Authors: Roxanne Holmes and Juan Carlos Zúñiga-Pflücker Corresponding author: [jczp@sri.utoronto.ca](jczp@sri.utoronto.ca) ### INTRODUCTION Differentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versatile, and efficient culture system that allows for the commitment, differentiation, and proliferation of T-lineage cells from different sources of stem cells. This article contains a series of protocols, the first of which describes the establishment, maintenance, and storage of OP9 and OP9-DL1 cells. Subsequent protocols detail how to co-culture the OP9 and OP9-DL1 cells with either ESCs or HSCs from fetal liver or bone marrow, leading to in vitro differentiation of the stem cells into lymphocytes. ### RELATED INFORMATION Preparation of the OP9 and OP9-DL1 cells should be started ~1 wk prior to initiating co-cultures with ESCs or HSCs. Protocols 3 and 4 use HSCs isolated from murine fetal liver or bone marrow. HSC isolation protocols are widely available (Klug and Jordan 2002; Schmitt et al. 2004; de Pooter et al. 2006; Bunting 2008). For a protocol to isolate and maintain mouse embryo fibroblasts (mEFs), see [Preparing Mouse Embryo Fibroblasts](http://cshprotocols.cshlp.org/lookup/doi/10.1101/pdb.prot4398) (Nagy et al. 2006a). For a protocol to derive or maintain ESCs, see [De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts](http://cshprotocols.cshlp.org/lookup/doi/10.1101/pdb.prot4403) (Nagy et al. 2006b). ### MATERIALS **Reagents** 1. Anti-CD24 monoclonal antibody (mAb) (J11d clone) (for Protocol 3) - *Use either culture supernatant that contains anti-CD24 mAb or purified anti-CD24 mAb (see Step 55)*. - BDPharmLyse (red blood cell lysing reagent; BD Biosciences 555899) (for Protocol 4) - Buffer for cell staining and cell sorting (for Protocol 4; see Step 72) - Complement, reconstituted from rabbit (Cedarlane CL3331) (for Protocol 3) - ESC medium (for Protocols 1, 2) - Freezing medium for OP9 cells (for Protocols 1, 2) - Leukemia inhibitory factor (LIF; 10 μg/mL; Chemicon LIF2010) (for Protocol 2; see Step 20) - Lympholyte-M (Cedarlane CL5120) (for Protocol 3) - Mice, 4-8 wk old (for Protocol 4; see Step 69) - Mice, fetal, at embryonic day 13-15 (for Protocol 3; see Step 52) - Mouse embryonic fibroblast (mEF) cells, irradiated and growing in a 6-cm dish (for Protocol 2) - *Alternatively, a gelatin-coated 6-cm dish can be used. For a protocol to isolate and maintain mEFs, see [Preparing Mouse Embryo Fibroblasts](http://cshprotocols.cshlp.org/lookup/doi/10.1101/pdb.prot4398) (Nagy et al. 2006a)*. - Mouse embryonic stem cells (ESCs), stored frozen in liquid nitrogen (for Protocol 2) - *R1 ESCs can be obtained from ATCC (SCRC-1036). For a protocol to derive or maintain ESCs, see [De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts ](http://cshprotocols.cshlp.org/lookup/doi/10.1101/pdb.prot4403)(Nagy et al. 2006b)*. - OP9 or OP9-DL1 cells, stored frozen in liquid nitrogen - *OP9 cells can be obtained from the Riken Laboratory Cell Repository (Japan), and then transduced with a retroviral construct encoding Delta-like-1 to generate OP9-DL1 cells (Schmitt and Zúñiga-Pflücker 2002), or they can be requested from the Zúñiga-Pflücker laboratory*. - OP9-DL1 or OP9 cells growing on a 6-well plate (for Protocol 2; see Step 39) - *OP9-DL1 cells are used to generate T-lineage cells, and OP9 cells are used to generate B-lineage and myelo-erythroid cells*. - OP9 medium - Phosphate-buffered saline (PBS; Hyclone SH30256) - Recombinant human Flt-3/Flk-2 ligand (R&D; Systems 308-FK) (for Protocols 2-4) - Recombinant murine IL-7 (Peprotech 217-17) (for Protocols 2-4) - Trypsin solution, 0.25% (Invitrogen 15090046) (for Protocols 1, 2) - *Dilute stock trypsin to 0.25% with PBS*. ### Equipment 1. Biosafety cabinet - Cell strainers (40-μm pore size; BD Falcon 352340) (for Protocols 2-4) - Centrifuge - Flow cytometer (for Protocols 2-4) - Forceps, 4-in. straight tips (Electron Microscopy Sciences 72991-4S) (for Protocol 4) - Forceps, curved fine points Dumont #7 (Electron Microscopy Sciences, 72800-D), and super fine points Dumont #5 (Electron Microscopy Sciences, 72700-D) (for Protocol 3) - Glass stopper, No. 24 (for Protocol 4) - Incubator, humidified (37°C and 5% CO2) - Liquid-nitrogen cell storage system - Magnetic assisted cell sorter (MACS) (optional; see Protocol 4, Step 73) - Microscope - Plunger from a 3-mL syringe (for Protocol 3) - Scissors, dissecting (Electron Microscopy Sciences 72940) (for Protocols 3, 4) - Tissue culture plasticware - *Carry out all procedures using standard aseptic technique with sterile plasticware (tissue-culture-treated 10-cm and 6-well plates, 15-mL and 50-mL centrifuge tubes, 2-mL cryovials, serological pipettes, and pipette filtered tips)*. - Water bath pre-set to 37°C (for Protocols 1, 2) ### METHOD **Protocol 1: Preparation of OP9 Cells for Co-culture** **Thawing OP9 or OP9-DL1 Cells** 1. Add 12 mL of OP9 medium to a 15-mL centrifuge tube. - Thaw OP9 or OP9-DL1 cells quickly in a 37°C water bath. - Pipette the cell suspension slowly and gently from the cryovial tube, and transfer the contents to the 15-mL tube containing medium. - Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. Resuspend the cells in 10 mL of OP9 medium. - Transfer the resuspended cells into a 10-cm dish, and place the dish in an incubator. **Maintaining OP9 or OP9-DL1 Cells** 6.To passage the cells, remove the medium from the 10-cm dish. *The cells will become confluent in the dish within 2-3 d, depending on the method used to freeze the cells. Passage the cells before they reach ~80% confluency (Fig. 1)*.  **Figure 1**. Photomicrographs of ESC/OP9 co-culture. (a) Undifferentiated ES cells on mEF. (b) Monolayer of OP9 cells. (c) Day 0 ESC/OP9 co-culture. (d,e) Day 5 ESC/OP9 mesoderm-like colonies. (f) Day 8 ESC/OP9 small, round clusters of cells. (g) Day 12 ESC/OP9-DL1 hematopoietic cells. (h) Day 16 ESC/OP9-DL1 hematopoietic and early T-lineage cells. (i) Day 20 ESC/OP9-DL1 T-lineage cells. 7.Wash the plate with 4 mL of PBS. Discard the PBS. 8.Trypsinize the cells with 4 mL of 0.25% trypsin solution, and incubate the cells for 5 min at 37°C. 9.Disaggregate the cells from the dish by pipetting them up and down, and transfer the cell suspension into a 15-mL tube containing 4 mL of OP9 medium. 10.Wash the plate with PBS to remove any remaining cells, and transfer these cells to the same 15-mL tube. 11.Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 4 mL of OP9 medium. 12.Transfer 1-mL aliquots of cells to four 10-cm dish each containing 9 mL of OP9 medium. *Maintain the cells by splitting them at a ratio of 1-to-4, and passaging the cells every 2 d. Do not keep the cells in continuous culture for more than 6 wk*. *The cells can be used in subsequent protocols; e.g., Steps 28, 59, and 74*. **Freezing OP9 or OP9-DL1 Cells** 13.Passage the cells as described in Steps 6-11, except resuspend the cells (~8-10 × 10e5 cells), in 2 mL of freezing medium for OP9 cells per 10-cm dish. *Preferably, freeze cells within the first two to three passages*. 14.Aliquot 1 mL of cell suspension per cryovial. 15. Freeze the cells at -80°C, and then transfer them to liquid nitrogen for storage. **Protocol 2: In Vitro Generation of T-Lymphocytes from ESCs** **Thawing ESCs** 16.Prepare a 15-mL tube containing 12 mL of ESC medium. 17.Thaw the ESCs quickly in a 37°C water bath, and transfer the thawed cells slowly into the 12 mL of ESC medium. 18.Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend in 3 mL of ESC medium. 19.Seed ESCs onto a 6-cm dish containing irradiated mEF cells or a gelatin-coated 6-cm dish. 20.To keep the ESCs from differentiating, add 10 ng/mL of LIF when grown on mEF cells or 20 ng/mL of LIF when grown on gelatin. *The mEF cells can be irradiated up to 2 d before using them as feeder cells. It is important that the mEF cell layer completely covers the surface of the tissue culture dish, because ESCs will begin to undergo differentiation within mEF cell-free areas*. **Maintaining ESCs** 21.The following day, change the ESC medium and again add the appropriate concentration of LIF. 22.Passage the ESCs the next day with trypsin-mediated disaggregation. *Trypsin-mediated passage is used to break up large ESC colonies, allowing the culture to expand*. - i. Remove the medium from the 6-cm dish. Wash the plate with 3 mL of PBS. Discard the PBS. - ii. Trypsinize the cells with 4 mL of 0.25% trypsin solution, and incubate the cells at 37°C for 5 min. - iii. Disaggregate the cells from the dish by pipetting them up and down, and transfer the cell suspension into a 15-mL tube containing 3 mL of ESC medium. - iv. Wash the plate with PBS to remove any remaining cells, and transfer these cells to the same 15-mL tube. - v. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 3 mL of ESC medium. 23.Seed ESCs in 3 mL of ESC medium onto irradiated mEF cells or gelatin-coated plates, and add LIF. 24.Repeat Steps 22 and 23 until the ESCs are needed for co-culturing. *The ESCs grow as colonies. If they become too crowded, passage the cells more sparsely (Fig. 1a)*. **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 × 10e5 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*.  **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 × 10e4 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. 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 × 10e5 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 enough cells 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 by forceful 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 the incubator. *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*.  **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** 50.Repeat Steps 41-43. **Day 20 and Onward, at 4-d to 5-d Intervals: No-Trypsin Passage** 51. Repeat Steps 44-47. *The differentiating lymphocytes expand rapidly (Fig. 1i), requiring daily monitoring to determine whether to simply change medium or to passage/split the co-cultures into additional dishes. For ESCs cultured on OP9, the majority of the cells will be CD19+ B-lineage cells. For ESCs cultured on OP9-DL1, the cells are mostly CD4+- and CD8+-expressing T-lineage cells (Fig. 3)*. **Protocol 3: Fetal Liver-Derived HSC Differentiation on OP9-DL1 Cells Day 0: Initiation of Fetal Liver Co-culture** 52.Isolate liver tissue from eight to 10 fetal mice at embryonic day 13-15 (E13-E15), using scissors and curved forceps. 53.Force the fetal livers through a 40-μm cell strainer using the rubber end from a 3-mL syringe plunger, and wash the strainer with OP9 medium. 54.Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 4 mL of OP9 medium in a 15-mL tube. 55.To enrich for HSCs, add 1 mL of reconstituted rabbit complement and either 5 mL of culture supernatant containing anti-CD24 mAb (J11d clone) or 5 ml of OP9 medium containing 1 ng of purified anti-CD24 mAb per 10e6 cells. *CD24 is expressed in all Lineage+ cells in E13-E15 fetal liver cells*. 56.Incubate for 30 min at 37˚C. 57.Slowly underlay 7 mL of Lympholyte-M to the bottom of the cell suspension, and centrifuge at 580g (1800 rpm) for 10 min at room temperature. 58.Carefully transfer the interphase layer to a 50-mL tube, fill it with OP9 medium, and centrifuge again at 450g (1600 rpm) for 10 min at room temperature. *These cells will contain ~80%-95% CD117+ hematopoietic progenitor cells, including an enriched fraction of CD117+ Sca-1+ Lineageneg cells. These CD24-depleted fetal liver cells can be seeded directly onto OP9 or OP9-DL1 cells for co-culture. Alternatively, the HSC-enriched fetal liver cell suspension can be stained and sorted for CD117+ Sca-1hi Linneg cells by flow cytometric cell sorting*. 59.Seed 4-6 × 10e4 cells in 10 mL of OP9 medium per 10-cm dish of 80%-90% confluent OP9 or OP9-DL1 cells, and add 5 ng/mL Flt-3L and 1 ng/mL IL-7. Place the dishes in the incubator. *The OP9 and OP9-DL1 cells were prepared in Steps 1-12*. **Day 4: Medium Change** *Steps 60-62 are optional if low numbers (6 wk) before initiating a co-culture. Extended periods of culturing will increase the likelihood that the cells will deteriorate and become less effective. Thus, it is highly recommended that a co-culture be started within 2 wk of thawing the OP9 cells, which has the added advantage that the same OP9 cells can be used for the entire co-culture period. Another variable to manage is the FBS used in the co-cultures, which can affect the growth and proliferation of the co-cultures. Test several lots of serum to determine the one that performs well and yields results similar to those shown in these protocols (Figs. 3, 4, and 5). The differentiation of lymphocytes from ESCs can be readily observed 10-12 d after the initiation of the co-cultures (Fig. 3). Prior to this stage, the majority of the cellular subsets are comprised of erythro-myeloid cell lineages. Beyond the second week of co-culture, B- or T-lineage cells (OP9 or OP9-DL1 co-cultures, respectively) predominate and expand rapidly. These cells display and follow a normal pattern of lineage differentiation, such as lineage-specific differentiation checkpoint events, to yield functional lymphocytes (Schmitt and Zúñiga-Pflücker 2002; Schmitt et al. 2004; de Pooter et al. 2006). As seen in Figures 4 and 5, the differentiation of fetal liver HSCs to the B- or T- cell lineage takes place with faster temporal kinetics than that of bone marrow HSCs. Additionally, ESC co-cultures, as expected, require a much longer period of time to yield lymphocytes because of the additional time involved in the initial hematopoietic induction and differentiation steps when starting from an ESC (Fig. 3). Although different stem cells give rise to lymphocytes with different kinetics, the overall differentiation steps follow a similar pattern. Additionally, if lymphocyte progenitors (CD117+ CD127+) from the bone marrow or thymus or more downstream progenitors such as pre-B- (CD117+ CD19+) or pre-T-cells (CD44+ CD25+ CD3− CD4− CD8−) are used, then the differentiation kinetics are accelerated from those shown in Figures 4 and 5. ### ACKNOWLEDGMENTS We thank the many members of the Zúñiga-Pflücker lab who over the years have helped to develop, test, and optimize the methods listed above. In particular, we are grateful to James Carlyle, Sarah Cho, Renée de Pooter, Dzana Dervovic, Ross La Motte-Mohs, Thomas Schmitt, Gladys Wong, and John Xu. Additionally, we thank Korosh Kianizad and Tina Wang for their helpful comments. We also appreciate the kind assistance and generosity of Tasuko Honjo and Toru Nakano for initially sharing the OP9 cells and expertise with us. These protocols were developed with support from the Canadian Institutes of Health Research and with funds from the Canadian Cancer Society. ### REFERENCES 1. 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