Generation of Human Neutrophils from Induced Pluripotent Stem Cells in Chemically Defined Conditions Using ETV2 Modified mRNA

Summary This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019).


SUMMARY
This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019).
CRITICAL: RNase-free reagents and RNase inhibitor should be used to stabilize synthesized RNA and prevent degradation. RNase contamination is the most common cause of poor mmRNA recovery. Usually 30-40 mg of mmRNA can be synthesized using 500-1,000 ng of template per a 20 mL reaction. Control template (provided in the kit) can be performed in parallel for troubleshooting. d) ETV2 mmRNA transfection master mix: i) Transfer aliquot of ETV2 mmRNA from À80 C and place on ice ( Figure 4B). ii) Place TransITâ-mRNA Kit on ice from 4 C. iii) For each well of a 6-well plate, combine the following reagents in a sterile 1.5 mL microcentrifuge tube at room temperature ( Figure 4B): 100 mL TeSR TM -E8 TM complete media 0.4 mL TransIT reagent (Provided in TransITâ-mRNA Kit) 0.4 mL mRNA boost reagent (Provided in TransITâ-mRNA Kit) 200 ng ETV2 mmRNA Mix by gently pipetting and keeping at room temperature for 3 min before adding into respective well ( Figure 4C).

Production of Matrigel-Coated Plates
Timing: $60 min 3. Use Matrigel for culturing of hiPSCs. Matrigelä, Growth Factor Reduced is frozen at À20 C to À80 C. Thaw overnight on ice at 4 C.

PCR Cycling Conditions
Step Temperature Time Cycles Initial Denaturation 98 C 3 0 s 1 4. Open the metal seal on the Matrigelä bottle and carefully remove the rubber stopper. Place bottle on ice and aliquot 0.5 mg into prechilled microcentrifuge tubes and store at À80 C for up to 12 months. 5. In a sterile 15 mL conical tube add cold 11 mL of 13 DPBS. 6. Remove one 0.5 mg Matrigelä aliquot from the freezer. Use a prechilled 1,000 mL pipet tip to add 1 mL of cold PBS to the Matrigelä aliquot. 7. Gently pipet up and down to thaw and dissolve the Matrigelä. Immediately transfer it to the 15 mL conical tube with 11 mL of DPBS and pipet to mix. 8. Immediately plate 2 mL into each well of a 6-well plate. Store the plates at 4 C for up to 7 days. 9. Prior to use, incubate a Matrigelä coated plate for at least 1-2 h at 37 C. 10. Aspirate the Matrigelä and wash once with 1 mL of TeSR TM -E8 TM complete media. 11. Add 2 mL of TeSR TM -E8 TM complete media.

Production of Collagen IV-Coated Plate
Timing: $60 min 12. To make 2 mg/mL of Collagen IV, dissolve 5 mg Collagen IV powder in 6.25 mL of acetic acid and 2.5 mL of sterile water and store overnight at 4 C. Aliquot 60 mL (2.4 mg/mL) into microcentrifuge tubes and store at À80 C for up to 12 months. 13. Remove one 60 mL aliquot from storage. Use a micropipette with a 1,000 mL tip to add 1 mL of ddH 2 O into the 60 mL aliquot of collagen IV and take the full mixture into 49 mL of ddH 2 O. It is enough for 436-well plates. 14. Immediately plate 2 mL in each well of a 6-well plate. Store the plates at 4 C for up to 7 days. 15. Prior to use, incubate for at least 1 h at 37 C. 16. Aspirate the collagen solution and wash once with 1 mL TeSR TM-E8 TM complete media. 17. Add 2 mL TeSR TM -E8 TM complete media to each well.

Dissociation Solution for hiPSCs
Mix the following reagents, filter sterilize using a 0.22 mm membrane filter, and aliquot 10 mL/tube 500 mL 0.5 M EDTA pH8.0 500 mL DPBS Store at 2 C-8 C for up to one month.

hiPSC Freezing Media
Combine the following reagents to prepare hiPSC freezing media and then aliquot 50 mL/tube 450 mL of TeSR TM -E8 TM Basal medium 50 mL of DMSO Store at 2 C-8 C for up to 2 weeks.

Hemogenic Endothelium Media
Mix the following reagents, filter sterilize using a 0.22 mm membrane filter, and aliquot 12 mL/tube 50 mL of Stemlineâ II Hematopoietic Stem Cell Expansion Medium 10 mL of 100 ng/mL Human FGF-2 (Final concentration of 20 ng/mL) [Reconstitution of Human FGF2: Centrifuge the vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute in 5 mM Tris, pH 7.6. according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at À80 C until needed for use.] Store at 4 C for up to 2 weeks.

Hematopoietic Differentiation Media
Mix the following reagents, filter sterilize using a 0.22 mm membrane filter, and aliquot 12 mL/tube 50 mL of Stemlineâ II Hematopoietic Stem Cell Expansion Medium 10 mL of 100 ng/mL Human FGF-2 (Final concentration of 20 ng/mL) 12.5 mL of 100 ng/mL Human GM-CSF (Final concentration of 25 ng/mL) [Reconstitution of Human GM-CSF: Centrifuge the vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute in sterile water according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at À80 C until needed for use.] 0.714 mL of 3.5 mM UM171 (Final concentration of 50 nM) [Reconstitution of small molecule UM171: Centrifuge the vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute in DMSO according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at À80 C until needed for use.] Store at 4 C for up to 2 weeks.
Note: UM171 does not increase the number of myeloid cells, but significantly improves neutrophil generation.

Neutrophil Differentiation Media
Combine the following reagents, filter sterilize using a 0.22 mm membrane filter, and aliquot 10 mL/tube 494 mL StemSpanä H3000 medium 5 mL GlutaMAXä-I 1003 (Final concentration 13) 1 mL 100% EX-CYTE (Final concentration 0.2%) 750 mL 100 ng/mL Human G-CSF (Final concentration 150 ng/mL) [Reconstitution of Human G-CSF: Centrifuge the vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute in sterile water according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at À80 C until needed for use.] 125 mL 10 mM Am580 retinoic acid agonist (Final concentration 2.5 mM) [Reconstitution of small molecule Am580: Centrifuge the vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute in DMSO according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at À80 C until needed for use.] Gentamycin solution Store at 2 C-8 C for up to 2 weeks.

Flow Cytometry Buffer
Combine the following reagents, filter sterilize using a 0.22 mm membrane filter, and aliquot 10 mL/tube 488 mL 13 DPBS 10 mL FBS 0.25 g Sodium azide (NaN 3 ) 2 mL 0.5 M EDTA (pH 8.0) Store at 4 C up to 6 months.
These cell lines are provided by WiCell (Madison, WI).

Maintenance of hiPSC
Timing: $25 min hiPSCs are maintained under feeder-free conditions on Matrigel in TeSR TM -E8 TM complete media ( Figure 2).
1. Retrieve a Matrigel solution containing 6-well plate from 4 C storage and incubate at 37 C for minimum 30 min to 1-2 h before thawing the iPSCs.
Note: Store Matrigel solution containing plates in 4 C refrigerator for overnight. These plates can be stored in refrigerator for 1 month.
2. Aspirate the Matrigel from each well and put 1 mL of prewarmed TeSR TM -E8 TM complete media in each well. 3. Add 5 mL of TeSR TM -E8 TM complete media to a 15 mL conical tube. 4. Thaw cryovial of hiPSC from liquid nitrogen storage in a 37 C water bath. CRITICAL: Proceed to step 5 immediately after complete thaw. Avoid keeping cryovial in water bath for extended period time, which will compromise cell viability.
5. Add 1 mL of TeSR TM -E8 TM complete media into the cryovial, gently mix and collect the total 6 ml cell suspension in the 15 mL tube from step 3.
CRITICAL: Mix cells very gently. Avoid vigorous mixing.
6. Centrifuge at 1503g for 5 min at room temperature and aspirate the supernatant. 7. Resuspend the cell pellet with TeSR TM -E8 TM complete media with 10 mM Rock inhibitor Y-27632 Dihydrochloride. 8. Gently rock the plate back-and-forth to coat the cells evenly over the wells, and place in 37 C, 5% CO 2 , incubator. 9. Change the media with fresh TeSR TM -E8 TM complete media one day after seeding to remove Rock inhibitor. 10. Replenish the media every 48 h with TeSR TM -E8 TM complete media.
CRITICAL: The cells should be passaged every 5-6 days . Do not allow hiPSC cultures to become more than 80% confluent.
14. Incubate at 37 C, 5% CO 2 , for 3 min and slowly aspirate the dissociation solution. 15. Add 2 mL of prewarmed TeSR TM -E8 TM complete media into each well of a 6-well plate and break the colonies into small aggregates by gentle pipetting.
CRITICAL: Do not use excessive mechanic force while pipetting. Note: Centrifugation speed is higher than that of the speed during thawing of hiPSCs. During thawing procedure cells are in very labile condition in DMSO containing freezing media. So, centrifugation speed is low for not to harm the cells.
17. Resuspend the cells in 6 mL of TeSR TM -E8 TM complete media. 18. Plate 1 mL of cell suspension in each well of a 6-well plate containing 1 mL of TeSR TM -E8 TM. complete media.
Note: Split the cells from one well of 6-well plate into 6 wells of a 6-well plate with TeSR TM -E8 TM complete media without Rock inhibitor.
19. Gently rock the plate side-to-side to coat the cells evenly over the wells and place in 37 C, 5% CO 2 , incubator. 20. Replenish the media every 48 h with TeSR TM -E8 TM complete media.
Note: The undifferentiated hiPSC colonies must be composed of tightly packed cells with prominent nucleoli. They should have well-defined sharp edges (Figure 3). Then they are ready to use for differentiation. Passage no lower than 30 is recommended to use for differentiation. If cells are not having well-defined sharp edges, then these cells are considered as bad hiPSCs. These cells do not show good mmRNA transfection efficiency.

Cryopreservation of hiPSCs
Timing: $30 min Cryopreservation of hiPSCs is performed in hiPSC freezing media to maintain the batch of hiPSCs for future differentiation.
21. When hiPSCs cell density reaches 60%-80%, aspirate the media and add 1 mL prewarmed dissociation solution (0.5 mM EDTA/13 PBS). 22. Incubate at 37 C, 5% CO 2 , for 3 min and slowly aspirate the dissociation solution. 23. Add 2 mL of prewarmed TeSR TM -E8 TM complete media into each well of a 6-well plate and break the colonies into small aggregates by gentle pipetting. 24. Collect the cells into a 15 mL conical tube and centrifuge at 3503g for 5 min at room temperature and aspirate the supernatant. 25. Resuspend the cells in 6 mL of hiPSC freezing media and distribute 1 ml of cell solution in each cryovials.
26. Store cryovials are in isopropanol containing coolant in À80 C for overnight.
Note: Do not keep cryovials at À80 C not more than a week.
27. Transfer the cryovials into liquid nitrogen tank for long term storage. To induce hemogenic endothelium, hiPSCs are transfected with ETV2 mmRNA in TeSR TM -E8 TM complete media using TransIT reagent and mRNA boost.
28. On day 0, retrieve collagen IV-coated 6-well plate from 4 C storage and keep at 37 C for at least 30 min before starting the differentiation of iPSCs. 29. Aspirate the collagen and put 2 mL prewarmed TeSR TM -E8 TM complete media supplemented with 10 mM Y-27632 Dihydrochloride into each well. 30. When iPSCs are 60% in confluency after 3 days of culture (Figure 3), aspirate the media and add 1 mL prewarmed HyQTase for single cell suspension of the iPSCs ( Figure 4A).

OPEN ACCESS
Optional: HyCloneä HyQTaseä has since been discontinued by GE Healthcare Life Sciences. We therefore recommend to use Accutaseâ (Innovative Cell Technologies, Inc., Cat. #AT-104) instead.
34. To remove residual HyQTaseä, resuspend the cells in 2 mL of TeSR TM -E8 TM complete media. 35. Centrifuge at 3503g for 5 min at room temperature and aspirate the supernatant. 36. Resuspend the cells in 1 mL of TeSR TM -E8 TM complete media. 37. Determine cell viability by trypan blue staining and a hemocytometer. 38. Plate 2310 5 cells into each well of a 6-well plate containing 2 mL of TeSR TM -E8 TM complete media with 10 mM Rock inhibitor and gently rock the plate side-to-side and back-and-forth to spread the cells evenly across the well.
CRITICAL: Cell number should be highly accurate as transfection efficiency is highly dependent on cell density and viability.
39. Incubate the plate at 37 C, 5% CO 2 , for 30 min to adhere the cells. 40. Place the TransIT reagent and mRNA boost (TransITâ-mRNA Kit, Mirus) and ETV2 mmRNA aliquot on ice ( Figure 4B). 41. Prepare ETV2 mmRNA transfection master mix in a sterile 1.5 mL microcentrifuge tube. 42. Mix the components by gentle pipetting and add the $100 mL transfection mixture to each well of the 6-well plate ( Figure 4C).
CRITICAL: Add the transfection master mix drop by drop into each well evenly. Rock the plate after addition to properly distribute.
CRITICAL: We recommend not to use an iPSC cell line beyond passage 30, after which, hematopoietic differentiation capacity reduces.

Expansion of Hemogenic Endothelial Cells
Timing: $2 days After ETV2 mmRNA transfection, cells are cultured in hemogenic endothelium medium (StemLine II with FGF2) to support formation and expansion of hemogenic endothelial cells.
44. On day 1, after 24 h of transfection, observe cell morphology under phase contrast microscopy ( Figure 3) and then aspirate the media and add 2 mL of prewarmed hemogenic endothelium media.
47. On day 3, prepare hematopoietic differentiation media and observe cell morphology under phase contrast microscopy ( Figure 3). 48. Aspirate the supernatant and add 2 mL of prewarmed hematopoietic differentiation media.
Place at 37 C, 5% CO 2 . 49. Add 1 mL hematopoietic differentiation media on the top of the existing media without removing the media every 48 h up to day 8. 50. On day 9, observe the morphology of the cells under phase contrast microscopy ( Figure 3). 51. Collect floating myeloid progenitors on day 9 into a 15 ml conical tube and transfer to neutrophil differentiation conditions.
Note: After the first collection of floating cells on day 9, adherent cells continue to produce myeloid progenitors. Adding of 2 mL hematopoietic differentiation media every 2 days to the remaining adherent cells allows for additional two rounds of myeloid progenitor collection between days 16 and 23.
Note: Adherent cells are hemogenic endothelial cells, which give rise to floating myeloid progenitors. UM171 is one of the important small molecules in hematopoietic differentiation media. It does not increase the number myeloid cells. However, it does significantly improve neutrophil output.

Neutrophil Differentiation
Timing: $8 days Myeloid progenitors are differentiated into fully functional neutrophils in a neutrophil differentiation medium (StemSpan H3000 with G-CSF and retinoic acid receptor a agonist Am580).
52. Centrifuge collected floating myeloid progenitor cells at 3503g for 5 min at room temperature and aspirate the supernatant. 53. Resuspend the cells in 2 mL of neutrophil differentiation media to remove any residual hematopoietic differentiation media. 54. Centrifuge at 3503g for 5 min at room temperature and aspirate the supernatant. 55. Resuspend the cells in 1 mL of neutrophil differentiation media. 56. Determine the cell viability by using trypan blue and a hemocytometer. 57. To induce neutrophil differentiation, plate 5310 5 cells in one well of a 6-well plate containing 4 mL neutrophil differentiation media and gently rock the plate side-to-side, back-and-forth to spread the cells evenly across the wells. 58. After 4 days, add fresh 2 mL of fresh neutrophil differentiation media on the top of the existing culture. 59. After 8 days of differentiation in neutrophil differentiation media observe the morphology of the cells under phase contrast microscopy on day 17 (Figure 3). 60. Harvest the neutrophils from the supernatant, leaving adherent progenitors and macrophages.
Note: Neutrophils have to be generated and used for functional analysis fresh. Although neutrophils do not survive freezing in standard freezing media, myeloid progenitors generated in ll OPEN ACCESS step 51 can be cryopreserved in standard conditions and used for neutrophil generation after thawing.

Colony-Forming Cell Assay of (CFC) Myeloid Progenitors
Timing: $13 days Colony-Forming Cell (CFC) assay is used to determine formation of myeloid progenitors in cultures on day 9 of differentiation. 61. Thaw 3 ml aliquots of CFC assay media (MethoCultä H4435 Enriched). 62. Suspend 3310 3 floating myeloid progenitor cells to a 3 ml aliquot of Methocult TM H4435. 63. Vortex vigorously. Let the tube stand still for 15 min at 37 C water bath. 64. Attach a 16-gauge blunt-end needle to a 3 ml syringe and draw up 2.2 ml. Do not draw up large bubbles; expel them at the beginning by pushing out a couple of times. Push out 1.1 ml each into two 30 mm non-treated dish and spread out the mixture evenly by rotating. 65. Place duplicate plates in a 100 mm plate together with a water dish containing 3 ml sterile water.
Culture for 13 days. 66. Characterize and score the colonies according to their morphology with a bright field microscope in a culture dish marked with a scoring grid.

Flow Cytometric Analysis of Myeloid Progenitors and Neutrophils
Timing: $60 min
These neutrophils can be tested in the following ways:

Morphology
Undifferentiated hiPSC colonies composed of tightly packed cells with prominent nucleoli and have well-defined sharp edges (Figure 3). After transfection of singularized iPSCs with ETV2 mmRNA and culture in endothelial expansion medium, cells acquire a typical endothelial morphology within 72 h (Figure 3). During the process of differentiation, hemogenic endothelial cells undergo endothelialto-hematopoietic transition and form floating myeloid progenitors (Figure 3). Neutrophil differentiation can be confirmed by morphologic analysis of Wright-Giemsa stained cytospins prepared from floating cells. Neutrophils have a characteristic multilobed nucleus with cytoplasm containing purplish granules ( Figure 6A).

Hematopoietic Colony-Forming Potential
The differentiation ability of myeloid progenitors can be determined by Colony-Forming Cell (CFC) assay. In this protocol myeloid progenitors produce CFU-Macrophage (CFU-M) ( Figure 5D

Other Evaluations
Functionality of generated neutrophils can be assessed by analyzing phagocytosis, reactive oxygen species (ROS) generation, migration and neutrophil extracellular trap (NET) formation assay.

LIMITATIONS
Our protocol produces a relatively homogenous population of neutrophils from different hESC and iPSC lines. However, differentiation efficacy can vary between lines. Optimization of ETV2 mmRNA transfection, cell densities and time frames at which differentiation steps are initiated, may all be required when other PSC lines are used. Using iPSCs beyond passage 30 is not recommended as the differentiation capability is reduced in feeder-free conditions. Transfection of mmRNA requires adequate skills and experience. Thus, preliminary evaluation of mmRNA transfection efficacy using eGFP mmRNA is recommended. Neutrophils generated by this protocol phagocytose bacteria and efficiently produce ROS. However, they are closer to fetal than adult neutrophils, and their NET production in response to PMA and chemotactic response to IL-8 is somewhat impaired in comparison with adult peripheral blood neutrophils.  [Link: Step-By-Step Method Details steps 40-42] Potential Solution 1 My mmRNA transfection failed sometimes due to higher than passage no 30. So, it is important to use low passage iPSCs for differentiation in this feeder-free condition. Also, my experiment failed sometimes due to incubation of the mmRNA transfection master mix more than 3 min at room temperature. Therefore, the incubation of mmRNA with transfection master mix at room temperature should not exceed more than 3 min. On the other hand, one of the reasons behind the failure, could be the addition of the mmRNA transfection master mix at the center of the plate without mixing. It is critical to distribute master mix into plate equally over the plate by adding drop by drop while rocking the plate side-by-side and back-and-forth.
Problem 2 Cell Density.

Potential Solution 2
It is crucial to maintain proper cell density. Cells should be plated as a single cell suspension and cell density must be maintained at 2310 5 cells in each well of a 6-well plate. More than 2310 5 cells can hamper transfection efficiency of mmRNA.
Problem 3 mmRNA stability. Potential Solution 3 It is important to make aliquots of mmRNA and store them at À80 C. It is necessary to take out the aliquots on ice to maintain mmRNA integrity. Avoid freeze-thaw by making aliquot volumes small enough for single use.

Potential Solution 4
It is recommended to use eGFP mmRNA as a positive control to assess the transfection efficiency with mmRNA. More than 90% of GFP-positive cells should be observed in iPSC cultures 24-48 h after transfection with eGFP mmRNA.

Potential Solution 5
It is advisable to analyze the ETV2 protein expression level after 24 h of ETV2 mmRNA transfection by Western Blot.

Potential Solution 6
Cytokines must be stored at À80 C for long term storage. It is necessary to take out the aliquots on ice. Avoid freeze-thaw by making aliquot volumes small enough for single use.

Lead Contact
Further information and requests for resources should be directed to the Lead Contact Igor Slukvin (islukvin@wisc.edu).

Materials Availability
This protocol did not generate any new data or code.

Data and Code Availability
This protocol did not generate any new data or code.