TITLE: Differentiation of iPSC into Microglia-Like Cells (iMGL)
AUTHORS: Fabia Filipello, Jacob Marsh, Rj Martinez, Celeste Karch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the derivation of Hematopoietic Progenitor Cells and differentiation of iMGLs using iPSC cultures. This protocol is modified the following papers.</div></div>

[STEPS]
?. [iPSCs Culture]
Thaw and culture iPSC line per the following protocol:
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?. [iPSCs Aggregate Plating]
Once iPSCs are 70-80% confluent in 2-3 wells of a 6-well tissue culture plate, passage and plate the iPSCs as aggregates
Aggregates should be approximately 100-200µm in diameter
?. [iPSCs Aggregate Plating]
Coat a 6-well tissue culture plate with Matrigel for a least  prior to passaging cells
A single 6-well tissue culture plate will yield approximately 1-1.5 million HPCs
?. [iPSCs Aggregate Plating]
Set the following media out to warm to :DispaseDMEM/F12PBSmTesR1
0 Room temperature
?. [iPSCs Aggregate Plating]
After plate has been coated for  and media has warmed to , proceed to passage aggregates as described below:
0 Room temperature
?. [iPSCs Aggregate Plating]
Aspirate media from well.
?. [iPSCs Aggregate Plating]
Wash cells with  per well
[of PBS]
?. [iPSCs Aggregate Plating]
Aspirate PBS from well.
?. [iPSCs Aggregate Plating]
Add Dispase to cells .
[per well]
?. [iPSCs Aggregate Plating]
Incubate at  for between  and .
37 °C
Edges of iPSC clusters should begin to lift off the Matrigel coated plate
Cells will not completely  dissociate from Matrigel coated plate. The majority of cells will remain attached after the incubation.
?. [iPSCs Aggregate Plating]
Aspirate the Dispase from the wells.
?. [iPSCs Aggregate Plating]
Gently wash cells 2-3 times with  per well, aspirating after each wash.
[of DMEM/F12]
The major goal of this step is to remove the Dispase without substantially disturbing the cells.
?. [iPSCs Aggregate Plating]
Add  to each well.
[of DMEM/F12]
?. [iPSCs Aggregate Plating]
Using the StemProEZPassage Disposable Stem Cell Passaging Tool, cut the iPSCs into aggregates that are approximately 100-200µm in diameter.
To accomplish this, the StemPro EZPassage Disposable Stem Cell Passaging Tool should be rolled throughout the entirety of the well horizontally (twice) and vertically (twice). This will result in the appropriate sized aggregates for plating.
?. [iPSCs Aggregate Plating]
Transfer the detached aggregates with a serological pipette into a 15 ml conical tube.
Check tissue culture plate under microscope to ensure that the majority of aggregates have been transferred from the plate.
If aggregates still remain, wash well with 2mL of DMEM/F12 and transfer to 15mL conical tube
?. [iPSCs Aggregate Plating]
Centrifuge 15 ml conical tubes containing cell aggregates at 750 rpm for .
?. [iPSCs Aggregate Plating]
Aspirate supernatant from cell pellet.
?. [iPSCs Aggregate Plating]
Resuspend pellet gently in
[of mTesR1]
Obtaining a uniform suspension of aggregates approximately 100-200µm in diameter is optimal. The StemPro EZPassage Disposable Stem Cell Passaging Tool should be used to ensure these aggregate sizes.
It is essential that pellet is resuspended by pipetting only one time, this ensures a larger degree of cell survival – as cells are incredibly delicate at this stage.
?. [iPSCs Aggregate Plating]
Perform triplicate aggregate counts to determine the average number of cell aggregates.
?. [iPSCs Aggregate Plating]
Pipette  into three individual wells of a 96-well flat bottom tissue culture plate.
[pf DMEM/F12]
?. [iPSCs Aggregate Plating]
Pipette  to each well.
[of aggregate suspension]
?. [iPSCs Aggregate Plating]
Manually count the number of aggregates in each well.
Do not count aggregates smaller than 100µm
?. [iPSCs Aggregate Plating]
Calculate the average number of aggregates per well.
Add the number of aggregates per well and then divide by 3 to find the average number of aggregates per well
?. [iPSCs Aggregate Plating]
Next calculate the Concentration of Aggregates or Aggregates/uL.
Take the average number of aggregates per well and divide by 5 (the dilution factor) to obtain the number of aggregates per microliter.
?. [iPSCs Aggregate Plating]
Determine the number of aggregates to plate in a 6-well tissue culture plate.
It is recommended plating 130-160 aggregates per well (optimal for control iPSC lines). This density typically results in 20-38 colonies per well after 24 hours of incubation.
The number of aggregates plated can be adjusted depending on how particular iPSC lines behave.
Plating more aggregates at this stage does not result in more HPCs obtained at the end of the protocol.
?. [iPSCs Aggregate Plating]
Calculating the plating volume of the cell aggregate mixture is accomplished by dividing the number of aggregates to plate by the concentration of the cell aggregates. For example:1. Plate 130 aggregates per well2. Concentration of Cell Aggregates =3 Aggregates/µL3. 130 Aggregates per Well / 3 Aggregates per µL= Plate ~43µL of Aggregate Mixture per Well
?. [iPSCs Aggregate Plating]
After calculating the number of aggregates to plate per well, aspirate Matrigel from previously coated 6-well plate(s) (refer to Step 2), and add  containing 2.5µM Rock Inhibitor to each well.
[of mTesR1]
?. [iPSCs Aggregate Plating]
Gently mix the cell aggregate mixture by inverting 15mL conical tube.
Do not vortex or pipette mixture.
?. [iPSCs Aggregate Plating]
Add the previously calculated volume of aggregate suspension to each well in the 6-well plate.
?. [iPSCs Aggregate Plating]
Place the plate containing mTesR1 and aggregate suspension in incubator at  for .
37 °C
Prior to placing plate in incubator, perform a “t” shaped motion with the plate to ensure that aggregates are distributed evenly throughout the wells.
?. [iPSCs Aggregate Plating]
After 24 hours, carefully aspirate mTesR1 containing 2.5µM Rock Inhibitor from each well.
?. [iPSCs Aggregate Plating]
Gently wash cells with  per well.
[of PBS]
?. [iPSCs Aggregate Plating]
Aspirate PBS.
?. [iPSCs Aggregate Plating]
Add  to each well.
[of pre-warmed fresh mTesR1]
?. [iPSCs Aggregate Plating]
Visualize plate under microscope and manually count the number of adhered aggregates per well.
Aim for at least 20 adhered aggregates per well of a 6-well tissue culture plate
Do not proceed further if  40 aggregates have adhered to 6-well tissue culture plate as differentiation will be compromised
?. [iPSCs Induction into Hematopoietic Stem Cells]
Prepare Medium A per the following recipe:
Medium A can be prepped and stored for a maximum of three days
?. [iPSCs Induction into Hematopoietic Stem Cells]
Prepare Medium B per the following recipe:1. Add Supplement B to Hematopoietic Basal Medium at a concentration of 1:200
Medium B can be prepped and stored for a maximum of three days
?. [iPSCs Induction into Hematopoietic Stem Cells]
Change media on the cell aggregates using the following schedule.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 0 - Aspirate medium from wells and add  per well.
[of Medium A]
Day 0 starts 24 hours after aggregate plating
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 2 - Gently remove  from each well and gently replace with  per well.
[of Medium A]
[of fresh Medium A]
It is best to use a serological pipette or a 1mL micropipette to perform the media removal.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 3 - Aspirate Medium A from wells and gently add  per well.
[of Medium B]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 5 - Gently remove  from each well and gently replace with per well.
[of Medium B]
[of fresh Medium B]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 7 - Gently remove  from each well and gently replace with per well.
[of Medium B]
[of fresh Medium B]
At this point, floating cells can often be seen in culture and they will continue to increase in number for the remainder of the protocol.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Day 10 - Gently remove  from each well and gently replace with per well.
[of Medium B]
[of fresh Medium B]
As the number of floating cells in the culture increases, it is imperative that media changes are done slowly and gently to avoid removing a large number of the floating cells.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Harvesting Cells for FACS Sorting:
?. [iPSCs Induction into Hematopoietic Stem Cells]
Floating and adherent cells should be harvested for FACS sorting on the twelfth day of culture for presence of the following cellular markers:1) CD432) CD343) CD45
?. [iPSCs Induction into Hematopoietic Stem Cells]
Begin harvesting floating cells using a serological pipette or 1mL micropipette, vigorously pipette media and cells up and down approximately 2-3 times in the well to break up floating cell aggregates.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Transfer floating cells and media to appropriately sized conical tube.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Wash well with  and transfer to same collection tube, this will ensure the majority of floating cells have been collected.
[of DMEM/F12]
Be sure that collected cells are kept on ice to avoid cell death.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Centrifuge the collection tube at 300 x g for  at .
4 °C
?. [iPSCs Induction into Hematopoietic Stem Cells]
Aspirate supernatant.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Re-suspend pellet in .
[of sterile FACS Buffer (PBS and 2% FBS)]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Filter the suspension through a 40µm filter into collection tubes.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Centrifuge the collection tube at 300 x g for  at .
4 °C
?. [iPSCs Induction into Hematopoietic Stem Cells]
Aspirate supernatant.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Re-suspend pellet in .
[of sterile FACS Buffer (PBS and 2% FBS)]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Begin harvesting Adherent Cells by first adding  to each well.
[of Accutase]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Incubate at  for.
37 °C
?. [iPSCs Induction into Hematopoietic Stem Cells]
Add  to each well.
[of DMEM/F12]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Triturate vigorously to achieve a single cell suspension.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Transfer cell suspension to appropriately sized collection tube.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Centrifuge the collection tube at 300 x g for  at .
4 °C
?. [iPSCs Induction into Hematopoietic Stem Cells]
Aspirate supernatant.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Re-suspend pellet in .
[of sterile FACS Buffer (PBS and 2% FBS)]
?. [iPSCs Induction into Hematopoietic Stem Cells]
Filter the suspension through a 40µm filter into collection tubes.
?. [iPSCs Induction into Hematopoietic Stem Cells]
Centrifuge the collection tube at 300 x g for  at .
4 °C
?. [iPSCs Induction into Hematopoietic Stem Cells]
Re-suspend pellet in .
[of sterile FACS Buffer (PBS and 2% FBS)]
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
To stain cells for FACS sorting, add the following antibodies to the filtered cell suspension (cells and FACS Buffer) in the noted concentrations:CD34-FITC (1:200) CD43-APC (1:200) CD45 – Alexa Fluor700 (1:200) (optional)CD41-PE (1:200) (optional)
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
Incubate cells and antibodies   in the dark for .
on ice
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
After incubation, add  to each tube and centrifuge at 300 x g for .
[of FACS Buffer]
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
Aspirate supernatant.
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
Re-suspend pellet in .
[of FACS Buffer]
?. [FACS Sorting CD43+ CD34+ CD45+ Cells]
Sort the CD34+ and CD43+cell population using a Becton Dickinson FACSAria II.
Sorting has to be performed in sterile conditions.
In order to obtain high quality HPCs, it is suggested to sort only the CD34+ and CD43+ double positive cell population, discarding the single or double negative cells.
?. [Freezing Down Sorted Hematopoietic Stem Cells]
Centrifuge positively sorted cells at 300 x g for  at .
4 °C
?. [Freezing Down Sorted Hematopoietic Stem Cells]
Aspirate supernatant.
?. [Freezing Down Sorted Hematopoietic Stem Cells]
Re-suspend cells at a concentration of 1 million cells per .
[of Cryostor CS10]
?. [Freezing Down Sorted Hematopoietic Stem Cells]
Aliquot  per cryovial.
[of cell and freezing medium suspension]
?. [Freezing Down Sorted Hematopoietic Stem Cells]
Place cells in  for approximately .
-80 °C
?. [Freezing Down Sorted Hematopoietic Stem Cells]
After 48 hours, cells can be transferred to liquid nitrogen for long-term storage.
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
Using previously sorted cryopreserved cells (Freezing Down Sorted Hematopoietic Stem Cells Section), place frozen vial of cells in  for quick thaw.
[water bath]
Thaw should take less than one minute, remove cells from water bath prior to complete thaw.
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
Transfer contents of cryovial to a conical tube containing .
[of DMEM/F12 supplemented with 2% FBS]
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
Centrifuge concial tube at 300 x g for .
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
Aspirate supernatant.
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
Re-suspend cell pellet in iMGL Diff Complete Medium at a concentration of ~500,000 cells per .
2 mL
?. [Deriving iMGLs - Thawing Hematopoietic Stem Cells]
iMGL Diff Complete Medium Recipe:  ABCDE1VendorCetalog #Dilution2iMGL Diff Base Medium3IL-34 (100 ng/ml)Peprotech200-341:50004TGFb-1 (50 ng/ml)Peprotech100-211:20005M-CSF (25 ng/ml)Peprotech300-251:4000
ABCDE1VendorCetalog #Dilution2iMGL Diff Base Medium3IL-34 (100 ng/ml)Peprotech200-341:50004TGFb-1 (50 ng/ml)Peprotech100-211:20005M-CSF (25 ng/ml)Peprotech300-251:4000
iMGL Diff Base Medium can be kept in storage at 4°C for approximately one month. It is important to make fresh iMGL Diff Complete Medium (addition of fresh cytokines to the base medium) every time you need to feed the culture
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 2 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 4 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 6 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 8 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 10 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
Use extreme caution as plate is nearly full with media.
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 12 - Collect all but  from wells and centrifuge at 300 x g for  at .
[of media]
0 Room temperature
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Aspirate the media and add  per well to re-suspend cell pellet.
[of iMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 14 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 16 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 18 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 20 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 22 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 24 - Add  per well of a 6-well tissue culture plate.
[of IMGL Diff Complete Medium]
Use extreme caution as plate is nearly full with media.
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 25 - Collect all but  from wells and centrifuge at 300 x g for  at .
[of media]
0 Room temperature
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Aspirate the media and add  per well to re-suspend cell pellet.iMGL Maturation Media Recipe:  ABCDE1VendorCatalog #Dilution2iMGL Complete Medium3CD200 (100 ng/ml)100 ug/mlNovoproteinC311-50 ug1:10004Cx3CL1 (100 ng/ml)100 ug/mlPeprotech300-311:1000
[of iMGL Maturation Medium]
ABCDE1VendorCatalog #Dilution2iMGL Complete Medium3CD200 (100 ng/ml)100 ug/mlNovoproteinC311-50 ug1:10004Cx3CL1 (100 ng/ml)100 ug/mlPeprotech300-311:1000
iMGL Diff Base Medium can be kept in storage at 4°C for approximately one month. It is important to make fresh iMGL Diff Complete Medium (addition of fresh cytokines to the base medium) every time you need to feed the culture
?. [Differentiation of Hematopoietic Stem Cells into iMGLs]
Day 28 - Cells should have reached maturity by this step.
Continue feeding cells with iMGL Maturation Media. Mature Microglia-Like Cells can be used for approximately 2-3 weeks.
Mature iMGLs can be also be detached with Accutase (  at  ) and replated on plastic or glass culture ware that has been pre-coated with Matrigel.