The Op9-Dl1 System: Generation Of T-Lymphocytes From Embryonic Or Hematopoietic Stem Cells In Vitro

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-cu