10
?. [Culturing hiPSCs in Feeder-Dependent System]
Remove the gelatine solution from the plate, seed the MEFs, and place them in  incubator . One vial of MEF (2 × 106 cell) is sufficient for a whole 6-well plate or one 100 mm dish.
37 °C
?. [Culturing hiPSCs in Feeder-Dependent System]
Lift off the iPSC colonies growing under feeder-free conditions as described in Subheading "hiPSC Culture in Feeder-Free System".
?. [Culturing hiPSCs in Feeder-Dependent System]
Remove medium and any unattached MEFs from the feeder plate and transfer iPSC colonies onto the feeder layer. Usually a splitting ratio of 1:10 works well at this stage.
?. [Culturing hiPSCs in Feeder-Dependent System]
After  a majority of colonies will have attached to the feeder layer. Remove medium and add fresh human iPS base medium supplemented with   but no Rock inhibitor. Change medium every day until colonies are ready to passage (see Note 10).
[bFGF]
?. [Culturing hiPSCs in Feeder-Dependent System]
For passaging, thaw collagenase, dispase aliquots at , and mix them in 1:1 ratio in a tube.
37 Room temperature
?. [Culturing hiPSCs in Feeder-Dependent System]
Remove medium from iPSCs growing on feeder layer, wash once with D-PBS, and add collagenase-dispase mix on the dish to cover the whole surface.
We recommend   for each well of a 6-well feeder plate and   for 100 mm dish.
?. [Culturing hiPSCs in Feeder-Dependent System]
Incubate at  for – until pluripotent colonies start to lift off but feeder layer and any differentiated cells remain attached. Start checking under a microscope for floating colonies after  and every – subsequently.
37 °C
The duration of incubation varies between iPSC lines and can take up to 90 min depending on colony size and level of differentiation.
It is important not to overdigest to prevent colonies from breaking up into small fragments.
?. [Culturing hiPSCs in Feeder-Dependent System]
Gently mix the collagenase-dispase solution up and down few times using a 10 mL stripette to dislodge loosely adherent colonies from the feeder layer. Collect collagenase-dispase mixture along with all floating colonies into a new 50 mL and add at least same volume of human iPSC basal medium to neutralize enzyme activity.
?. [Culturing hiPSCs in Feeder-Dependent System]
Centrifuge harvested colonies at  and discard the supernatant.
Centrifuge: 290 34, 3 min, 20 10
?. [Culturing hiPSCs in Feeder-Dependent System]
After the last wash, if required, break the colonies into smaller sizes, gently resuspend in desired volume of iPSC base medium supplemented with bFGF and Rock inhibitor, transfer to new feeder plates in a desired splitting ratio, and continue as before.
?. [Differentiation of iPSCs into Macrophages]
Once iPSC colonies are 70–80% confluent on feeder-dependent culture detach them from the feeder layer using collagenase-dispase method. Transfer all the floating iPSC colonies with enzyme mix into a fresh 50 mL tube and add at least twice the volume of iPSC base medium to neutralize enzyme function.
?. [Differentiation of iPSCs into Macrophages]
Keep the tube upright in a tube rack undisturbed for – to allow colonies to settle.
A large pellet of iPSC colonies will become visible at the bottom of the tube and medium on the top will be clearer. Do not centrifuge at this stage as fragmentation of colonies into smaller pieces will reduce the efficiency of EB formation in subsequent steps.
?. [Differentiation of iPSCs into Macrophages]
Aspirate medium from the top without disturbing iPSC colonies in the bottom of the tube.
?. [Differentiation of iPSCs into Macrophages]
Resuspend iPSC colonies in iPSC base medium without any cytokine supplement, and distribute them to desired numbers of 100 mm low-adherent bacteriological dishes.
We recommend dividing one 100 mm dish of feeder-dependent iPSC culture into four 100 mm low-adherent bacteriological dishes for EB formation (see Note 11).
?. [Differentiation of iPSCs into Macrophages]
Add extra medium as necessary to make the final volume of each 100 mm low-adherent bacteriological dish ~.
30 mL
?. [Differentiation of iPSCs into Macrophages]
On day 5, the EBs should have formed and be visible with the naked eye. Harvest them by collecting all the media into 50 mL tubes using a 10 mL stripette and leaving the tubes in an upright position for – to let the EBs settle by gravity.
Do not centrifuge at this stage.
?. [Differentiation of iPSCs into Macrophages]
After 4–5 days, the majority of EBs will attach to the gelatine-coated surface and start to spread out as a stromal layer around the EB. It is possible that some EBs are still not attached to the surface at this stage; in such a case add additional – and leave the EBs in culture for another 4–5 days (see Note 12).
12 mL
[cytokine-supplemented myeloid precursor base media]
At this stage many small apoptotic looking floating cells will arise in the medium; these are released from underlying EBs, but are not myeloid precursors and should be regularly removed from the culture.
?. [Differentiation of iPSCs into Macrophages]
Change the medium every 4–5 days for next 2–3 weeks by harvesting all media from the EB dish and pass through a 70 μm cell strainer. Discard the flow through as this will mostly contain contaminating small cells and debris. Any floating EBs will be retained by the cell strainer and should be transferred back into the culture dish; invert the cell strainer and directly pipette the medium onto the bottom surface of the strainer back into the same dish.
?. [Differentiation of iPSCs into Macrophages]
Approximately 3–4 weeks after transferring the EBs to gelatinized plates, the smaller apoptotic looking cells will disappear from the culture to be replaced by larger blast-like cells with dendrite-like structure. These are the myeloid precursor cells that could be further differentiated into mature macrophagesas described below. From  onwards this is a continuous culture; myeloid precursors can be harvested every 4–5 days and as long as cytokine-supplemented fresh medium are added to EBs they will produce further precursors. This process can be continued for 6–8 months after which precursor number drops significantly (see Notes 13–15).
?. [Differentiation of iPSCs into Macrophages]
Harvest the myeloid precursor cells by removing spent medium from EB plates.
?. [Differentiation of iPSCs into Macrophages]
Count cells and plate ~150,000 precursor cells in each well of a 6-well plates or 500,000 cells in a 100 mm tissue culture dish (see Note 16).
?. [hiPSC Culture in Feeder-Free System]
Thaw the vial at  in water bath. Gently transfer the defrosted cells into  and slowly invert the tube to evenly mix the cells and to dilute out DMSO.
37 °C
[E8 media]
DMSO is toxic for cells at  ; therefore the thawing process needs to be as quick as possible; keep frozen vial in water bath for minimum time required to defrost but do not warm up cells to  .
?. [hiPSC Culture in Feeder-Free System]
Resuspend the cell pellet into desired volume of E8 medium supplemented with .
[Rock inhibitor (final concentration)]
?. [hiPSC Culture in Feeder-Free System]
Plate  into each well of a 6-well plate. Content of one frozen vial (equivalent to 1 well of ~70–80% confluent 6-well plate or 1/10th of a 100 mm dish) can be distributed into 2–3 wells of a 6-well plate with .
[cell suspension]
[medium]
Alternatively, if a specific iPSC line shows good recovery and growth characteristics, cells can be seeded less densely—for example one vial can be directly seeded onto a 100 mm dish using  . Generally it is better to seed the colonies densely after thawing to ensure better recovery (see Notes 8 and 9).
?. [hiPSC Culture in Feeder-Free System]
Prepare vitronectin-coated maintenance plate at least 2 h before plating the hiPSCs. Thaw  at  and dilute it in . Add  into each well of 6-well tissue culture plates or add whole  into 100 mm tissue culture dish.
[aliquot of vitronectin]
4 Room temperature
[D-PBS]
[diluted vitronectin]
6 mL
?. [hiPSC Culture in Feeder-Free System]
Make sure that the vitronectin solution covers the whole surface of the plastic vessel and incubate at  for at least  or at  tightly sealed with Parafilm. If necessary, sealed plates can be stored at  for up to 5 days without removing the vitronectin solution (see Note 7).
4 Room temperature
4 °C
4 °C
?. [hiPSC Culture in Feeder-Free System]
Remove the spent media.
?. [hiPSC Culture in Feeder-Free System]
Wash the plate with an appropriate volume (~) of D-PBS. (1/2)
5 mL
?. [hiPSC Culture in Feeder-Free System]
Wash the plate with an appropriate volume (~) of D-PBS. (2/2)
5 mL
?. [hiPSC Culture in Feeder-Free System]
Resuspend the colonies in  supplemented with . If colonies are too big at this stage pipette up and down 2–3 times to break them into smaller clumps.
[fresh E8 media]
[Rock inhibitor]
?. [Culturing hiPSCs in Feeder-Dependent System]
Resuspend the cell pellet in desired volume of human iPSC base medium.
?. [Culturing hiPSCs in Feeder-Dependent System]
Resuspend these colonies into the required volume of human iPSC base medium supplemented with  and  (final concentrations).
[bFGF]
[Rock inhibitor]
?. [Culturing hiPSCs in Feeder-Dependent System]
Place the dish inside the incubator, redistribute colonies evenly on feeder layer, and keep undisturbed for at least .
?. [Culturing hiPSCs in Feeder-Dependent System]
Wash at least two times by resuspending colonies in iPSC base medium followed by centrifugation.
It is important to make sure that all traces of enzymes are removed before replating the cells onto a new feeder plate.
?. [Culturing hiPSCs in Feeder-Dependent System]
Resuspend colonies in iPSC base medium followed by centrifugation. (1/2)
?. [Culturing hiPSCs in Feeder-Dependent System]
Resuspend colonies in iPSC base medium followed by centrifugation. (2/2)
?. [Differentiation of iPSCs into Macrophages]
Wash 2–3 times by adding excess volumes of iPSC base media and by allowing the colonies to settle.
?. [Differentiation of iPSCs into Macrophages]
Wash by adding excess volumes of iPSC base media. (1/3)
?. [Differentiation of iPSCs into Macrophages]
Wash by adding excess volumes of iPSC base media. (2/3)
?. [Differentiation of iPSCs into Macrophages]
Wash by adding excess volumes of iPSC base media. (3/3)
?. [Differentiation of iPSCs into Macrophages]
Transfer the dishes to a humidified  incubator and leave undisturbed for  (4 days) to allow EB formation.
37 °C
?. [Differentiation of iPSCs into Macrophages]
Carefully aspirate the medium along with single cell and cell debris and gently resuspend EBs into desired volume of myeloid precursor base medium supplemented with IL-3 and M-CSF.
?. [Differentiation of iPSCs into Macrophages]
Distribute the EBs onto gelatine-coated 100 mm tissue culture-treated dishes with final volume of ~ in each dish.
[media]
?. [Differentiation of iPSCs into Macrophages]
Filter through a 70 μm cell strainer and collect the flow through in 50 mL tubes as it will contain the precursor cells.
?. [Differentiation of iPSCs into Macrophages]
Centrifuge at  and resuspend the pellet in macrophage differentiation base medium supplemented with .
Centrifuge: 290 34, 3 min, 20 10
[M-CSF]
?. [Differentiation of iPSCs into Macrophages]
Culture cells at  incubator for another  (6 days) to differentiate into mature macrophages.
37 °C
?. [Differentiation of iPSCs into Macrophages]
From day 7 onwards fully mature macrophages will be ready for phenotypic assessment or downstream functional assays.