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How to Differentiate hPSCs to Lymphoid-Competent CD34+ Hematopoietic Stem and/or Progenitor Cells Using a Scalable Suspension Protocol

How to Differentiate hPSCs to Lymphoid-Competent CD34+ Hematopoietic Stem and/or Progenitor Cells Using a Scalable Suspension Protocol

6 Parts 17 Materials Print

Large-scale differentiation of human pluripotent stem cells (hPSCs) into hematopoietic progenitors serves as a foundation for a wide range of immune cell research workflows, from modeling early hematopoiesis and optimizing NK or T cell differentiation, to enabling the development of consistent starting materials for cell therapy production. Traditional monolayer and embryoid body (EB) hPSC differentiation methods, while effective for small-scale applications, can introduce variability and limit culture scalability. The EB-based suspension culture approach provides a streamlined, feeder-free alternative that enhances throughput, reproducibility, and scalability, enabling researchers to efficiently generate CD34+ hematopoietic stem and/or progenitor cells suitable for downstream immune cell applications.

This protocol describes a scalable suspension culture method for the differentiation of hPSCs to lymphoid-competent CD34+ hematopoietic stem and/or progenitor cells. Prior to performing this protocol, users should familiarize themselves with protocols corresponding to the STEMdiff™ NK Cell Kit or STEMdiff™ T Cell Kit if intending to perform downstream differentiation into specific immune cell types. This protocol has been validated with suspension cultures of 2 - 100 mL, allowing the user to tune the culture volume to produce the desired number of CD34+ hematopoietic stem and/or progenitor cells for downstream applications. This protocol is compatible with 100 - 500 mL PBS-MINI MagDrive Bioreactor vessels. Refer to Table 1 and Figure 2 to select the appropriate vessel size for differentiating hPSCs to CD34+ cells. To determine the approximate yield of cells per culture volume, refer to Figure 5. The lymphoid potential of CD34+ cells generated from this protocol has been validated in five hPSC lines using the STEMdiff™ NK Cell Kit and in one cell line using the STEMdiff™ T Cell Kit (Figures 7 and 8). Myeloid potential of CD34+ cells generated from this protocol has been validated using the colony-forming unit (CFU) assay (Figure 6).

Materials

Required Materials
  • STEMdiff™ Hematopoietic - EB Basal Medium (Catalog #100-0171)
  • STEMdiff™ Hematopoietic - EB Supplement A (Catalog #100-0172)
  • STEMdiff™ Hematopoietic - EB Supplement B (Catalog #100-0173)
  • DMEM/F-12 with 15 mM HEPES (Catalog #36254)
  • Y-27632 (Dihydrochloride) (Catalog #72302)
  • Gentle Cell Dissociation Reagent (Catalog #100-0485)
  • D-PBS Without Calcium (Ca2+) and Magnesium (Mg2+) (Catalog #37350)
  • Culture vessel selected from Table 1:
  • Cell scraper
  • 37 µm Reversible Strainer - Large (Catalog #27250)
  • Falcon® tube for cell collection
  • Serological pipettes
  • Micropipette tips
  • EasySep™ Buffer (Catalog #20144), or D-PBS without calcium (Ca2+) and magnesium (Mg2+) containing 2% fetal bovine serum and 1 mM EDTA
Additional and Optional Materials
  • TeSR™-AOF 3D (Catalog #100-0720) for hPSCs maintained in 3D suspension culture
  • Orbital shaker for cultures in 2 - 60 mL volumes i.e. Celltron (Infors HT)
  • EasySep™ Human CD34 Positive Selection Kit II (Catalog #17856 for optional CD34+ cell enrichment
  • Materials for clump counting i.e. Nucleocounter® NC-250™ (ChemoMetec; Product #970-0251) Viability and Cell Count A100 and B Assay Protocol:
    • ChemoMetec A2 slides (ChemoMetec; Product #942-0001)
    • Reagent A100 (ChemoMetec; Product #910-0003)
    • Reagent B (ChemoMetec; Product #910-0002)
    • DAPI (4',6-diamidino-2-phenylindole) (Catalog #75004)
    • Eppendorf tubes
  • 70 µm Reversible Strainer - Large (Catalog #27260) for harvesting large volumes
Workflow diagram for suspension differentiation of hPSCs using the STEMdiff™ Hematopoietic - EB Progenitor Differentiation Protocol.

Figure 1. Differentiation of hPSCs Using the STEMdiff™ Hematopoietic - EB Suspension Differentiation Protocol

Human pluripotent stem cells (hPSCs) maintained in 2D culture using mTeSR™ Plus*, or in 3D suspension culture using TeSR™-AOF 3D, are compatible with this protocol. hPSC clumps are generated and seeded into a culture vessel containing Embryoid Body (EB) Formation Medium (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement A supplemented with 10 µM Y-27632) to induce EB formation and mesoderm induction. The culture vessel of choice (either a 6-well plate or filter-top bottle set on an orbital shaker or a PBS-MINI bioreactor vessel) should be agitated at the appropriate mixing speed, outlined in Figure 2. On Day 2, half of the medium is replaced with EB Medium A (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement A). On Day 3, another half-medium change using EB Medium B (STEMdiff™ Hematopoietic - EB Basal Medium + STEMdiff™ Hematopoietic - EB Supplement B) is performed to initiate hematopoietic lineage differentiation. Medium exchanges with EB Medium B are performed on Days 5, 7, and 10. On Day 12, hematopoietic stem and/or progenitor cells are collected by passing the culture through a 70 or 37 µm reversible cell strainer to remove EBs. Optionally, CD34+ cells can be enriched using the EasySep™ Human CD34 Positive Selection Kit II.
* 2D hPSC culture in mTeSR™1 and TeSR™-AOF has also been validated with this protocol.

Table 1. Culture Vessel Options for Differentiation

Suspension Vessel
Vendor/Catalog #
Culture Volume
Mixing Speed
Recommended Seeding Density (Viable Cells/mL)
6-Well Flat-Bottom Plate, Non-Treated*
STEMCELL Technologies/38040
2 mL
70 RPM
1 x 105 - 2 x 105
Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles*†
Thermo Fisher/455-0250
15 mL‡
30 mL‡
60 mL‡
40 RPM
55 RPM
65 RPM
1 x 105 - 2 x 105
PBS-MINI 0.1 MAG Single-Use Vessel
STEMCELL Technologies/100-1006
100 mL§
40 RPM
1 x 105 - 2 x 105
PBS-MINI 0.5 MAG Single-Use Vessel
STEMCELL Technologies/100-1007
500 mL§
35 - 45 RPM
1 x 105 - 2 x 105

*Note: 6-well plates and Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles should be placed on an orbital shaker with an orbital diameter of 2.5 cm.
†Note: As the Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles do not have vented caps, open the caps slightly when placing the bottles in the incubator to allow gas exchange.
‡Note: Agitation rates for the Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles are compatible with the recommended volume ± 4 mL. For example, at 40 RPM, volumes ranging from 11 - 19 mL are acceptable.
§Note: The PBS-MINI 0.1 has a working range of 80 - 100 mL, and the PBS-MINI 0.5 has a working range of 300 - 500 mL. However, operating conditions have been optimized for 100 mL and 500 mL cultures in the PBS-MINI 0.1 and 0.5, respectively.
¶Note: This is a recommended starting range and may have to be optimized for specific cell lines.

Recommendations for which culture volumes and mixing speeds to use with 6-well plates, Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles, or PBS-MINI bioreactors.

Figure 2. Culture Vessels and Recommended Parameters for Use in Differentiation

The culture volume for differentiating human pluripotent stem cells (hPSCs) to CD34+ hematopoietic stem and/or progenitor cells can be fine tuned to user needs. For estimating desired CD34+ cell output, Figure 5C can be used as a starting point. For culture volumes of 2, 15, 30, and 60 mL, either 6-well plates or Nalgene bottles are placed on an orbital shaker at the recommended agitation rates. Cultures in PBS-MINI Bioreactors are placed on the PBS-MINI Bioreactor Base Unit for mixing.

Culture Images from Days 0, 1, 5, and 12.

Figure 3. Culture Images from Days 0, 1, 5, and 12

On Day 0, differentiation is initiated by preparing and seeding human pluripotent stem cell (hPSC) clumps into EB Medium A, supplemented with 10 µM Rho kinase (ROCK) inhibitor Y-27632. Embryoid bodies (EBs) are formed in the culture by the next day and their growth progresses throughout the culture. By Day 12, single and loosely clustered hematopoietic stem and/or progenitor cells can be observed in suspension. Suspended cells are harvested for analysis and downstream applications. Scale bars = 500 µm.

Preparation of Reagents and Culture Medium

In this protocol, EB Medium A is required in Stage 1 (Day 0 - 2), and EB Medium B is required in Stage 2 (Day 3 - 12). Use sterile technique to prepare the media as indicated in Table 2.

  1. Thaw STEMdiff™ Hematopoietic - EB Basal Medium at room temperature (15 - 25°C) or overnight at 2 - 8°C. Mix thoroughly.
    Note: If not used immediately, aliquot and store at -20°C. After thawing aliquots, use immediately or store at 2 - 8°C for up to 2 weeks. Do not re-freeze. Do not exceed the shelf life of the basal medium.
  2. Thaw STEMdiff™ Hematopoietic - EB Supplement A or B at room temperature or at 2 - 8°C until just thawed. Mix thoroughly. If necessary, centrifuge for 30 seconds to remove liquid from the cap.
    Note: If not used immediately, aliquot and store at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately or store at 2 - 8°C for up to 2 weeks. Do not re-freeze.
  3. Combine components as indicated in Table 2. Mix thoroughly. Warm to room temperature before use. Note: STEMdiff™ Hematopoietic - EB Supplement A is supplied as a 200X concentrate and STEMdiff™ Hematopoietic - EB Supplement B is supplied as a 10X concentrate.

Table 2. Preparation of STEMdiff™ Hematopoietic - EB Media

Medium Components Volume In-Use Storage/Stability
EB Medium A STEMdiff™ Hematopoietic - EB Basal Medium 3 mL* If not used immediately, store the complete medium at 2 - 8°C for up to 2 weeks. Do not exceed the shelf life of the basal medium or supplement.
STEMdiff™ Hematopoietic - EB Supplement A (supplied as a 200X concentrate) 15 µL*
EB Formation Medium EB Medium A 2 mL* Use immediately.
Y-27632 (10 µM final concentration) 4 µL* of 5 mM stock solution
EB Medium B STEMdiff™ Hematopoietic - EB Basal Medium 4.5 mL* If not used immediately, store the complete medium at 2 - 8°C for up to 2 weeks. Do not exceed the shelf life of the basal medium or supplement.
STEMdiff™ Hematopoietic - EB Supplement B (supplied as a 10X concentrate) 0.5 mL*

*Note: Indicated volumes correspond to the amount required for one well of a 6-well plate (2 mL final culture volume) for the entire protocol. If using multiple wells, multiply the volumes accordingly. For larger culture vessels, scale the media components based on the final culture volume for your selected format (e.g. 15, 30, or 60 mL for filter-top bottles; 100 or 500 mL for PBS-MINI bioreactor vessels). Ensure all components are scaled proportionally to maintain consistent media composition.

Table 3. Medium Requirements for EB Formation and Medium Exchanges; Days 0 - 10

Day Medium Medium Required for Seed/Medium Exchange (% of Total Culture Volume)
0 EB Formation Medium 100
2 EB Medium A 50
3 EB Medium B 50
5 EB Medium B 90 - 100
7 EB Medium B 50
10 EB Medium B 50

Preparation of Culture Medium (Day 0)

  1. Prepare EB Medium A and EB Formation Medium according to Table 2.
  2. Prepare medium for hPSC clump resuspension:
    1. For 2D-maintained hPSC culture, use DMEM/F12 supplemented with 10 µM Y-27632 for suspension of hPSC clumps.
    2. For 3D-maintained hPSC culture, use TeSR™ -AOF 3D supplemented with 10 µM Y-27632.
  3. Prepare EB Medium B according to Table 2.

Preparation of hPSCs for Differentiation (Day 0)

hPSCs should be dissociated into a clump suspension using the following protocols, depending on the status of your starting cells. Please follow the directions pertaining to either hPSCs cultured in 2D or hPSCs cultured in 3D before proceeding to the protocol for Cell Counting and Seeding of hPSC Clumps to Initiate Differentiation (Day 0).

Preparation of 2D-Maintained hPSCs for Differentiation (Day 0)

hPSCs cultured in 2D with mTeSR™ Plus, mTeSR™1, or TeSR™-AOF should be dissociated into a clump suspension using the following protocol; if using hPSCs cultured in 3D, skip to the protocol for Preparation of 3D-Cultured hPSCs for Differentiation (Day 0). This protocol is for harvesting cells from one 100 mm dish. If using other cultureware, adjust volumes accordingly.

  1. Pre-warm EB Formation Medium, in a 37°C incubator, with cap loosened.
    Note: For culture volumes smaller than 15 mL, pre-warming medium at room temperature is sufficient.
  2. Aspirate hPSC culture medium, and rinse with 10 mL D-PBS.
  3. Aspirate D-PBS and add 5 mL Gentle Cell Dissociation Reagent (GCDR).
  4. Allow 4 - 6 minutes for dissociation at room temperature.
    Note: Incubation times may vary when using different cell lines or other non-enzymatic cell dissociation reagents. Dissociation should be monitored under the microscope until the optimal time is determined.
  5. Aspirate GCDR and add 5 mL DMEM/F12 medium supplemented with 10 µM Y-27632.
  6. Use a cell scraper to gently lift hPSCs into suspension, then gently collect with a serological pipette, and transfer into a Falcon® tube. Collection of clumps will break them down to approximately the desired size. Viable clump size is similar to hPSC passaging: 50 - 100 µm in diameter. If clumps are too large, pipette the entire suspension slowly one time. Do not pipette more than twice as this may result in clumps that are too small or singularized cells, reducing cell viability and seeding efficiency.
  7. Proceed to Cell Counting and Seeding hPSCs to Initiate Differentiation (Day 0).
Preparation of 3D-Cultured hPSCs for Differentiation (Day 0)

hPSCs cultured in 3D using TeSR™-AOF 3D should be dissociated into a clump suspension using the following protocol; if using hPSCs cultured in 2D please follow the protocol for Preparation of 2D-Maintained hPSCs for Differentiation (Day 0) before proceeding to the protocol for Cell Counting and Seeding of hPSC Clumps to Initiate Differentiation (Day 0). This protocol is for the dissociation of aggregates from a 3D hPSC culture initiated in a 15 mL culture volume and reaching a ~15.9 mL culture volume by Day 3 or 4 of hPSC expansion. For other culture volumes, refer to Table 4 for volumes of GCDR and TeSR™-AOF 3D for clump resuspension. Refer to this passaging video for additional visuals.

  1. Pre-warm EB Formation Medium, in a 37°C incubator, with the cap loosened.
  2. Aliquot and pre-warm 5 mL of GCDR to 37°C.
  3. Prepare enough TeSR™ -AOF 3D medium supplemented with 10 µM Y-27632 to resuspend all conditions. See Table 4 for recommended resuspension volumes.
  4. Filter out non-aggregated single cells by passing the entire volume of the culture through a large 37 µm Reversible Strainer. To do this, place the strainer with the arrow pointing up into a 50 mL conical tube or media bottle for culture volumes < 45 mL.
    Note: For suspension cultures in a 6-well plate, the small 37 µm Reversible Strainer can be used with a 15 mL conical tube. Collect aggregates on the smaller side of the strainer with the arrow pointing up.
    Note: When passaging the PBS-MINI 0.5 MAG Single-Use Vessel, modifications may be required for this step due to the large number of aggregates. Two options for modifications are as follows:
    • Use multiple 37 µm Reversible Strainers to collect the aggregates, then wash all of the aggregates into the same conical tube in Step 5.
    • Settle the aggregates at the bottom of the culture vessel for 30 seconds to 2 minutes, remove the spent medium, and add 10 mL D-PBS to rinse. Transfer the aggregates to a new 50 mL conical tube (Step 6), settle the aggregates again, and remove the D-PBS, then resuspend in GCDR.
  5. Carefully flip the strainer onto a new 50 mL conical tube ensuring that the arrow is pointing down, and rinse with 5 mL of warm GCDR while gently tapping the strainer to dislodge all aggregates into the new tube.
  6. After the aggregates are rinsed off the strainer, flip the strainer onto another new 50 mL conical tube such that the arrow is pointing up, and set aside. This strainer can be used to dissociate aggregates into small clumps (Step 11).
  7. Incubate the conical tube containing the aggregates and GCDR in a 37°C water bath for 6 minutes (undisturbed).
    Note: In this step, the aggregates are partially dissociated by the GCDR in preparation for generation of small clumps (Step 11).
    Note: Optimal incubation time may vary depending on the cell line.
  8. Centrifuge the conical tube for 3 minutes at 100 x g to collect any aggregates that have not settled.
  9. Using a serological pipette, slowly aspirate the GCDR, leaving ~0.5 mL to avoid removing any aggregates.
  10. Add 5 mL of TeSR™-AOF 3D supplemented with 10 µM Y-27632 Medium to the tube. Flick or gently swirl the tube to resuspend the aggregates.
    Note: If the culture has expanded > 5-fold over the course of the passage and the aggregate density is high, consider doubling the volume of TeSR™-AOF 3D supplemented with 10 µM Y-27632 used to resuspend the aggregates prior to passing through the 37 µm strainer.
  11. Using a 25 mL serological pipette, remove the resuspended, partially dissociated aggregates from the tube and place the serological pipette in contact with the strainer on the conical tube that was set aside at Step 6. With the serological pipette in a vertical orientation, press the tip gently against the strainer mesh (Figure 4), and slowly force the partially dissociated aggregates through the strainer at the slowest setting on the Pipette-Aid (0.5 mL/second flow rate). This will generate clumps of appropriate size to initiate the differentiation.
    Note: If the strainer appears clogged, slide the pipette laterally on the strainer while maintaining direct contact with it. Alternatively, increasing the flow rate slightly can help to prevent clogging, but use the lowest flow rate possible to minimize damage to the cells.
  12. Optional: Rinse the tube from Step 10 with 5 mL of additional TeSR™-AOF 3D Seed Medium. Pass through the strainer into the tube used in Step 11 to collect any remaining aggregates from the conical tube after GCDR dissociation.
  13. Proceed to Cell Counting and Seeding hPSCs to Initiate Differentiation (Day 0).

Table 4. Dissociation of 3D-Cultured hPSCs

hPSC Initiation Culture Volume (mL) Day 4 Culture Volume (mL) Volume of GCDR (mL) Volume of TeSR™-AOF 3D for Clump Resuspension (mL)
2 2.12 1 1
15 15.9 5 5
60 63.6 15 20
100 106 20 25
500 530 30 40 - 50
3D-Cultured hPSC aggregates being pushed through a cell strainer to generate a clump suspension.

Figure 4. Methods for Dissociating 3D-Cultured hPSCs

Image showing human pluripotent stem cell (hPSC) aggregates being pushed through a cell strainer to generate a clump suspension in Step 11 of the protocol for Preparation of 3D-Cultured hPSCs for Differentiation (Day 0). Preparation of 3D-Cultured hPSCs for Differentiation (Day 0). The tip of the serological pipette is pressed lightly onto the strainer to ensure cell aggregates are forced through, breaking up the aggregates to generate the appropriate hPSC clump size.

Cell Counting and Seeding of hPSC Clumps to Initiate Differentiation (Day 0)

Determine viable cell counts and calculate the volume of clump suspension required for seeding at the density recommended for your culture vessel of choice.

Note: It is recommended to use automated viable cell counts (e.g. A100-B Assay on the ChemoMetec Nucleocounter® NC-250™) to reduce seeding variability.

    For counting using the A100-B Assay on the ChemoMetec Nucleocounter® NC-250™:

  1. To determine clump density: Add 99.5 µL of clump suspension into an Eppendorf tube, then add 0.5 µL DAPI, mix by flicking the tube; DO NOT triturate. Load 31 µL of this cell sample into chamber 2 of NC-Slide A2™.
  2. To determine total viable cell counts: In a second Eppendorf tube, mix 99 µL of Reagent A100 with 1 µL DAPI, then add 100 µL clump suspension and triturate 20 times. Add 100 µL of Reagent B and triturate 5 times. Load 31 µL of this cell sample into chamber 1 of NC-Slide A2™.
  3. Perform cell count using the Nucleocounter® NC-250™.

    (Alternatively, use your preferred clump and viable cell counting method in place of Steps 1 - 3.)
  4. Pre-fill the suspension culture vessel with pre-warmed EB Formation Medium.
    Note: Especially when using larger culture volumes, pre-fill the culture vessel with medium and place it into the incubator at the start of the protocol to equilibrate the medium.
  5. Seed cell clumps at the seeding density recommended in Table 1: 1 x 105 - 2 x 105 live cells/mL.
  6. Place plate on orbital shaker (2.5 cm throw) at 70 RPM or place PBS-MINI vessel onto the MagDrive at the speed specified in Table 1.
    Note: On Day 1, formation of many embryoid bodies (cell aggregates) should be observed, as shown in Figure 3. If successful EB formation was not achieved, see Table 5 for troubleshooting.

50% Medium Exchanges (Days 2, 3, 7, and 10)

A 50% medium exchange should be performed on Days 2, 3, 7, and 10 using the following protocols, depending on the culture vessel of choice. Please follow the directions pertaining to either 2 mL cultures in a 6-well plate or 15+ mL cultures in filter-top bottles/PBS-MINI bioreactor vessels before proceeding to the protocol for 90% Medium Exchange (Day 5).

50% Medium Exchanges for 2 mL Culture in 6-Well Plate (Days 2, 3, 7, and 10)

50% medium exchanges in a 6-well plate should be performed on Days 2, 3, 7, and 10 using the following protocol; if using a 15+ mL vessel, please skip to the protocol for 50% Medium Exchanges for 15+ mL Cultures (Days 2, 3, 7, and 10).

Note: On Day 2, feed with EB Medium A, and on Days 3, 7, and 10, feed with EB Medium B.
  1. Pre-warm EB Medium A or B at room temperature.
  2. Tilt the plate for 30 - 60 seconds so that the EBs sink to the bottom of the well.
  3. Using a P1000 micropipette, remove 800 µL of medium per well and discard. Remove medium from the surface of the culture, avoiding picking up the EBs.
    Note: When feeding cells on Day 10, single cells in the spent medium can be collected and replaced back into the culture. This step allows you to retain suspended CD34+ cells and increases yield. To do this, pipette the spent medium into a Falcon® tube and Centrifuge at 300 x g for 5 - 10 minutes. Carefully aspirate the supernatant. Use a small volume of fresh medium to transfer the cells back into culture.
  4. Add 1 mL/well fresh medium into the culture.
50% Medium Exchanges for 15+ mL Cultures (Days 2, 3, 7, and 10)

50% medium exchanges in a filter-top bottle or PBS-MINI bioreactor vessel should be performed on Days 2, 3, 7, and 10 using the following protocol; if using a 6-well plate, please follow the protocol for 50% Medium Exchanges for 2 mL Culture in 6-Well Plate (Days 2, 3, 7, and 10) before proceeding to the protocol for 90% Medium Exchange (Day 5).

Note: On Day 2, feed with EB Medium A, and on Days 3, 7, and 10, feed with EB Medium B. This variation allows you to feed the suspension culture by capturing EBs on a strainer and washing them back into the culture.
  1. Aliquot and pre-warm EB Medium A or B, in a 37°C incubator, with the cap loosened.
  2. Prepare a collection vessel, Falcon® tube, or sterile bottle large enough to collect spent medium.
  3. Prepare a 37 µm reversible strainer.
  4. Place the strainer, arrow up, on the collection vessel.
  5. Using a serological pipette, remove 50% of spent medium and pipette gently through the strainer into the collection vessel, capturing EBs on the strainer.
    Note: When feeding cells on Day 10, single cells in the spent medium can be collected and replaced back into the culture. This step allows you to retain suspended CD34+ cells and increases yield. To do this, pipette the spent medium into a Falcon® tube and centrifuge at 300 x g for 5 - 10 minutes. Reserve approximately 5 mL of fresh medium and continue with the feed as normal. After centrifuging the spent medium, carefully aspirate the supernatant. Use the reserved fresh medium to transfer the cells back into culture.
  6. Flip the strainer onto the cell culture vessel.
  7. Pipette fresh medium through the strainer and into the cell culture vessel, washing any captured EBs back into culture.

90% Medium Exchange (Day 5)

A 90% medium exchange using EB Medium B should be performed on Day 5 using the following protocols, depending on the culture vessel of choice. Please follow the directions pertaining to either 2 mL cultures in a 6-well plate or 15+ mL cultures in filter-top bottles/PBS-MINI bioreactor vessels before proceeding to the protocol for Harvesting CD34+ Hematopoietic Stem and/or Progenitor Cells.

90% Medium Exchanges for 2 mL Culture in 6-Well Plate (Day 5)

A 90% medium exchange in a 6-well plate should be performed on Day 5 using the following protocol; if using a 15+ mL vessel please skip to the protocol for 90% Medium Exchanges for 15+ mL Cultures (Day 5).

  1. Pre-warm EB Medium B at room temperature.
  2. Tilt the plate for 30 - 60 seconds so that the EBs sink to the bottom of the well.
  3. Using a P1000 micropipette, remove 800 µL of medium per well and discard. Repeat to remove another 800 µL. Remove medium from the surface of the culture to avoid picking up the EBs.
  4. Add 2 mL/well of fresh medium into culture.
0% Medium Exchanges for 15+ mL Cultures (Day 5)

A 90% medium exchange in a filter-top bottle or PBS-MINI bioreactor vessel should be performed on Day 5 using the following protocol; if using a 6-well plate, please follow the protocol for 90% Medium Exchanges for 2 mL Culture in 6-Well Plate (Day 5) before proceeding to the protocol for Harvesting CD34+ Hematopoietic Stem and/or Progenitor Cells.

  1. Aliquot and pre-warm EB Medium B, in a 37°C incubator, with the cap loosened.
  2. Prepare a collection vessel, Falcon® tube, or sterile bottle large enough to collect spent medium.
  3. Prepare a 37 µm reversible strainer.
  4. Place the strainer, with the arrow pointing up, onto the collection vessel.
  5. Using a serological pipette, remove 90% of the spent medium and pipette gently onto the strainer into the collection vessel, capturing EBs on the strainer.
  6. Flip the strainer onto the cell culture vessel.
  7. Pipetting through the strainer, add 100% of the culture volume of fresh medium into the cell culture vessel, washing all captured EBs back into culture.

Harvesting CD34+ Hematopoietic Stem and/or Progenitor Cells

Strainers will be used to separate single cells from EBs. Single cells are used for further differentiation and EBs are discarded. If you wish to dissociate the EBs, refer to the protocols corresponding to the STEMdiff™ NK Cell Kit or STEMdiff™ T Cell Kit.

  1. Image the culture if desired.
  2. Prepare collection tubes by placing 70 µm strainer(s) onto Falcon® tube(s).
    Note: A 37 µm strainer can be used here, but may result in clogging when large culture volumes are used.
  3. Pipette the cell suspension up and down 2 - 3 times to break up any small cell aggregates. Pipette the entire cell suspension onto the cell strainer.
    Note: When working with culture volumes of greater than 100 mL, it may be necessary to use multiple strainers to avoid clogging.
  4. Rinse the culture vessel with EasySep™ Buffer, RoboSep™ Buffer, or D-PBS without calcium (Ca2+) and magnesium (Mg2+) containing 2% fetal bovine serum and 1 mM EDTA. Pipette onto the strainer to ensure all cells are collected. Discard the strainer.
  5. Centrifuge the single cell suspension at 300 x g for 5 - 10 minutes.
  6. Aspirate the supernatant and resuspend the remaining cell pellet in EasySep™ Buffer or your preferred culture medium for counting and downstream assays.
  7. Optional: Enrich CD34+ cells using EasySep™ Human CD34 Positive Selection Kit II. Perform the CD34+ isolation using the protocol optimized for embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) (Table 2 in the EasySep™ PIS); to increase yield, reduce the number of separations in the magnet to two while retaining a sufficient CD34+ purity for further culture.
  8. Optional: Cryopreserve CD34+ cells in CryoStor® CS10 freezing medium.
  9. Optional: Assess cell phenotype using flow cytometry for hematopoietic markers such as: CD34, CD45, CD43.

Optional: Downstream Differentiation

The colony-forming unit (CFU) assay demonstrates that this suspension protocol can generate multipotent progenitors capable of myeloid and erythroid differentiation (Figure 6). Cells cultured using this protocol are suitable for downstream differentiation to various immune cell types, for example, using the following kits: StemSpan™ NK Cell Generation Kit generates high yields of CD56+ NK cells expressing CD16 as well as natural cytotoxicity receptors capable of cytotoxicity against cancer cell lines (Figure 7); StemSpan™ T Cell Generation Kit generates CD4+CD8+ double-positive T cells expressing CD3 and TCRɑβ (Figure 8).

Data Figures

Flow plots and bar graphs showing the yield of CD34+ HSPCs generated via scalable suspension differentiation of hPSCs.

Figure 5. Scalable Suspension Differentiation of hPSCs Generated High Yields of CD34+ Hematopoietic Progenitors

Human pluripotent stem cells (hPSCs) differentiated into CD34+ hematopoietic stem and/or progenitor cells using a 12-day suspension culture protocol (Figure 1) were assessed by flow cytometry for expression of hematopoietic stem and/or progenitor cells markers CD34, CD43, and CD45 on Day 12. Differentiation culture volumes of 2 mL, 60 mL, and 100 mL were used. (A) Representative flow cytometry plots of H9-derived and WLS-1C-derived CD34+ cells are shown. (B) The average frequency of viable CD34+ cells across all cell lines was 73 ± 1.9%. (C) The average CD34+ cell yield across all cell lines was 1.3 x 106 ± 1.3 x 105 cells per mL of culture volume. (E) On average, a yield of 7 ± 0.5 CD34+ cells was generated per input hPSC. Data represent mean ± SEM (H9, WLS-1C, SCTi003-A, SCTi004-A, BiPSC; n = 54).

Representative images and colony counts showing multilineage differentiation of suspension culture-derived CD34+ cells.

Figure 6. Suspension Culture-Derived CD34+ Cells Were Capable of Multilineage Differentiation in a CFU assay

Suspension cultures in 2 mL, 60 mL, and 100 mL were utilized to differentiate human pluripotent stem cells (hPSCs) to CD34+ hematopoietic stem and/or progenitor cells. To determine their myeloid potential, CD34+ hematopoietic stem and/or progenitor cells were plated in MethoCult™ H4435 Enriched. 14 days later, the formed colonies were imaged using STEMvision™. Images were used to manually identify and count colonies. (A) Representative images for BFU-E (top), CFU-G/M (middle), and CFU-GEMM (bottom) colonies. Scale bars are 300 µm. (B) CFU assay data from 4 hPSC lines. The total average number of colonies formed from 5,000 hPSC-derived CD34+ cells is 116 ± 14. Data represents mean ± SEM (H9 n = 3, WLS-1C n = 1, SCTi003-A n = 3, BiPSC n = 2).

Flow plots and bar graphs showing the yield of functional NK cells generated from suspension-culture derived HSPCs.

Figure 7. High Yields of Functional NK Cells Can Be Generated from Suspension Culture-Derived CD34+ Hematopoietic Stem and/or Progenitor Cells

Suspension cultures in 2 mL, 60 mL, and 100 mL were utilized to differentiate human pluripotent stem cells (hPSCs) to CD34+ hematopoietic stem and/or progenitor cells. (A) hPSC-derived CD34+ hematopoietic stem and/or progenitor cells were differentiated into natural killer (NK) cells in static cultures using StemSpan™ NK Cell Generation Kit reagents and assessed by flow cytometry for the expression of NK cell markers CD56, CD16, NKp44, and NKG2D on Days 21 - 24. Representative flow cytometry plots are shown for (B) H9-derived NK cells and (C) SCTi004-A-derived NK cells. (D) The average frequency of viable CD56+ NK cells across all cell lines on Days 21 - 24 was 80 ± 2.4%; data represent mean ± SEM (n = 25). (E) The average yield of CD56+ NK cells was 2356 ± 492 cells per input hPSC (n = 25). (F) The average percentage of K562 cell killing by H9-derived NK cells at E:T ratios of 0.3:1, 1:1, and 3:1 was 20 ± 10%, 40 ± 16%, and 64 ± 14%, respectively (n = 4), which was comparable to that of peripheral blood (PB)-isolated NK cells (18 ± 6.4%, 43 ± 13%, and 79 ± 7.0%, respectively; n = 6). (G) At an E:T ratio of 3:1, SKOV3 cell killing by H9- and WLS-1C-derived NK cells (21% and 25%, respectively) was comparable to PB-isolated NK cells (23%); at 10:1 cytotoxicity by H9- and WLS-1C-derived NK cells (43% and 48%, respectively) was reduced relative to PB-isolated NK cells (69%; n = 2).

Flow plots and bar graphs showing the efficient generation of double-positive T cells from suspension culture-derived CD34+ HSPCs.

Figure 8. Double-Positive T Cells Can Be Efficiently Generated from Suspension Culture-Derived CD34+ Hematopoietic Stem and/or Progenitor Cells

Suspension cultures in 2 mL, 60 mL, and 100 mL vessels were utilized to differentiate human pluripotent stem cells (hPSCs) to CD34+ hematopoietic stem and/or progenitor cells. (A) Suspension culture-derived CD34+ hematopoietic stem and/or progenitor cells were differentiated into CD4+CD8+ double-positive (DP) T cells in static cultures using StemSpan™ T Cell Generation reagents and StemSpan™ Lymphoid Differentiation Coating Material, and were analyzed by flow cytometry for the expression of T cell markers CD4, CD8, CD3, and TCRαβ. (B) Representative flow cytometry plots of H9 embryonic stem (ES) cell-derived T cells. (C) The average frequency of viable DP T cells on Days 21 - 24 was 38 ± 6.6%. (D) The average yield of DP T cells was 146 ± 50 cells per input hPSC. (E) The average frequency of CD3+TCRαβ+ cells within the DP T cell population was 17 ± 3.7%. Data represent mean ± SEM (n = 10).

Table 5. Troubleshooting for Generation of CD34+ Cells in Suspension Culture

Problem Possible Solution
Embryoid Body (EB) formation is not observed on Day 1 Ensure seeded clumps are not too small or over-triturated. Viability of clump suspension should be at least 60%. Ensure that Day 0 culture is supplemented with 10 µM Y-27632.
Increase hPSC clump seeding density by 0.5 - 1 × 105 live cells/mL.
For 2D-maintained hPSCs, feed cultures with mTeSR™ Plus for 1 passage cycle prior to seeding if not already in mTeSR™ Plus.
EBs are too large or merged together Ensure that EBs are not allowed to settle for longer than 5 minutes. If feeds take longer, swirl the culture manually as needed.
Reduce hPSC clump size at seeding on Day 0 by increasing incubation in Gentle Cell Dissociation Reagent and/or increasing triturations up to 2 triturations.
Lower than expected CD34+ cell yield Increase hPSC clump seeding density equivalent to 2 × 105 live cells/mL. This may be particularly beneficial in vessels of 100 mL and larger.
If a high frequency of CD34-CD45+ cells is observed on Day 12, an earlier cell harvest may be beneficial.
CD34+ cells have poor lymphoid differentiation potential If cell culture has a high density of suspended CD34+ cells by Day 12 (i.e. >2 × 106 live CD34+ cells/mL), add an additional 50% medium exchange with EB Medium B on Day 11. Cells can be harvested on Days 10 - 11.
If cell culture density exceeds 2 x 106 live CD34+ cells/mL by Day 12, reduce seeding density of hPSC clumps by 5 × 104 - 1 × 105 live cells/mL in subsequent experiments.
If CD34+ cell frequency is <50% at Day 12, enrich for CD34+ cells using EasySep™ Human CD34 Positive Selection Kit II. Perform the CD34+ isolation using the protocol optimized for ESC or iPSC cultures (see Table 2 in the EasySep™ PIS); to increase yield, reduce the number of separations in the magnet to two while retaining a sufficient CD34+ purity for further culture.
Review troubleshooting steps in the protocol for the STEMdiff™ NK Cell Kit. Additionally, it may be beneficial to optimize timing of the Lymphoid Progenitor Stage of NK cell differentiation. A reduction of the Lymphoid Progenitor Stage from 14 days to 7 - 10 days may be beneficial for improving NK cell purity and yield.

Acknowledgements

We would like to acknowledge Dr. Deepta Bhattacharya from the University of Arizona for kindly providing the B cell-derived iPSC cell line (BiPSC).


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