Jul 25, 2024

Public workspaceHigh-Yield Monocyte/Macrophage Differentiation from hiPSC

DOI
dx.doi.org/10.17504/protocols.io.14egn37oql5d/v1
  • 1Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada;
  • 2Faculté de Pharmacie, Univeristé de Montréal, Montréal, Québec, Canada
Lilia Rodriguez
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DOI: dx.doi.org/10.17504/protocols.io.14egn37oql5d/v1
Protocol Citation: Lilia Rodriguez, Florence Petit, Janelle Drouin-Ouellet 2024. High-Yield Monocyte/Macrophage Differentiation from hiPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn37oql5d/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: July 30, 2023
Last Modified: July 25, 2024
Protocol Integer ID: 85699
Keywords: Differentiation of iPSCs to Hematopoietic Progenitor Cells, Differentiation of HPC to monocytes, Differentiation of iPSC-derived monocytes into macrophages, Macrophage activation, ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000525
Abstract
Here, a highly efficient method for differentiating monocytes/macrophages from hiPSC is described. The process utilizes commercially-available materials to derive CD34+ progenitor cells that are apically released from a hemogenic endothelium. Subsequently, the hemogenic endothelium gives rise to highly pure (>95%), CD34-CD14+ monocytes in 19-23 days and yields more monocytes by day 35 when compared to previous methods. These monocytes are further differentiated to macrophages after 7 days. The efficient workflow and increase in monocyte output boost feasibility for high throughput studies and enables clinical-scale iPSC-derived manufacturing processes.
Guidelines
Summary
Here, a highly efficient method for differentiating monocytes is described. The process utilizes commercially-available materials to derive CD34+ progenitor cells that are apically released from a hemogenic endothelium. Subsequently, the hemogenic endothelium gives rise to highly pure (>95%), CD34-CD14+ monocytes in 19-23 days and yields more monocytes by day 35 when compared to previous methods. The efficient workflow and increase in monocyte output boost feasibility for high throughput studies and enables clinical-scale iPSC-derived manufacturing processes.
Materials
Materials:

  1. mTeSRTM Plus cGMP (StemCell, Catalog #100-0276)
  2. Rock inhibitor (Y-27632, Miltenyi Biotech, 130-106-538)
  3. Gentle Cell Dissociation Reagent (StemCell, Catalog # 100-0485)
  4. Corning® Matrigel® hESC-Qualified Matrix (Fisher Scientific, 08-774-552)
  5. Falcon® 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate (Corning Catalog # 353046)
  6. Suspension Flat Bottom 6-well Cell Culture Plate (Sarstedt, Catalog # 83.3920.500)
  7. DPBS (Without Mg2+ or Ca2+) (Gibco, Catalog # 14190144)
  8. STEMdiff Hematopoietic Kit (StemCell, Catalog # 05310)
  9. Fisherbrand™ Sterile Cell Strainers 70 µM (Fisher, 22-363-548)
  10. Cell Dissociation Solution Non-enzymatic 1x (Sigma, C5914-100ML)
  11. Monocyte factory medium:
  • X-VIVOTM 15 Serum-free Hematopoietic Cell Medium (Lonza, Catalog #: 04-418Q)
  • 50 mM 2-Mercaptoethanol (Thermo-Fisher, 31350010)
  • GlutaMAX Supplement 1X (Thermo-Fisher, 35050061)
  • 100 ng/ml recombinant human M-CSF (Peprotech, 300-25)
  • 25 ng/ml recombinant human IL-3 (Peprotech, 200-03)

12. Macrophage differentiation media (MDM):
  • X-VIVOTM 15 Serum-free Hematopoietic Cell Medium
  • 50 mM 2-Mercaptoethanol
  • GlutaMAX Supplement 1X
  • 100 ng/ml human recombinant M-CSF

13. LPS-EB, LPS from E. coli O111:B4 (Invivogen, tlrl-3pelps)
14. rhIFNγ (Thermo-Fisher PHC4031)

ReagentmTeSR™ PlusSTEMCELL Technologies Inc.Catalog #100-0276

ReagentStemMACS™ Y27632Miltenyi BiotecCatalog #130-106-538

ReagentGentle Cell Dissociation ReagentSTEMCELL Technologies Inc.Catalog #100-0485

ReagentCorning™ Matrigel™ hESC-Qualified MatrixFisher ScientificCatalog #08-774-552

ReagentFalcon® 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate with Lid Individually WraCorningCatalog #353046

ReagentCell culture plate 6 well surface: Suspension flat baseSarstedtCatalog #83.3920.500

ReagentGibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144

ReagentSTEMdiff™ Hematopoietic KitSTEMCELL Technologies Inc.Catalog # 05310

ReagentFisherbrand™ Sterile Cell StrainersFisher ScientificCatalog #22-363-548

ReagentCell Dissociation Solution Non-enzymatic 1xMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5914-100ML

ReagentGibco™ 2-Mercaptoethanol (50 mM)Thermo Fisher ScientificCatalog #31350010

ReagentGlutaMAX™ SupplementThermo Fisher ScientificCatalog #35050061

ReagentRecombinant Human M-CSFpeprotechCatalog #300-25

ReagentRecombinant Human IL-3peprotechCatalog #200-03

ReagentLPS-EB (LPS from E. coli O111:B4)InvivoGenCatalog #tlrl-3pelps

ReagentIFN-γ Recombinant Human ProteinThermo FisherCatalog #PHC4031

Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
1h 5m
1h 5m
Start with large cultures of pluripotent stem cells around 50–70% confluence cultured on Matrigel hESC.
Coat 6-well plates with fresh Matrigel (Amount1 mg /plate) 1 h to DurationOvernight at Temperature37 °C .

1h
Overnight
Prepare iPSC maintenance medium (mTeSRplus) with Concentration10 micromolar (µM) Rock inhibitor (concentration depending on your line). Add Amount1.5 mL of medium supplemented with rock inhibitor to each well of the Matrigel coated 6-well plate.
Pipetting
Prepare 96-well plate for counting cell clusters. Add Amount40 µL DPBS to 3 empty wells of the 96-well plate.
Dissociate iPSC cultures with GCDR:

Remove iPSC medium and wash Amount1 mL DPBS to remove dead cells.
Wash
Treat cells with Amount1 mL GCDR to each well of a 6-well plate for 5-6 min.

Remove GCDR from the well.
Scrape the cells in Amount1 mL mTeSRplus + Rock inhibitor and transfer to a 15 mL Falcon. Using a 5 ml pipette, gently pipette up and down 2-3 times in order to achieve clusters of ~ 50-100 cells.

Pipette Amount5 µL of cell cluster mixture into each well of the 96-well plate (n = 3 wells) for counting.
Note
Important! It is recommended to pipett Amount5 µL of cells with a p20 pipette to get a more accurate representation of cell cluster size that will be plated. Use of a p10 pipettor may triturate cells too far. Additionally, Amount5 µL volume can be adjusted if there are too many cell clusters to easily and accurately count or cell cluster number.

Critical
Count all cell clusters of ~100 cells (100–200 μm diameter). Determine # of clusters per μL (total count of correct sized clusters in 3 wells/15 μL total cell cluster suspension).
Note
Important! Do not count clusters that are too big or too small. Clusters that are too small can be ignored as they likely will not survive passage, and the few that remain will not differentiate. However, clusters that are too large should be avoided. If there are many large clusters, triturate the cell suspension and recount. It can also be helpful to let larger clusters settle to the bottom of the tube if there are only several in suspension and trituration would create too many smaller clusters.

Critical
Using cluster #/μL calculations, plate 20–40 clusters per well. The goal is to produce 10–20 surviving clusters of ~100 cells each per well the next day. Swirl plate in incubator to evenly distribute the clusters throughout the plate.
Note
Important! After counting, flick or invert cell suspension tube as clusters of cells will have settled to the bottom. To guarantee success on preliminary runs, it can be helpful to plate several wells of various densities from 20 to 40 clusters per well. If your cell line is particularly robust, you can plate lower densities such as 10–20 clusters per well. With quick-growing cell lines, you will also want to err toward lower densities as these colonies will be larger than slow-growing iPSC lines.

Critical
24 h after plating, count colonies and choose wells to begin with. Do not blindly start differentiations in all wells. Choose only wells with 10–20 undifferentiated iPSC clusters of ~100 cells each.
Representative morphology of KOLF iPSC line at day 0. Due to ROCK inhibitor, colonies might look with more loose packing.

Note
Critical step: Smaller clusters may be ignored and these wells may still be used (if all clusters are small but density is good, cells can be cultured in mTeSR plus 1–2 days prior to starting experimentation). Wells that have 2 or more large clusters of iPSCs cannot be used as these larger clusters will interfere with proper differentiation. Wells with more than 30 clusters cannot be used as these will result in significantly decreased yield. Wells with less than 10 clusters may be used, but final yield will be diminished.

Critical
Prepare Medium A from StemDiff Hematopoietic kit by diluting Supplement A 1:200 in basal medium. You will need Amount3 mL Medium A per well for the entire differentiation.
Remove mTeSRplus and add Amount2 mL Medium A per well (Day 0).
48 h later, supplement cultures with additional Amount1 mL Medium A per well (Day 2).



Pipetting
48 h later, remove all medium A and add Amount2 mL Medium B (1:200 dilution of supplement B into basal medium) per well (Day 4).

Pipetting
Repeat addition of Amount1 mL medium B every 48 h three times (Day 6, 8, 10).
Note
Important! Do not remove medium B during the differentiation process ever, the differentiating cells are secreting crucial cytokines into the medium and removal of these will hinder the differentiation process. Over the course of differentiation, the phenol red in the medium will begin to turn yellow—this is expected. By day 6–7 you should be able to see round, phase-bright cell clusters forming within the endothelial cell layers. If these do not occur, the differentiation is not likely to be successful. Common issues are that clusters were originally plated at too high a density or too close to each other.


Critical
On Day 10, collect round, floating, phase-bright cells by agitating the plate gently and collecting all medium with a serological pipette.
Note
Shaking plate too vigorously or washing/rinsing the well may result in lift-off of immature or non-CD43+ cells leading to contaminated cultures. Be gentle when collecting cells.

Centrifuge Centrifigation300 x g, 00:05:00 . To continue collecting cells on day 12, replace Amount2 mL supernatant (conditioned medium) per well (add to Amount1 mL fresh medium).
5m
Centrifigation
At this point cells may be analyzed for HPC markers by flow cytometry or ICC (cells should always be >95% CD43+).
Analyze
Differentiation of HPC to monocytes
Differentiation of HPC to monocytes
5m
5m
On Day 12, CD34+ cells are harvested from the plate leaving behind a hemogenic endothelium.
Note
The CD34+ cells can be cryopreserved at 106 cells/mL in Cryostor CS10 (STEMCELL Technologies) according to the manufacturer’s protocol and the remaining hemogenic endothelium is cultured in Amount2 mL of monocyte factory medium. The hemogenic endothelium begins producing monocytes over the next 8-9 days and for 30+ weeks.

On Day 19 monocytes are harvested by gently rinsing monocytes off each well before passing through a Thikness70 µm nylon filter and centrifuging at Centrifigation300 x g, Room temperature, 00:05:00 .
5m
Centrifigation
CD45+, CD14+, CD11b+ cells are checked by flow cytometry. We usually obtain >85% CD14+ cells.
The medium in each harvested well is immediately replaced with Amount2 mL of fresh monocyte factory medium.
Note
iPSC-derived monocytes are used immediately.

Differentiation of iPSC-derived monocytes into macrophages
Differentiation of iPSC-derived monocytes into macrophages
5m
5m
Freshly harvested monocytes are plated at 2.5-3x105 per well of a 6-well plate ultra-low attachment plate in Amount2 mL of macrophage differentiation media (MDM) Day 0 (Day 19 from beginning).
On Day 4, Amount1 mL of media is removed and each well is supplemented with Amount2 mL of MDM.
Between days 7 and 10 (Day 26-29), monocyte-derived macrophages are gently dissociated from the plate using Cell Dissociation Buffer, enzyme-free, pelleted at Centrifigation300 x g, Room temperature, 00:05:00 , resuspended in fresh MDM medium, and plated for experiments. CD11b+, CD68+ cells are checked by flow cytometry.
5m
Centrifigation
Macrophage activation
Macrophage activation
iPSC-derived MDM are plated at 5x105 cells per well in a 6 well plate in Amount2 mL of MDM medium without M-CSF.
Cells are left untreated or activated with Amount500 ng/mL of LPS, Amount20 ng/mL of rhIFNγ, or both LPS and rhIFNγ.
After 24 hours of activation, supernatants are harvested and assayed for human IL-6, TNF, and IL-1β via enzyme linked immunosorbent assay (ELISA).

Table 1. iPSC lines used.
ABC
LineReprogramming methodCell source
KOLFCRISPR Cas9 ARID2Fibroblast male 55-59 yrs
AIW002-02CytoTune™-iPS 2.0 Sendai Reprogramming Kit to generate iPS (non integrative system)PBMC male 37 yrs
A18945episomal reprogramming to generate iPScFemale CD34 + cord blood using a three-plasmid < 1 yr

Protocol references
This protocol was adapted from
Amanda McQuade and Mathew Blurton-Jones, Human Induced Pluripotent Stem Cell-Derived Microglia (hiPSC-Microglia). Methods in Molecular Biology (2022) 2454: 473–482. DOI 10.1007/7651_2021_429
Lucas H. Armitage, Mohsen Khosravi-Maharlooei, Amy Meacham, Edward J. Butfiloski, Ryan Viola, Dieter Egli, Megan Sykes, Mark A. Wallet, Clayton E. Mathews. High-Yield Monocyte, Macrophage, and Dendritic Cell Differentiation from Induced Pluripotent Stem Cells. https://doi.org/10.1101/2021.04.29.441947
Beren Aylan, Laure Botella, Maximiliano G. Gutierrez, Pierre Santucci. High content quantitative imaging of Mycobacterium tuberculosis responses to acidic microenvironments within human macrophages. FEBS Open Bio (2022) DOI: 10.1002/2211-5463.13537