EasySep™小鼠TIL（CD45）正选试剂盒
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
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
- 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:
- 6-Well Flat-Bottom Plate, Non-Treated (Catalog #38040)
- Nalgene™ Rapid-Flow™ Sterile Filter Storage Bottles (Thermo Fisher; Catalog #455-0250)
- PBS-MINI Bioreactor Base Unit (Catalog #100-1005) with PBS-MINI 0.1 MAG Single-Use Vessel (Catalog #100-1006) or PBS-MINI 0.5 MAG Single-Use Vessel (Catalog #100-1007)
- 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
- 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
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
30 mL‡
60 mL‡
55 RPM
65 RPM
*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.
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.
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.
- 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.
- 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.
- 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)
- Prepare EB Medium A and EB Formation Medium according to Table 2.
- Prepare medium for hPSC clump resuspension:
- For 2D-maintained hPSC culture, use DMEM/F12 supplemented with 10 µM Y-27632 for suspension of hPSC clumps.
- For 3D-maintained hPSC culture, use TeSR™ -AOF 3D supplemented with 10 µM Y-27632.
- 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).
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.
- 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.
- Aspirate hPSC culture medium, and rinse with 10 mL D-PBS.
- Aspirate D-PBS and add 5 mL Gentle Cell Dissociation Reagent (GCDR).
- Allow 4 - 6 minutes for dissociation at room temperature.
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