Jul 07, 2023
iPSC differentiation into Macrophages
- 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
Protocol Citation: Narayana Yadavalli, Shawn M. Ferguson 2023. iPSC differentiation into Macrophages. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbympovpk/v1
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 25, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82433
Keywords: iPSC differentiation, macrophages, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes iPSC differentiation into macrophages.
Attachments
724-1820.docx
18KB
Guidelines
This protocol is slight modification from the original protocol published.
Shi J, Xue C, Liu W, Zhang H. Differentiation of Human-Induced Pluripotent Stem Cells to Macrophages for Disease Modeling and Functional Genomics. Curr Protoc Stem Cell Biol. 2019;48
This original published protocol involves differentiation of iPSCs into CD34 positive hematopoietic progenitors by embryoid body generation on low attachment plates. Embryoid body generation is laborious, time consuming and costly. This protocol also requires FACS sorting of hematopoietic progenitors.
The CD34 positive progenitors can be differentiated without the embryoid body generation step and FACS sorting by using STEMdiff™ Hematopoietic Kit.
Materials
Reagents required
STEMdiff™ Hematopoietic Kit 1 Kit
STEMCELL Technologies Inc.Catalog #5310 (The kit has 3 components)
- Base media, aliquot and freeze in -20 °C (3 mL and 5 mL aliquots).
- Supplement A (200X), aliquot and freeze in -20 °C , 15 µL aliquots (for 3 mL media).
- Supplement B (200X), aliquot and freeze in -20 °C , 25 µL aliquots (for 5 mL media).
FBS (#16140-071) MSR
Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135
Recombinant Human M-CSFpeprotechCatalog #300-25
Note
Received M-CSF is a lyophilized powder, spin down the vial and resuspend in 100 µL of sterile water followed by 900 µL of 0.1% BSA (BSA solution is prepared in sterile PBS and filter sterilized).
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-freeCorningCatalog #356230
Note
iPSCs-notes: A18945 Gibco iPSCs are the best for this differentiation, WTC iPSCs also differentiation but with low efficiency compared to A18945 iPSCs. NGN WTC iPSCs efficiency is much lesser. I do not know exact reason why this difference between these cell lines.
- 6 well plate
- 96 well plate
iPSC differentiation into Macrophages
iPSC differentiation into Macrophages
11m
11m
Note
The differentiation protocol involves two steps.
1. iPSC differentiation into CD34 positive hematopoietic progenitors
2. Hematopoietic progenitors’ differentiation into mature Macrophages
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.
Then add 1 mL of 0.5 micromolar (µM) EDTA to the well and leave in the incubator for about 00:05:00 . 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
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.
Now gently swirl the plate to mix EDTA solution containing cells and E8+Ri media. (Never pipet, this mechanical force will disrupt the clumps).
After swirling the plate pipet 1 mL of the cells with 5 ml pipet into 15 ml falcon tube.
Spin down the cells at 1 rcf, 00:03:00 .
Note
This slow spin is required for avoiding single cells smaller clumps.
3m
After this spin, take aspirate the supernatant and gently tap the cell pellet.
Then add 3 mL of E8+Ri media and tap gently once again to mix the clumps.
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.
Now bring the Matrigel coated plate, aspirate the Matrigel and add 1.5 mL of E8+Ri media (3 well were coated on day 0).
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.
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:
- 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.
- 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 (2 mL base media + 10 µL supplement A).
Aspirate E8+Ri media and add 2 mL Media A and leave plate in incubator for 2 days.
Day 2
Add 1 mL A (1 mL base media + 5 µL supplement A).
Day 3
Prepare Media B (2 mL base media + 10 µL supplement B).
Aspirate media A and add 2 mL Media B.
Day5, 7,9,10
supplement the cell with 1 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 3 rcf, 00:03:00 .
3m
Remove the supernatant and resuspend the cell pellet in Macrophage differentiation media. (RPMI+ 20% FBS+100ng/ML M-CSF).
Count the cell by using hemocytometer and plate 100,000 in one well of 6 well plate.
Day 15 and 18
Supplement with 2 mL of Macrophage differentiation media.
Day 19
you will have mature IBA1 positive macrophages ready for the experiments.