TheOP9DL1System:GenerationofTLymphocytesfromEmbryonic2

• Freezing ESCs 
  

• 25. Passage the ESCs as described in Step 22. Resuspend the cells in 3 mL of freezing medium for OP9 cells. 
  
  

• 26. Aliquot 1 mL of cell suspension per cryovial (~3-6 x 10⁵ cells/cryovial). 
  
  

• 27. Freeze the cells at -80°C, and then transfer them to liquid nitrogen for storage. 
  

Establishing ESC/OP9-DL1 Cell Co-cultures

• Day 0: Initiation of Co-culture 
  See Figure 2 for a schematic of the ESC/OP9 co-culture. 
  
  

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  Figure 2. Schematic overview of ESC/OP9(-DL1) co-culture system, with key steps highlighted.

• 28. For each 10-cm dish containing 80%-90% confluent OP9 cells, remove the medium and replace it with 9 mL of fresh OP9 medium. 
  The OP9 cells were prepared in Steps 1-12. 
  
  

• 29. Harvest ESCs as a single cell suspension by trypsin-mediated disaggregation (Step 22). Resuspend the cells to a concentration of 5 x 10⁴ cells per mL of OP9 medium. 
  
  

• 30. Seed 1 mL of ESCs per 10-cm dish of OP9 cells, and return the dishes to the incubator (Fig. 1c). 
  Use OP9 cells (not ectopically expressing Dll1) to initiate the culture, because a more robust population of mesoderm colonies grows on these stromal cells as opposed to OP9-DL1 cells. The cells will be transferred onto OP9-DL1 cells at day 8 to induce T-lineage differentiation (Fig. 2). 
  See Troubleshooting. 
  

• Day 3: Medium Change 
  

• 31. Remove the old medium, replace it with 10 mL of OP9 medium, and return cells to the incubator. 
  The ESCs should have less defined borders and should start forming mesoderm colonies. 
  

• Day 5: Trypsin-Mediated Passage and Pre-plating 
  

• 32. Remove the medium and trypsinize the cells (see Steps 7 and 8). 
  Mesoderm colonies should be visible (Fig. 1d,e), and some colonies will appear three-dimensional with a wagon wheel shape, whereas others will appear like small cells clustered in a pile or as bunches. These colonies will become visible without the aid of a microscope and will appear as white circles in the dish. 
  
  

• 33. Resuspend the trypsin-disaggregated cells with 4 mL of OP9 medium. 
  
  

• 34. Return the dish to the incubator for 30 min. 
  This pre-plating step will allow OP9 cells to readhere to the dish, which will limit the number of OP9 cells that are transferred. 
  
  

• 35. Wash the non-adherent cells off the dish, filter the cells through a 40-µm cell strainer, and centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. 
  
  

• 36. Resuspend the cells in OP9 medium, seed 5 x 10⁵ cells per 10-cm dish containing ~80% confluent OP9 cells in 10 mL of OP9 medium, and add 5 ng/mL of recombinant human Flt-3L.Return the dish to the incubator. 
  The number of 10-cm dishes to be seeded should be equal to, or greater than, the number of analysis time points required. This ensures that the cultures remain undisturbed and there are enough samples for analysis (e.g., flow cytometry). Each 10-cm dish at day 5 yields enoughcells to seed six to eight 10-cm dishes. 
  

• Day 8: Harvesting Semiadherent Hematopoietic Cells 
  

• 37. Using the medium that is in the dish, wash the cells gently enough to keep the OP9 cell monolayer intact, but use enough force to remove the semiadherent cells. 
  Clusters of round, shiny cells should be visible as small groups, either semiattached to the OP9 cells or in suspension (Fig. 1f). Check the dish under the microscope to ensure that all the round, shiny cells have been removed. 
  See Troubleshooting. 
  
  

• 38. Filter the cells through a 40-µm cell strainer, and centrifuge at 400g (1500 rpm) for 5 min at 4°C. 
  
  

• 39. Resuspend the cells in 3 mL of OP9 medium per 10-cm dish initially seeded on day 5, and seed 3 mL per well of a 6-well plate containing either OP9-DL1 cells to generate T-lineage cells or OP9 cells to generate B-lineage and myelo-erythroid cells. 
  
  

• 40. Add cytokines to each well; final concentrations are 5 ng/mL Flt-3L and 1 ng/mL. 
  

• Day 10: Medium Change 
  

• 41. Remove the medium carefully so as to not disturb the co-culture. 
  Large clusters of round, shiny cells should be present, with some cells detached in suspension. Collect all of the medium (which may contain differentiating lymphocytes) and centrifuge these cells at 400g (1500 rpm) for 5 min at 4°C, thus limiting cell loss during the medium change. 
  
  

• 42. Centrifuge the medium at 400g (1500 rpm) for 5 min at 4°C. Resuspend the cells present in the spent medium with 3 mL of OP9 medium containing 5 ng/mL Flt-3L and 1 ng/mL IL-7. 
  
  

• 43. Transfer the medium and cells to the same well, and return the plates to the incubator. 
  

• Day 12: No-Trypsin Passage 
  

• 44. Disaggregate the cells without the use of trypsin by simply pipetting them up and down to create a cell suspension. 
  Large clusters of round, shiny cells should be present throughout the dish, with some cells detached in suspension (Fig. 1g). The OP9 cells may lift as a sheet, which will be broken up byforceful pipetting. 
  
  

• 45. Filter the cells through a 40-µm cell strainer. 
  
  

• 46. Wash the well with 3 mL of PBS, filter the suspension through the same cell strainer, and centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. 
  
  

• 47. Resuspend the cells in 3 mL of OP9 medium, and seed the cells onto a new well of OP9 or OP9-DL1 cells, with the addition of 5 ng/mL Flt-3L and 1 ng/mL IL-7. Return the plates to theincubator. 
  This is a good time point to analyze by flow cytometry to determine the presence of different lympho-hematopoietic cell populations. As shown in Figure 3 , for ESCs cultured on OP9 cells, the majority of cells will be erythro-myeloid Ter119⁺ or CD11b⁺, while for ESCs cultured on OP9-DL1 cells, most of the cells will resemble DN1 (CD44⁺ CD25^-) stage thymocytes, although some DN2 (CD44⁺ CD25⁺) and DN3 (CD44^- CD25⁺) stage cells may be present. 
  
  

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  Figure 3. Flow cytometry analysis for the indicated cell surface markers of ESC/OP9 co-cultures and ESC/OP9-DL1 co-cultures at the indicated time points and culture conditions.

• Day 14: Medium Change 
  

• 48. Repeat Steps 41-43. 
  

• Day 16: No-Trypsin Passage 
  

• 49. Repeat Steps 44-47. 
  By this time point, the dish should be replete with differentiating lymphocytes (Fig. 1h). For ESCs cultured on OP9, CD19⁺ cells will be starting to emerge. For ECSs cultured on OP9-DL1, cells that are mostly at the DN3 and DN4 stage of thymocyte development will be present, and some CD4⁺ CD8⁺ (double positive, DP) may start to appear (Fig. 3). 
  

• Day 18 and Onward, at 2-d to 4-d Intervals: Medium Change