TITLE: High-Yield Monocyte/Macrophage Differentiation from hiPSC
AUTHORS: Lilia Rodriguez, Florence Petit, Janelle Drouin-Ouellet

[DESCRIPTION]
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.

[STEPS]
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
1. Start with large cultures of pluripotent stem cells around 50–70% confluence cultured on Matrigel hESC.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
2. Coat 6-well plates with fresh Matrigel (1 mg/plate) 1 h to 60 min at 37 °C.

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
3. Prepare iPSC maintenance medium (mTeSRplus) with 10 micromolar (µM) Rock inhibitor (concentration depending on your line). Add 1.5 mL of medium supplemented with rock inhibitor to each well of the Matrigel coated 6-well plate.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
4. Prepare 96-well plate for counting cell clusters. Add 40 µL DPBS to 3 empty wells of the 96-well plate.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
5. Dissociate iPSC cultures with GCDR:

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
6. Scrape the cells in 1 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.

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
7. Pipette 5 µL of cell cluster mixture into each well of the 96-well plate (n = 3 wells) for counting. 
 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
8. 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). 
 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
9. 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.
 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
10. 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. 
 

 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
11. Prepare Medium A from StemDiff Hematopoietic kit by diluting Supplement A 1:200 in basal medium. You will need 3 mL Medium A per well for the entire differentiation.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
12. Remove mTeSRplus and add 2 mL Medium A per well (Day 0).
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
13. 48 h later, supplement cultures with additional 1 mL Medium A per well (Day 2).

 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
14. 48 h later, remove all medium A and add 2 mL Medium B (1:200 dilution of supplement B into basal medium) per well (Day 4).

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
15. Repeat addition of 1 mL medium B every 48 h three times (Day 6, 8, 10).
 


SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
16. On Day 10, collect round, floating, phase-bright cells by agitating the plate gently and collecting all medium with a serological pipette. 
 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
5.1. Remove iPSC medium and wash 1 mL DPBS to remove dead cells. 
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
5.2. Treat cells with 1 mL GCDR to each well of a 6-well plate for 5-6 min. 

SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
5.3. Remove GCDR from the well.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
16.1. Centrifuge 300 x g, 5 min. To continue collecting cells on day 12, replace 2 mL supernatant (conditioned medium) per well (add to 1 mL fresh medium). 
SECTION: Differentiation of HPC to monocytes
17. On Day 12, CD34+ cells are harvested from the plate leaving behind a hemogenic endothelium. 
 

SECTION: Differentiation of HPC to monocytes
18. On Day 19 monocytes are harvested by gently rinsing monocytes off each well before passing through a   nylon filter and centrifuging at 300 x g, 5 min. 
SECTION: Differentiation of HPC to monocytes
20. The medium in each harvested well is immediately replaced with 2 mL of fresh monocyte factory medium. 
 

SECTION: Differentiation of iPSC-derived monocytes into macrophages
21. Freshly harvested monocytes are plated at 2.5-3x105 per well of a 6-well plate ultra-low attachment plate in 2 mL of macrophage differentiation media (MDM) Day 0 (Day 19 from beginning).
SECTION: Differentiation of iPSC-derived monocytes into macrophages
22. On Day 4, 1 mL of media is removed and each well is supplemented with 2 mL of MDM.
SECTION: Differentiation of iPSC-derived monocytes into macrophages
23. 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 300 x g, 5 min, resuspended in fresh MDM medium, and plated for experiments. CD11b+, CD68+ cells are checked by flow cytometry.
SECTION: Macrophage activation
24. iPSC-derived MDM are plated at 5x105 cells per well in a 6 well plate in 2 mL of MDM medium without M-CSF. 
SECTION: Macrophage activation
25. Cells are left untreated or activated with 500 ng/mL of LPS, 20 ng/mL of rhIFNγ, or both LPS and rhIFNγ. 
SECTION: Macrophage activation
26. 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.
 

SECTION: Differentiation of HPC to monocytes
19. CD45+, CD14+, CD11b+ cells are checked by flow cytometry. We usually obtain >85% CD14+ cells.
SECTION: Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
16.2. At this point cells may be analyzed for HPC markers by flow cytometry or ICC (cells should always be >95% CD43+).