Differentiation of iPSC into Microglia-Like Cells (iMGL)

Celeste M M. Karch, Jacob Marsh, Rj Martinez, Abhirami Kannan Iyer, Emma Danhash, Fabia Filipello

Published: 2022-06-28 DOI: 10.17504/protocols.io.q26g7bwqklwz/v3

hematopoietic progenitor cells

Abstract

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

Citation

McQuade A, Coburn M, Tu CH, Hasselmann J, Davtyan H, Blurton-Jones M 2018 Development and validation of a simplified method to generate human microglia from pluripotent stem cells. Molecular neurodegeneration https://doi.org/10.1186/s13024-018-0297-x

Citation

Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CHH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M 2017 iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron https://doi.org/10.1016/j.neuron.2017.03.042

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)

Attachments

Differentiation_of_iPSC_into_Microglia_Protocol_JAM_FINAL_03302020[2].docx

Steps

iPSCs Culture

Thaw and culture iPSC line per the following protocol:

iPSC Cell Culture – Maintenance and Expansion

iPSCs Aggregate Plating

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

Note

Aggregates should be approximately 100-200µm in diameter

Coat a 6-well tissue culture plate with Matrigel for a least 1h 0m 0s prior to passaging cells

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% CO₂until aggregates are ready to be plated.

Set the following media out to warm to Room temperature:

ReLeSR
DMEM/F12
PBS
mTesR1

After plate has been coated for 1h 0m 0s and media has warmed to Room temperature, proceed to passage aggregates as described below:

6.1.

Aspirate media from well.

6.2.

Wash cells with 2mL per well

6.3.

Aspirate PBS from well.

6.4.

Add ReLeSR to cells 1mL.

6.5.

Incubate at 37Room temperature for between 0h 1m 0s and 0h 1m 30s.

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 0h 5m 0s to 0h 7m 0s.

Note

At approximately 4.5 minutes, iPSC appear to be lifted from the base of the wells

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

Note

The amount of iPSC aggregates released after ReLeSR treatment from one well of a 6-well plate is sufficient to perform aggregate plating in 1, 6-well plate or more depending on aggregate count. You may also notice some unattached aggregates after tapping, you can repeat this process and harvest another round, if required.

Perform triplicate aggregate counts to determine the average number of cell aggregates.

7.1.

Pipette 100µL into three individual wells of a 96-well flat bottom tissue culture plate.

7.2.

Pipette 5µL to each well.

7.3.

Manually count the number of aggregates in each well.

Note

A uniform suspension of aggregates (50-200µm size) is optimal. Do not count aggregates smaller than 100µm

7.4.

Calculate the average number of aggregates per well.

Note

Add the number of aggregates per well and then divide by 3 to find the average number of aggregates per well

7.5.

Next calculate the Concentration of Aggregates or Aggregates/uL.

Note

Take the average number of aggregates per well and divide by 5 (the dilution factor) to obtain the number of aggregates per microliter.

Determine the number of aggregates to plate in a 12-well or 6-well tissue culture plate.

Note

For a 12 well tissue culture plate it is recommended to plate 40 - 60 aggregates/well (10 aggregates/cm²) to achieve 16 colonies/well (4 - 10 colonies/cm²) adhered to the culture ware after 24 hours of incubation; however, multiple plating densities may need to be tested.

Note

For a 6 well tissue culture plate it is recommended to plate 40-60 aggregates/well to 6-well plates with mTESR1 + ROCKi media as prepared in Step 4. Multiple plating densities may need to be tested for each donor-derived iPSC line Prior to plating tap the conical tube once or twice to dislodge the pelleted aggregates (earlier wells may have bigger aggregates than later ones).

Place the plate in a 37°C, 6% CO₂incubator. 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.

Note

Usually the aggregates attach in about 6 hours, we have also performed aggregate plating early in the morning and after confirmation of attached aggregates, switched to Medium A on the same day in the evening

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.

Note

To facilitate counting, aspirate medium, wash with PBS and replace with fresh mTeSR™1.

Note

CRITICAL: Do not proceed if cultures have < 16 colonies or > 40 colonies per well, as differentiation will be compromised

11.

Prepare Medium A per the following recipe:

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

Note

Medium A can be prepped and stored for a maximum of three days

12.

Prepare Medium B per the following recipe:

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

Note

Medium B can be prepped and stored for a maximum of three days

Note

Medium A and Medium B can be prepared by adding Supplement A or B into STEMdiff Hematopoietic Basal Medium and stored frozen as 50mL aliquots in -20C until use

13.

Change media on the cell aggregates using the following schedule for a 6 well tissue culture plate.

13.1.

Day 0 - Aspirate medium from wells and add 2mL per well and incubate at 37°C, 6% CO₂.

Note

Day 0 starts 24 hours after aggregate plating

13.2.

Day 2 - Gently add 1mL to each well and incubate at 37°C, 6% CO₂

13.3.

Day 3 - Aspirate Medium A from wells and gently add 2mL per well.

13.4.

Day 5 - Gently add 1mL to each well and incubate at 37°C, 6% CO₂

13.5.

Day 7 - Gently add 1mL to each well and incubate at 37°C, 6% CO₂

Note

At this point, floating cells can often be seen in culture and they will continue to increase in number for the remainder of the protocol.

13.6.

Day 10 - Gently add 1mL to each well and incubate at 37°C, 6% CO₂

Note

If desired, cells may be harvested now as described for Day 12. The cell yield and proportion of CD34+CD45+ cells will be much lower at Day 10 than at Day 12.

Note

Between Days 7 and 10 add 1mL of Medium B to each well if media changes color to orange-yellowish

14.

Harvesting Cells for FACS Sorting:

14.1.

Pre-coat a 6 or 12 well tissue culture plate with Matrigel 1h 0m 0s prior to harvesting cells for FACS Sorting

Note

Harvest both the floating cells (>90% of these are CD43+ HPCs) and the adherent cells (10-70% are CD43+ HPCs). Recommend for every step to be done in sterile conditions.

Note

Keep HPCs cold on ice or at 4°C throughout the process of harvesting, staining for flow-sorting, collection after sorting and until ready to freeze them down if not proceeding with microglia differentiation immediately

14.10.

Begin harvesting Adherent Cells by first washing the well with 1mL and discarding the wash

14.11.

Add 1mL to each well.

14.12.

Incubate at 37°C for0h 20m 0s.

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.

14.14.

Transfer the single-cell suspension to a collection tube containing 2mL

14.15.

Wash the well with an additional 1mL of DMEM/F12. Add wash to the collection tube. Repeat.

14.16.

Centrifuge the collection tube at 300 x g for 0h 5m 0s at 4Room temperature.

14.17.

Aspirate supernatant.

14.18.

Re-suspend pellet in 300µL and keep on ice.

14.19.

Filter the suspension through a 40µm filter before antibody staining or transfer cells into Filter FACS Tubes (Falcon 352235)

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:

CD43
CD34
CD45

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.

14.4.

Transfer floating cells and media to appropriately sized conical tube.

14.5.

Wash well with 1mL, triturate, and transfer to same collection tube, this will ensure the majority of floating cells have been collected. Repeat at least one more time.

14.6.

Centrifuge the collection tube at 300 x g for 0h 5m 0s at 4Room temperature.

14.7.

Aspirate supernatant.

14.8.

Re-suspend pellet in 300µL and keep on ice.

Note

If smaller cell pellet, resuspend in lower volume. If larger cell pellet, resuspend in larger volume

14.9.

Filter the suspension through a 40µm filter before antibody staining or transfer cells into Filter FACS Tubes (Falcon 352235)

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 )

16.

Incubate cells and antibodies On ice in the dark for 0h 20m 0s.

17.

After incubation, add 2mL to each tube and centrifuge at 300 x g for 0h 5m 0s.

18.

Aspirate supernatant.

19.

Re-suspend pellet in 500µL.

20.

Sort the CD34⁺ and CD43⁺cell population using a Becton Dickinson FACSAria II and collect the selected population in sterile tubes.

Note

Sorting has to be performed in sterile conditions.

Note

In order to obtain high quality HPCs, it is suggested to sort only the CD34⁺, CD45⁺ and CD43⁺ triple positive cell population, discarding the single or double negative cells.

Note

If you wish to continue with the iMGL protocol from freshly sorted cells, skip the Freezing Down Sorted Hematopoietic Stem Cells steps. Re-suspend cell pellet in iMGL Diff Complete Medium at a concentration of ~200,000/300,000 cells per well of a 6 well tissue culture plate

Freezing Down Sorted Hematopoietic Stem Cells

21.

Centrifuge positively sorted cells at 300 x g for 0h 10m 0s at 4°C.

22.

Aspirate supernatant.

23.

Re-suspend cells at a concentration of 1 million cells per 1mL.

24.

Aliquot 1mL per cryovial.

25.

Place cells in -80°C for approximately 24h 0m 0s.

26.

After 24 hours, cells must be transferred to liquid nitrogen for long-term storage.

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.

Note

Thaw should take less than one minute, remove cells from water bath prior to complete thaw.

28.

Transfer contents of cryovial to a conical tube containing 8mL.

29.

Centrifuge concial tube at 300 x g for 0h 5m 0s.

30.

Aspirate supernatant.

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

31.1.

iMGL Differentiation Basal Medium (per 500 mL)

A	B	C	D	E	F
Component	Stock Concentration	Final Concentration	Vendor	Catalog #	Volume
Phenol-free DMEM/F12 (1:1)			Thermofisher	11039021	462.4 mL
Insulin (0.02 mg/mL), Holo-transferrin (0.011 mg/mL), Sodium selenite (13.4 ug/mL) (ITS-G Solution)	100X	2X	Thermofisher	41400045	10 mL
B27	50X	2% V/V	Thermofisher	17504044	10 mL
N2	100X	0.5% V/V	Thermofisher	17502048	2.5 mL
Monothioglycerol	11.5M	400 uM	Sigma Aldrich	M1753-100ML	17.4 uL
Non-Essential Amino Acids (NEAA)	100X	1X	Thermofisher	11140050	5 mL
Glutamax	100X	1X	Thermofisher	35050061	5 mL
Pen/Strep	100X	1X	Thermofisher	15140-122	5 mL
Recombinant Human Insulin	20 mg/mL	5 ug/mL	Sigma Aldrich	I2643	125 uL

31.2.

iMGL Diff Complete Medium Recipe:

A	B	C	D	E
		Vendor	Cetalog #	Dilution
iMGL Diff Base Medium
IL-34 (100 ng/ml)	500 ug/mL in H20	Peprotech	200-34	1:5000
TGFb-1 (50 ng/ml)	100 ug/mL in 10mM Citric Acid	Peprotech	100-21	1:2000
M-CSF (25 ng/ml)	100 ug/mL in H20	Peprotech	300-25	1:4000

Note

iMGL differentiation basal medium is made at the start of each round of differentiation and more if required is made subsequently

Note

Aliquots of cytokines are stored at -80°C and added fresh to an aliquot of required volume of basal medium on teh day of media addition to iMGLs. Remaining volume of aliquots are stored at 4°C and used up first before thawing new aliquots for further days of media addition

Note

iMGL differentiation complete medium + 1x cytokines refers to required volume of basal medium + 1:5000 hIL-34 + 1:2000 hTGF-β1 + 1:4000 hM-CSF while complete medium + 2x cytokines refers to required volume of basal medium + 1:2500 hIL-34 + 1:1000 hTGF-β1 + 1:2000 hM-CSF

Note

For splitting steps, N refers to existing no. of wells and N’ refers to newer wells pre-coated with Matrigel at least for 1 h or O/N

Note

During splitting, all collected supernatants also will contain floating iMGLs while some are left behind attached to the wells

Note

IL-34, hTGF-β1 and M-CSF are cytokines that promote iMGL survival and homeostasis and are referred to as maintenance cytokines while CD200 and CX3CL1 cytokines that induce iMGL maturation and are accordingly referred to as maturation cytokines

Differentiation of Hematopoietic Stem Cells into iMGLs

32.

Day 2 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

33.

Day 4 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

34.

Day 6 - Split and add 1mL + 2X Cytokines per well of a 6-well tissue culture plate.

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 0h 6m 0s at 37Room temperature

34.2.

While above centrifugation is ongoing, split 3 existing wells (N) of iMGLs into 1 new well (N')

Note

There should be ~1 mL of cells in each well after harvesting supernatant in prior step. To split the cells, ~300 µL from each of the 3 existing wells is transferred to one new Matrigel-coated well for each line. iMGLs tend to be more confluent around the center of each well, therefore while taking up ~300 µL for splitting into new well, ensure to collect cells from the center of each well and pipette up/down one time around the center and then transfer the ~300 µL to the new well.

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)

35.

Day 8 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

36.

Day 10 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

Note

Use extreme caution as plate is nearly full with media.

37.

Day 12 - Split and add 1mL + 2X Cytokines per well of a 6-well tissue culture plate (Refer to Step 34)

38.

Day 14 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

39.

Day 16 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

40.

Day 18 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

41.

Day 20 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

42.

Day 22 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

43.

Day 24 - Add 1mL + 1X Cytokines per well of a 6-well tissue culture plate.

Note

Use extreme caution as plate is nearly full with media.

Note

At any point during the culture from Days 12 to 36, if cells look stressed either due to thawing, centrifugation or other reasons, add iMGL Differentiation Complete Medium + 2X Cytokines

44.

Split and add 1mL + 2X Cytokines per well of a 6-well tissue culture plate (Refer to Step 34)

iMGL Maturation Media Recipe:

A	B	C	D	E
		Vendor	Catalog #	Dilution
iMGL Complete Medium
CD200 (100 ng/mL)	100 ug/mL	Novoprotein	C311-50ug	1:1000
CX3CL1 (100 ng/mL)	100 ug/mL	Peprotech	300-31	1:1000

Note

Every 2 days, supplement cells with 1mL per well of iMGL Maturation Media (after day 37 feeding, maturation media + 1x maintenance and maturation cytokines unless if cells look stressed, in which case feed maturation media + 2x maintenance cytokines + 1x maturation cytokines)

45.

Day 28 - Cells should have reached maturity by this step and are ready for experimental use.

Note

Continue feeding cells with iMGL Maturation Media . Mature Microglia-Like Cells can be used for approximately 2-3 weeks.

Differentiation of iPSC into Microglia-Like Cells (iMGL)

Abstract

Before start

Attachments

Steps

iPSCs Culture

iPSCs Aggregate Plating

iPSCs Induction into Hematopoietic Stem Cells

FACS Sorting CD43+ CD34+ CD45+ Cells

Freezing Down Sorted Hematopoietic Stem Cells

Deriving iMGLs - Thawing Hematopoietic Stem Cells

Differentiation of Hematopoietic Stem Cells into iMGLs

推荐阅读