Aug 04, 2023

Public workspaceiPSC differentiation into Microglia

DOI
dx.doi.org/10.17504/protocols.io.261ge3qpwl47/v1
  • Narayana Yadavalli1,2,3,4,5,
  • Shawn M. Ferguson1,2,3,4,6,5
  • 1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 3Program in Cellular Neuroscience, Neurodegeneration and Repair;
  • 4Wu Tsai Institute Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 6Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Berrak Ugur
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DOI: dx.doi.org/10.17504/protocols.io.261ge3qpwl47/v1
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. iPSC differentiation into Microglia. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3qpwl47/v1
Manuscript citation:


License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 24, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82378
Keywords: iPSC differentiation, microglia, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes iPSC differentiation into microglia.
Attachments
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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
Materials
Reagents required

ReagentSTEMdiff™ Hematopoietic Kit 1 Kit STEMCELL Technologies Inc.Catalog #5310

ReagentMatrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-freeCorningCatalog #356230

ReagentGibco™ DMEM/F-12 HEPESFisher ScientificCatalog #11-330-032

ReagentMEAA (MEM Non-Essential Amino Acids)Gibco - Thermo FischerCatalog #11140050

Glutamax (Gibco, )

ReagentGibco™ N-2 Supplement (100X)Thermo Fisher ScientificCatalog #17502048

ReagentGibco™ B-27™ Supplement (50X) serum freeFisher ScientificCatalog #17-504-044

ReagentRecombinant Human M-CSFpeprotechCatalog #300-25

ReagentRecombinant Human TGF-β1 (HEK293 derived)peprotechCatalog #100-21

ReagentRecombinant Human IL-34peprotechCatalog #200-34

ReagentRecombinant Human Fractalkine (CX3CL1)peprotechCatalog #300-31

CD200 (Novo protein #C311)

Reagent1-ThioglycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M6145

ReagentInsulin-Transferrin-Selenium (ITS -G) (100X)Thermo FisherCatalog #41400045

ReagentInsulinMerck MilliporeSigma (Sigma-Aldrich)Catalog #I2643-25MG

Base media:
  • DMEM/ F12
  • 2X insulin-transferrin-selenite
  • 2X B27, 0.5X N2
  • 1X glutamax
  • 1X non-essential amino acids
  • 400μM monothioglycerol
  • 5 μg/mL insulin

3 cytokine media:
Base media
mCSF 25 ng/ml
IL34  100 ng/ml
TGFbeta50 ng/ml
5 Cytokine media:
Base media 
mCSF 25 ng/ml
IL34 100 ng/ml
TGFbeta  50 ng/ml
CX3CL1 100 ng/ml
CD200 100 ng/ml
Steps for iPSC differentiation into CD34 positive hematopoietic progenitors
Steps for iPSC differentiation into CD34 positive hematopoietic progenitors

Note
Note: This differentiation protocol requires healthy iPSCs approximately 50-60% confluency with nice tight colonies.
Day-2: Coat 3 wells of a 6 well pates with Matrigel. (1 hour coating also works)
Day-1:
Bring iPSC maintenance to plate cell culture hood, remove the media and rinse once with PBS.
Wash
Then add Amount1 mL of Concentration0.5 micromolar (µM) EDTA to the well and leave in the incubator for about Duration00:05:00 . By this time, you will see colonies lifting from the plates. If not leave plate in the incubator for few more minutes.
Note
Note: Do not tap or pipet to lift off the cells from the plate. This protocol requires a clumps off iPSC colonies for good yield of hematopoietic progenitors.

5m
Incubation
Pipetting
Once the 50 to 60% of colonies come off the plate, bring plate into the hood and neutralize the reaction by adding Amount2 mL of E8+Ri media.
Pipetting
Now gently swirl the plate to mix EDTA solution containing cells and E8+Ri media.
Note
Never pipet, this mechanical force will disrupt the clumps.

After swirling the plate pipet Amount1 mL of the cells with 5 ml pipet into 15 ml falcon tube.
Pipetting
Spin down the cells at Centrifigation1 rcf, 00:03:00 .
Note
This slow spin is required for avoiding single cells smaller clumps.

3m
Centrifigation
After this spin, take aspirate the supernatant and gently tap the cell pellet.
Then add Amount3 mL of E8+Ri media and tap gently once again to mix the clumps.
Pipetting
For counting the clump number take Amount5 µL of this mix into a 96 well plate, add Amount100 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.
Pipetting
Imaging
Now bring the Matrigel coated plate, aspirate the Matrigel and add Amount1.5 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.
Note
*Since counting of clumps is arbitrary, it is never perfect. This exercise is essential till you get very good experience in plating the clumps.

Pipetting
Day 0
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.
Note
Note:
  1. Each clump should have 20-40 cells. If you have smaller colonies change media to E8 only and let the colonies grow for 1-2 days.
  2. Above 30 clumps number differentiation may not work because mesodermal cells require space to migrate and differentiate into hematopoietic progenitors.

Choose on well with desired number of colonies and you can discontinue maintaining remaining wells.
After achieving desired colony number prepare Media A (Amount2 mL base media + Amount10 µL supplement A).
Aspirate E8+Ri media and add Amount2 mL Media A and leave plate in incubator for Duration48:00:00 .

2d
Incubation
Pipetting
Day 2
Add Amount1 mL A (Amount1 mL base media + Amount5 µL supplement A).
Pipetting
Day 3
Prepare Media B (Amount2 mL base media + Amount10 µL supplement B).

Aspirate media A and add Amount2 mL Media B.
Pipetting
Day 5, 7,9,10
supplement the cell with Amount1 mL of Media B.
Day 12: Collection
By day 12 you will see lot of floating hematopoietic progenitor cells.
Collect hematopoietic progenitor cells by gently swirling the plate with a 5 ml pipet into 15 ml falcon tube.
Spin down the cells Centrifigation3 rcf, 00:03:00 .
3m
Centrifigation
Remove the supernatant and resuspend the cell pellet in Macrophage differentiation media. (RPMI+ 20% FBS+Amount100 ng/mL M-CSF).
Count the cell by using hemocytometer and plate 100,000 in one well of 6 well plate.
Steps for Microglia differentiation from hematopoietic progenitors
Steps for Microglia differentiation from hematopoietic progenitors
6m
6m
Day 12:
Plate 100,000 hematopoietic progenitor on Matrigel coated 6 well plate in 3 cytokine media.
On days 14,16,18,20 and 22 supplement with Amount1 mL of 3 cytokine media.

Day 24
Collect Amount6 mL of cells + media into 15 ml falcon by leaving Amount1 mL conditioned media in the plate. Spin down at Centrifigation3 rcf, 00:03:00 and remove the supernatant.
3m
Centrifigation
Resuspend the pellet in Amount2 mL of fresh 3 cytokine media and plate back into the same well containing conditioned media.
Day 26,28,30,32,34,36 supplement with Amount1 mL of 3 cytokine media.
Day 37
Collect Amount6 mL of cells + media into 15 ml falcon by leaving 1ml conditioned media in the plate.
Spin down at Centrifigation3 rcf, 00:03:00 and remove the supernatant.
3m
Centrifigation
Resuspend the pellet in Amount2 mL of fresh 5 cytokine media and plate back into the same well containing conditioned media.

Day 39: supplement cells with Amount1 mL of 5 cytokine media.
Day 41: Collect cells for experiment.