TITLE: Differentiation of iPSC into Microglia-Like Cells (iMGL)
AUTHORS: Abhirami Kannan  Iyer, Emma  Danhash, Fabia  Filipello, Jacob  Marsh, Rj  Martinez, Celeste M M. Karch

[DESCRIPTION]
This protocol outlines the derivation of Hematopoietic Progenitor Cells and differentiation of iMGLs using iPSC cultures. This protocol is modified the following papers.
 
 
 


[BEFORE_START]
Derivation of Hematopoietic Progenitor Cells and Differentiation of iMGLs – Timeline
iPSCs Culture (2-3 Days)
iPSCs Aggregates Plating (1 Day) Critical: Go/No-Go Decision 
iPSCs Induction into Hematopoietic Stem Cells (12 Days) Critical: Go/No-Go Decision 
FACs Sorting CD43+CD34+ CD45+ Cells (1 Day)
Freezing Down Sorted Hematopoietic Stem Cells (1 Day)
Thawing Hematopoietic Stem Cells (1 Day)
Differentiation of Hematopoietic Stem Cells into Induced Microglia (28 Days)

[STEPS]
SECTION: iPSCs Culture
1. Thaw and culture iPSC line per the following protocol:
 

SECTION: iPSCs Aggregate Plating
2. Once iPSCs are 70-80% confluent in 2-3 wells of a 6-well tissue culture plate, passage and plate the iPSCs as aggregates
 

SECTION: iPSCs Aggregate Plating
3. Coat a 6-well tissue culture plate with Matrigel for a least 60 min prior to passaging cells



SECTION: iPSCs Aggregate Plating
5. Set the following media out to warm to Room temperature:
ReLeSR
DMEM/F12
PBS
mTesR1

SECTION: iPSCs Aggregate Plating
6. After plate has been coated for 60 min and media has warmed to Room temperature, proceed to passage aggregates as described below:

SECTION: iPSCs Aggregate Plating
6.1. Aspirate media from well.
SECTION: iPSCs Aggregate Plating
6.2. Wash cells with 2 mL per well

SECTION: iPSCs Aggregate Plating
6.3. Aspirate PBS from well.
SECTION: iPSCs Aggregate Plating
6.4. Add ReLeSR to cells 1 mL.
SECTION: iPSCs Aggregate Plating
6.5. 
Incubate at 37 Room temperature for between 1 min and 90 s.



SECTION: iPSCs Aggregate Plating
6.6. Aspirate the ReLeSR from the wells using a p1000 micropipette and allow cells to continue sitting without any reagent in the wells for 5 min to 7 min. 

 


SECTION: iPSCs Aggregate Plating
6.7. Add 1mL per well of mTesR1 + 5-10µM by gently allowing to trickle down the wall of the well. Tap the plate to release lifted cells to form aggregates of various sizes. Following this, use a p1000 to transfer 1mL of aggregates from each well to a 15mL conical tube without pipetting up and down 
 


SECTION: iPSCs Aggregate Plating
7. Perform triplicate aggregate counts to determine the average number of cell aggregates.
SECTION: iPSCs Aggregate Plating
7.1. Pipette 100 µL into three individual wells of a 96-well flat bottom tissue culture plate.

SECTION: iPSCs Aggregate Plating
7.2. Pipette 5 µL to each well.

SECTION: iPSCs Aggregate Plating
7.3. Manually count the number of aggregates in each well.
 

SECTION: iPSCs Aggregate Plating
7.4. Calculate the average number of aggregates per well.
 

SECTION: iPSCs Aggregate Plating
7.5. Next calculate the Concentration of Aggregates or Aggregates/uL.
 

SECTION: iPSCs Aggregate Plating
8. Determine the number of aggregates to plate in a 12-well or 6-well tissue culture plate.
 

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
11. Prepare Medium A per the following recipe:

Add Supplement A to Hematopoietic Basal Medium at a concentration of 1:200

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
12. Prepare Medium B per the following recipe:

1. Add Supplement B to Hematopoietic Basal Medium at a concentration of 1:200

 

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
13. Change media on the cell aggregates using the following schedule for a 6 well tissue culture plate.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.1. Day 0 - Aspirate medium from wells and add 2 mL per well and incubate at 37°C, 6% CO2.

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.2. Day 2 - Gently add 1 mL to each well and incubate at 37°C, 6% CO2 


SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.3. Day 3 - Aspirate Medium A from wells and gently add 2 mL per well.

SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.4. Day 5 - Gently add 1 mL to each well and incubate at 37°C, 6% CO2 
SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.5. Day 7 - Gently add 1 mL to each well and incubate at 37°C, 6% CO2
 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
13.6. Day 10 - Gently add 1 mL to each well and incubate at 37°C, 6% CO2
 

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14. Harvesting Cells for FACS Sorting:
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.2. Floating and adherent cells should be harvested for FACS sorting on the twelfth day of culture for presence of the following cellular markers:
1) CD43
2) CD34
3) CD45
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.3. 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.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.4. Transfer floating cells and media to appropriately sized conical tube.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.5. Wash well with 1 mL, triturate, and transfer to same collection tube, this will ensure the majority of floating cells have been collected. Repeat at least one more time. 



SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.6. Centrifuge the collection tube at 300 x g for 5 min at 4 Room temperature.

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.7. Aspirate supernatant.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.8. Re-suspend pellet in 300 µL and keep on ice.

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.9. Filter the suspension through a 40µm filter before antibody staining or transfer cells into Filter FACS Tubes (Falcon 352235) 
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.10. Begin harvesting Adherent Cells by first washing the well with 1 mL and discarding the wash

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.12. Incubate at 37 °C for20 min.

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.14. Transfer the single-cell suspension to a collection tube containing 2 mL 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.13. Triturate vigorously with a 1mL pipette tip to dislodge the adherent cells and create a single-cell suspension. Do not scrape residual colonies from the tissue culture plate surface, as these clumps will not further dissociate. 
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.15. Wash the well with an additional 1mL of DMEM/F12. Add wash to the collection tube. Repeat. 
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.16. Centrifuge the collection tube at 300 x g for 5 min at 4 Room temperature.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.17. Aspirate supernatant.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.18. Re-suspend pellet in 300 µL and keep on ice.
SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.19. Filter the suspension through a 40µm filter before antibody staining or transfer cells into Filter FACS Tubes (Falcon 352235) 
SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
15. 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)
CD41-PE (1:200) (optional)
SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
16. Incubate cells and antibodies on ice  in the dark for 20 min.

SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
17. After incubation, add 2 mL to each tube and centrifuge at 300 x g for 5 min.

SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
18. Aspirate supernatant.
SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
19. Re-suspend pellet in 500 µL.
SECTION: FACS Sorting CD43+ CD34+ CD45+ Cells
20. Sort the CD34+ and CD43+cell population using a Becton Dickinson FACSAria II and collect the selected population in sterile tubes.
 

 

 

SECTION: Freezing Down Sorted Hematopoietic Stem Cells
21. Centrifuge positively sorted cells at 300 x g for 10 min at 4 °C.
SECTION: Freezing Down Sorted Hematopoietic Stem Cells
22. Aspirate supernatant.
SECTION: Freezing Down Sorted Hematopoietic Stem Cells
23. Re-suspend cells at a concentration of 1 million cells per 1 mL.

SECTION: Freezing Down Sorted Hematopoietic Stem Cells
24. Aliquot 1 mL per cryovial.

SECTION: Freezing Down Sorted Hematopoietic Stem Cells
25. Place cells in -80 °C for approximately 1440 min.

SECTION: Freezing Down Sorted Hematopoietic Stem Cells
26. After 24 hours, cells must be transferred to liquid nitrogen for long-term storage.
SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
27. Using previously sorted cryopreserved cells (Freezing Down Sorted Hematopoietic Stem Cells Section), place frozen vial of cells in 37 °C for quick thaw.
 


SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
28. Transfer contents of cryovial to a conical tube containing 8 mL.

SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
29. Centrifuge concial tube at 300 x g for 5 min.
SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
30. Aspirate supernatant.
SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
31. Re-suspend cell pellet in iMGL Diff Complete Medium at a concentration of ~500,000 cells per well of a 6 well tissue culture plate 


SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
31.2. iMGL Diff Complete Medium Recipe:
 

 

 

 

 

 

 




SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
32. Day 2 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
33. Day 4 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
34. Day 6 - Split and add 1 mL + 2X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
35. Day 8 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
36. Day 10 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
 

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
37. Day 12 - Split and add 1 mL + 2X Cytokines per well of a 6-well tissue culture plate (Refer to Step 34)

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
38. Day 14 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
39. Day 16 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
40. Day 18 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
41. Day 20 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
42. Day 22 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
43. Day 24 - Add 1 mL + 1X Cytokines per well of a 6-well tissue culture plate.
 

 

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
44. Split and add 1 mL + 2X Cytokines per well of a 6-well tissue culture plate (Refer to Step 34) 

iMGL Maturation Media Recipe:


 


 

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
45. Day 28 - Cells should have reached maturity by this step and are ready for experimental use.
 


SECTION: iPSCs Aggregate Plating
4. Prepare desired volume of mTesR1 and 5-10µM ROCK Inhibitor (ROCKi = 1:2000 or 1:1000). After 1 hour of Matrigel coating, aspirate and replace with 2mL per well mTesR1 + Desired Concentration of ROCK Inhibitor. Pre-warm plates with media at 37°C and 6% CO2 until aggregates are ready to be plated. 
SECTION: iPSCs Aggregate Plating
9. Place the plate in a 37°C, 6% CO2 incubator. Move the plate in several quick, short, back-and-forth, and side-to-side motions to distribute the cell aggregates. Do not disturb the plate for 24 hours.

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
10. After 24 hours, confirm that 20- 38 colonies/well (6-well plate) or 16 - 40 colonies/well (p12 well-plate) are adhered to the plate. Ensure to count all colonies, including tiny colonies with only a few cells.

 

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.1. Pre-coat a 6 or 12 well tissue culture plate with Matrigel 60 min prior to harvesting cells for FACS Sorting

 

 

SECTION: iPSCs Induction into Hematopoietic Stem Cells
14.11. Add 1 mL to each well.
SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
34.1. Collect all but 1mL of media from each well and transfer to a 50mL conical tube. Spin this supernatant media at 300 xg for 6 min at 37 Room temperature 

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
34.2. While above centrifugation is ongoing, split 3 existing wells (N) of iMGLs into 1 new well (N')

 

SECTION: Differentiation of Hematopoietic Stem Cells into iMGLs
34.3. After spin step, aspirate supernatant after freezing an aliquot ( ~500 µL) for future ELISA experiments and resuspend cells in (N+N’) mL of iMGL differentiation complete medium + 2X cytokines and evenly distribute 1ml/well to N+N’ wells (this means you continue the culture in existing wells in addition to expansion into new wells with every splitting performed)
SECTION: Deriving iMGLs - Thawing Hematopoietic Stem Cells
31.1. iMGL Differentiation Basal Medium (per 500 mL)