TITLE: iPSC differentiation into Microglia
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes iPSC differentiation into microglia.

[GUIDELINES]
This protocol is adapted from the below article.
McQuade A, Coburn M, Tu CH, Hasselmann J, Davtyan H, Blurton-Jones M. Development and validation of a simplified method to generate human microglia from pluripotent stem cells. Mol Neurodegener. 2018;13(1):67.

The differentiation protocol involves two steps. 
1. iPSC differentiation into CD34 positive hematopoietic progenitors 
2. Hematopoietic progenitors’ differentiation into mature Microglia

[STEPS]
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
8. Day 12:
Plate 100,000 hematopoietic progenitor on Matrigel coated 6 well plate in 3 cytokine media.
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9. On days 14,16,18,20 and 22 supplement with 1 mL of 3 cytokine media.

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1. 
 
Day-2: Coat 3 wells of a 6 well pates with Matrigel. (1 hour coating also works)
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2. Day-1: 
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.1. Bring iPSC maintenance to plate cell culture hood, remove the media and rinse once with PBS.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.2. Then add 1 mL of 0.5 micromolar (µM) EDTA to the well and leave in the incubator for about 5 min. By this time, you will see colonies lifting from the plates. If not leave plate in the incubator for few more minutes. 
 

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.3. Once the 50 to 60% of colonies come off the plate, bring plate into the hood and neutralize the reaction by adding 2 mL of E8+Ri media.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.4. Now gently swirl the plate to mix EDTA solution containing cells and E8+Ri media.
 

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.5. After swirling the plate pipet 1 mL of the cells with 5 ml pipet into 15 ml falcon tube.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.6. Spin down the cells at 1 rcf, 3 min. 
 

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.7. After this spin, take aspirate the supernatant and gently tap the cell pellet.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.8. Then add 3 mL of E8+Ri media and tap gently once again to mix the clumps.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.9. For counting the clump number take 5 µL of this mix into a 96 well plate, add 100 µL of E8 media. Gently tap the plate and count the colonies under the microscope. Repeat this in 3 wells of 96 well plate and average the clump number.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
2.10. Now bring the Matrigel coated plate, aspirate the Matrigel and add 1.5 mL of E8+Ri media. (3 well were coated on day0) Now plate 10 clumps in 1st well, 20 clumps in 2nd well and 30 clumps in 3rd well of the 6 well plate.*  Shake the plate up and down and to sides, return the plate to the incubator. 
 

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
3. Day 0
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
3.1. Count the colonies in in each well, ideal colony number is between 10-30. It doesn’t have to be perfect number. Wells with as low as 5 colonies and max up to 30 can also be used. Anything above 40 should be avoided. 
 

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
3.2. Choose on well with desired number of colonies and you can discontinue maintaining remaining wells.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
3.3. After achieving desired colony number prepare Media A (2 mL base media + 10 µL supplement A).
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
3.4. Aspirate E8+Ri media and add 2 mL Media A and leave plate in incubator for 2880 min.

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
4. Day 2
Add 1 mL A (1 mL base media + 5 µL supplement A).
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
5. Day 3
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5.1. Prepare Media B (2 mL base media + 10 µL supplement B).

SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
5.2. Aspirate media A and add 2 mL Media B.
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6. Day 5, 7,9,10
supplement the cell with 1 mL of Media B.
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7. Day 12: Collection
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
7.1. By day 12 you will see lot of floating hematopoietic progenitor cells.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
7.2. Collect hematopoietic progenitor cells by gently swirling the plate with a 5 ml pipet into 15 ml falcon tube.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
7.3. Spin down the cells 3 rcf, 3 min.
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
7.4. Remove the supernatant and resuspend the cell pellet in Macrophage differentiation media. (RPMI+ 20% FBS+100 ng/mL M-CSF).
SECTION: Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
7.5. Count the cell by using hemocytometer and plate 100,000 in one well of 6 well plate.
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
10. Day 24
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
10.1. Collect 6 mL of cells + media into 15 ml falcon by leaving 1 mL conditioned media in the plate. Spin down at 3 rcf, 3 min and remove the supernatant.
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
10.2. Resuspend the pellet in 2 mL of fresh 3 cytokine media and plate back into the same well containing conditioned media. 
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11. Day 26,28,30,32,34,36 supplement with 1 mL of 3 cytokine media.
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
12. Day 37
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12.1. Collect 6 mL of cells + media into 15 ml falcon by leaving 1ml conditioned media in the plate.
Spin down at 3 rcf, 3 min and remove the supernatant.
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12.2. Resuspend the pellet in 2 mL of fresh 5 cytokine media and plate back into the same well containing conditioned media.

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13. Day 39: supplement cells with 1 mL of 5 cytokine media.
SECTION: Steps for Microglia differentiation from hematopoietic progenitors
14. Day 41: Collect cells for experiment.