TITLE: Microglia differentiation
AUTHORS: Riana Lo Bu, Frank Soldner

[DESCRIPTION]
This protocol has been refined to differentiate microglia cells from hESC adapted to feeder free culture systems as described in the following protocol:
 
Protocol Overview

A. Flask Preparation
B. Poly-D-Lysine (PDL) plate coating
C. Media recipes
D. Cell passage and differentiation
E. Microglia precursor isolation and platting for maturation

General Notes

A list of reagents and relevant vendor information can be found in the table listed under the materials tab.


[STEPS]
SECTION: Flask preparation
1. Thaw matrigel   on ice inside a 4 °C fridge. Chill all flasks, pipettes and materials to be used for flask preparation under the same conditions and keep everything as cold as possible throughout the procedure (do not use a freezer as it can cause the matrigel solution to freeze while platting).
SECTION: Flask preparation
2. Dilute matrigel in cold DMEM/F12 as described by the manufacturer and add into the T75 flasks (~6 mL/flask total volume). Incubate at 37 °C for at least 60 min before use. Flasks can be stored in the incubator for several weeks before use.

SECTION: Poly-D-Lysine (PDL) plate coating
3. Prepare PDL (0.5 µg/ml) in sterile water and add it to each well and incubate 360 min to   at 37 °C. The day after, wash each well 4 times with sterile water to remove excess PDL (at this point the plates are ready to be used for culture - do not let the wells dry at any step as it can cause PDL crystallization (cytotoxic)).

SECTION: Media preparation
4. The below media recipes are used in the remainder of the protocol:
SECTION: Media preparation
4.1. STEP1 media: mTeSR media + Penicillin-Streptomycin (100U/ml)+ 80ng/ml BMP4

SECTION: Media preparation
4.2. STEP2 media: StemPro-34 SFM media + 2 mM GlutaMAX + 80 ng/ml VEGF, 25 ng/ml FGF + 100 ng/ml SCF
SECTION: Media preparation
4.3. STEP3 media: StemPro-34 SFM media + 50 ng/ml SCF + 50 ng/ml IL-3 + 5 ng/ml TPO + 50 ng/ul M-CSF + 50 ng/ul Flt3
SECTION: Media preparation
4.4. STEP4 media: StemPro-34 SFM media + 50 ng/ml SCF + 50 ng/ml IL-3 + 5 ng/ml TPO + 50 ng/ul M-CSF + 50 ng/ul Flt3
4.5. Microglia maturation media: Neurobasal media + N2 Neuroplex (1x final concentration) + GEM21 Neuroplex (1x final concentration) + 20% AlbuMAX I (0.2% final concentration) + NaCl (5M) (50mM final concentration) + sodium pyruvate 100x (1x final concentration) + glutaMAX 100x (1x final concentration) + Penicillin-Streptomycin (100U/ml) + 50 ng/ml TGF-β1 + 100 ng/ml  IL-34 + 12.5 ng/ml  M-CSF

SECTION: Cell passage and differentiation
5. 
 

 

Manually dissect undifferentiated hPSC colonies and transfer 40-60 small aggregates into a matrigel coated flask.
SECTION: Cell passage and differentiation
6. Maintain undifferentiated cells in mTeSR media until undifferentiated colonies reach an average colony size of 1 mm. Usually, 7 mL of media/flask and media change every other day works well to maintain the cells until they reach the right size. 

SECTION: Cell passage and differentiation
7. STEP1 (days 0-3): Once the hPSC colonies reach the correct size, microglia differentiation is initiated (day 0) by culturing the undifferentiated cells in 6 mLof STEP1 media. STEP1 media should be replaced with fresh media on day 2.

SECTION: Cell passage and differentiation
8. STEP2 (days 4-5): Culture cells in 6 mL of STEP2 media. No media change until the next step.

SECTION: Cell passage and differentiation
9. STEP3 (days 6-13): Culture cells in 6 mL of STEP3 media. Media should be changed on day 10.

SECTION: Cell passage and differentiation
10. STEP4 (days 14-28): Culture cells in 6 mL of STEP4 media. Media should be replaced every 5760 min.


SECTION: Microglia precursor isolation and platting for maturation
11. 
 

 

 

 

Once microglia precursor differentiation is validated on day 28 or later, microglia progenitors are collected from STEP4 flasks by transferring the supernatant to a conical tube followed by   centrifugation at 150 rcf for 4 min. The cell pellet is resuspended in  microglia maturation media and plated at 75k cells/cm2 (Primaria or PDL-coated plates) in microglia maturation media. Media should be changed every 4 days for at least 2 weeks to allow the cells to mature.