Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.
The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.
A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture “microenvironment” that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals.
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this “day 5” passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.
1. Preparation of Culture Media, Cytokines and Gelatinized Plates
2. Preparation and Maintenance of Feeder Cells and Mouse Embryonic Stem Cells (mESC)
NOTE: Incubate all cells in a humidified 37 °C incubator with 5% CO2.
3. OP9-DL1 Co-culture Procedure
When grown on MEFs in the presence of LIF, mESCs can be maintained in an undifferentiated state. Under ideal conditions, they appear as compact colonies of cells surrounded by a shiny halo in phase contrast microscopy (Figure 1). These cultures must be monitored daily. Depending on the confluence of the cells, media can be changed or cells can be split. Neighboring mESC colonies should not come to the point of contact with one another. A normal, healthy culture of undifferentiated mESCs is an essential starting point for in vitro T cell differentiation. Upon initiating co-culture, it is recommended that cells be fully confluent without being overcrowded. This is so the majority of cells harvested for seeding cells onto OP9 monolayers are mESC (rather than feeder cells). If major differences in confluence are observed among the various mESC clones to be differentiated at day 0, then it would be best to remove MEFs by pre-adherence to tissue culture dishes (as in step 3.4.3-3.4.6 using complete ES cell media) before counting the mESCs for co-culture seeding.
OP9 cells must be well maintained prior the co-culture initiation (as described in section 2.4). In addition, it is thought that OP9 cells lose some of their properties with prolonged culture and/or pronounced increases in their proliferation rate.7 Avoiding split ratios beyond 1:5 during maintenance of OP9 cells, and discarding continuous OP9 cultures after six weeks, will help avoid these pitfalls.
At initial cell seeding and cell transfer steps of the co-culture (day 0, 5, 8, 12, 16), OP9 monolayers should be within an optimal range of confluence. Figure 2 shows the low (Figure 2A) and high (Figure 2B) ends of the range of OP9 cell confluence advisable for use as co-culture monolayers. An example of an over-confluent plate is shown in Figure 2C. Failure to maintain optimal confluence may induce formation of adipocytes (Figure 2D) that, in large amounts, can negatively affect the co-culture.
On day 0, the co-culture is started on OP9 cells rather than OP9-DL1 cells since mesoderm formation is favored on OP9 cells. Cells are plated on OP9-DL1 cells monolayers beginning on day 8 to induce T cell production. While some mESC clones will visually display robust and quantitative (~90%) formation of mesoderm-like colonies by day 5 of co-culture (Figure 3A-B), others can display delays at this step (Figure 3C-F). Under the microscope, the mesoderm-like colonies derived in this assay have been variably described as resembling wagon wheels or craters (Figure 4A) or, starbursts or florets (Figure 4B).
While the published OP9-DL1 protocol reflects a norm of quantitative mesoderm formation by day 5 of co-culture,7 we find that not all ESC clones achieve that norm within this time frame. In these cases, we change the medium on day 5 and wait an additional 1-2 days before conducting the “day 5” cell harvest/transfer protocol. The goal of this delay is to allow ~80-90% of ES cell colonies to visually become mesodermal prior to transfer. It is important to make clear that this 80-90% figure reflects a strictly visual criterion discernible by simple phase contrast microscopic inspection of colony morphology in the co-culture dishes. It refers only to the proportion of ESC colonies that resemble those shown in Figure 4. It is possible that some ESC clones will not reach this visual criterion, even after a two-day delay. In such a case, it is possible to enrich for blood forming mesodermal precursors using flow cytometry cell sorting for Flk-1+ cells, as has been previously described.6,11 In any case, for the sake of nomenclature convenience, we “reset the clock” and refer to the day of mesoderm-like colony transfer as “day 5.”
When multiple ESC clones are being differentiated in parallel, it is possible that some co-cultures will be ready for the day 5 passage on time, while others lag behind. In this case, it is advisable to delay the passage of all the co-cultures until the slower clones catch up to the others. The delay seems not to do any harm to the “on time” co-cultures, but benefits the subsequent differentiation of the slower clones a great deal.
Day 8 (i.e., three days after mesodermal colony transfer) sees the emergence and accumulation of HPCs. HPCs are visible as shiny clusters of cells that are loosely adhered to the OP9 feeder cells (Figure 5). A careful washing procedure, using a pipette and the media on the dish, will detach and collect the HPCs while leaving the OP9 monolayer mostly intact (Figure 6A-C). This is perhaps the most technique-sensitive step of the protocol. It takes some practice to find the appropriate motions and media ejection pressure to achieve the desired balance of maximal harvest of HPCs with minimal disruption of the feeder layer. It is acceptable if some of the OP9 monolayer lifts off, since the majority of any stray monolayer will be captured in the 40 μm nylon mesh after filtering. Figure 6D shows a monolayer after excessive pipetting leading to greater monolayer disruption than necessary. During this washing step, it is important to check under the microscope whether or not all the HPCs have been collected, and adjust the washing pressure accordingly.
Progress of the co-culture can be monitored by flow cytometry. To specifically analyze the non-erythroid hematopoietic progeny of the ESC, it is important to first gate on live CD45+ cells. This step screens out the OP9 cells from the analyses, while use of DAPI or other nuclear staining dye enables the exclusion of nonviable cells. By day 12, many clusters of small, round, shiny cells should be visible. It is not necessary to count the cells at this stage. But we have observed that live, gated flow cytometry event counts are typically in the range of 1-3 x 105 per day 12 co-culture well. Flow cytometry analyses of live-gated cells differentiated on the OP9 monolayer will yield erythroid (CD45neg, TER119+) and monocytic (CD45+, CD11bhi) lineages (Figure 7A). Analyses of live, CD45+ cells differentiating on OP9-DL1 monolayers should reveal markers characteristic of CD4/CD8 double negative (DN)-1 (CD44+, CD25neg, CD4neg, CD8neg) and DN2 (CD44+, CD25+, CD4neg, CD8neg) stage T cells (Figure 7B). By day 16, there is a significant increase in the amount of small, round, shiny cells in the cultures. On OP9-DL1s, T cell differentiation products progress to the DN3 (CD44neg, CD25+, CD4neg, CD8neg) and DN4 (CD44neg, CD25neg, CD4neg, CD8neg) stages. Some DP (CD4+, CD8+) T cells can also begin emerging by day 16 (Figure 7B). HPCs seeded on OP9 cells yield B cells (CD19+) by day 16. By day 20 of OP9-DL1-mESC co-culture, there will be large amounts of DP T cells and single positive (SP) CD8+ T cells present (Figure 7B). Figure 8 provides a diagram summarizing the key steps of the above procedures, and the suggested analysis time points during co-culture.
Figure 1. Phase contrast microscopy image of undifferentiated mESCs (sharp edged colonies) growing on top of a MEF monolayer (100X magnification).
Figure 2: Phase contrast microscopy images of OP9 monolayer confluency. (A) Lowest and (B) highest confluency levels advisable for co-culture passage/seeding (40X magnification). (C) Example of an over-confluent OP9 monolayer (40X magnification). (D) Over-confluent OP9 monolayer (200X magnification) with adipocyte formation (large, vesicle containing, cells).
Figure 3: Phase contrast microscopy images of mesoderm-like colony formation at “day 5” of co-culture. (A) 40X and (B) 100X views of co-culture plates with >90% mesodermal colony differentiation. These plates are ready for day 5 passage. (C-F) Examples of Day 5 co-culture plates that require 1-2 day postponing before transfer (C&E, 40X magnification, D&F, 100X magnification).
Figure 4: Phase contrast microscopy images of the variety of mesoderm-like colony formations at day 5 of co-culture. (A) The colony morphology referred to as “craters” or “wagon wheel.” (B) The colony morphology referred to as “starburst” or “florets” (200X magnification).
Figure 5. Phase contrast microscopy image of day 8 co-culture plate with clusters of small, round, shiny HPCs on an OP9 monolayer (40X magnification).
Figure 6: OP9 monolayer after HPCs collection on co-culture day 8. (A-C) Appropriate range of disruption of OP9 monolayer after careful washing to harvest HPCs. (D) An example of an OP9 monolayer that has been over-disrupted by the day 8 HPC harvesting procedure.
Figure 7: Representative flow cytometry (FACS) analyses at particular time points during mESC differentiation in vitro. (A) Staining for erythroid (left), monocytic (middle) and B cell (right) products of mESC-OP9 co-culture at day 12 or 16 as indicated. (B) Staining for developing T cells at the indicated time points of mESC-OP9-DL1 co-culture. Please see text for detailed descriptions of the immunophenotypes.
Figure 8. Diagram of the steps of the mESC-OP9 co-culture procedure. (A) The key cell transfer steps of the first eight days of co-culture. The approximate number of cells seeded on OP9 cells at days zero and five are indicated. (B) The day 8 transfer step and the expected cellular differentiation products detected by flow cytometry at key timepoints of co-culture. Please see text for detailed descriptions of the immunophenotypes.
The OP9-DL1 co-culture system has been utilized to study the role of various gene products during the development of blood cell types from stem cells.8,12,13 It has also proven an effective model to study the function of gene regulatory DNA during cellular differentiation.9,14 Using this approach as an alternative to whole mouse models can yield considerable savings in time and cost of experiments that address many basic questions in hematopoiesis. However, adaptation of this protocol for these purposes requires cognizance of the potential variability among mESC clones that would be used in these procedures. The timeline of the published protocol is expectable from the average, well-maintained, non-manipulated mESC line.7 In addition, mESC clones selected for the generation of chimeric mice usually are of higher quality and will perform very robustly in OP9 co-culture. On the other hand, mESC clones emerging directly from stable transfection with an ectopically integrated transgene will display varying degrees of initial differentiation efficiency ranging from average to below average. The protocol described here provides for a strategic 1-2 day delay of the day 5 passage step to even out these potential differences prior to induction of hematopoiesis in vitro.
The day 8 passage is the step at which the execution of this protocol has the most potential for error. Patience, practice and careful observation will enable one to develop the technique that optimizes HPC harvest while minimizing OP9 monolayer disruption. Beyond the key day 5 and day 8 steps, the critical parameters involve meticulous cell culture maintenance (particularly OP9 cells, as described above) and special attention to the reagents used in the protocol. The most critical reagent is the FBS. Distinct lots of FBS will vary markedly in their ability to support this procedure. Multiple lots must be tested in order to determine which will yield robust differentiation in this assay.
This co-culture system has its limitations. For example, this model does not support the differentiation of single positive CD4 cells. One reason for this is the lack of expression of the major histocompatibility complex (MHC) Class II antigen presentation infrastructure in the OP9 cells.1 Cell surface presentation of antigens by MHC Class II is required for proper development of CD4 SP cells. The CD8 SP cells that do emerge represent a mix of mature and immature CD8 SP thymocyte stages.1 Furthermore, successful transplantation of mESC derived T cell progenitors into a mouse requires prior passage through fetal thymic organ culture.1 Nevertheless, this technology has proven its promise as a powerful investigative approach to both cellular and molecular questions in blood and immune system development that were previously only possible to explore in vivo. Thus, aside from providing a novel way to make more rapid progress on these questions, wider adoption of the OP9-ESC co-culture system will have the broader impact of reducing the use of experimental animals.
The authors have nothing to disclose.
We thank Joon Kim for expert flow cytometry assistance. Research in the authors’ labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.
MATERIALS | COMPANY | CATALOG NUMBER | COMMENTS |
DMEM | Corning | 15-013-CV | |
Stem cell qualified FBS | Gemini | 100-125 | Heat inactivated |
Penicillin/Streptomycin | Corning | 30-002-CI | |
L-alanyl L-glutamine | Corning | 25-015-CI | |
HEPES buffer | Millipore | TMS-003-C | |
Non-Essential Amino Acids | ThermoScientific | SH30853.01 | |
Gentamicin Regent Solution (50 mg/mL) | Life Technologies | 15750-060 | |
β-mercaptoethanol (55 mM) in DPBS | Life Technologies | 21985-023 | |
Filter Unit | Millipore | SCGPU05RE | 0.22μm PES membrane |
Cell Culture Grade Water | Corning | 25-055-CM | |
α-MEM | Life Technologies | 12000-022 | Powder, reconstitute per manufacture recommendation |
Sodium bicarbonate | Sigma | S5761-500G | |
FBS | ThermoScientific | SH 30396.03 | Testing of individual lots required |
Dimethyl Sulphoxide | Sigma | D2650 | |
Recombinant Human Flt-3 Ligand | R&D Systems | 308-FK | |
Recombinant Murine IL-7 | PeproTech | 217-17 | |
LIF | Millipore | ESG1107 | |
Utrapure water with 0.1% gelatin | Millipore | ES-006-B | |
MEFs mitomycin C treated | Millipore | PMEF-CF | Any mitotically arresteded MEFs can be used |
DPBS | Corning | 21-031-CV | |
Trypsin EDTA, 1X | Corning | 25-053-Cl | |
Cell strainer (40 μm) | Fisher | 22363547 | |
Tissue culture dish 100 X 20 mm | BD Falcon | 353003 | |
Multiwell 6-well | BD Falcon | 353046 | |
1.5 ml microcentrifuge tubes | USA Scientific | 1615-5500 | |
15 ml centrifuge tubes | BD Falcon | 352096 | |
50 ml centrifuge tubes | BD Falcon | 352070 | |
5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap | BD Falcon | 352235 | Tubes for FACS |
ES R1 cells | ATCC | SCRC-1011 | |
OP9 cells | Cells can be obtained from the Riken Laboratory Cell Repository (Japan). | ||
OP9-DL1 cells | Cells can be requested from the Zúñiga-Pflücker laboratory. | ||
FlowJo software | Tree Star | FACS data analyses | |
Flow Cytometer | BD | FACScan, FACSCalibur and FACSVantage have been used in our lab |