Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The overall goal of this procedure is to drive the quantitative differentiation of T cells from mouse embryonic stem cells or e ESCs in vitro. This is accomplished by first seeding the ESCs onto a monolayer of OP nine stromal cells. After five to seven days, the mesodermal like colony formations are harvested and replated onto a fresh OP nine cell monolayer.

Three days later, the small round hematopoietic progenitor cells or HPCs are harvested and transferred to a fresh OP nine monolayer for the generation of erythroid, monocytic and B cells, or to a fresh OP nine DL one monolayer to generate T cells. Ultimately, this method can be used as an in vitro model for studying the molecular, genetic and cellular events that regulate hematopoiesis and t lymphocyte development. This procedure was developed in the lab of Dr.Juan Carlos Sunga Fluker at the University of Toronto.

We learned the technique from his group so that we could apply it to our research on gene regulation during T-cell development at Hunter College. The main advantage of this technique over existing methods is that it allows for the experimental manipulation and observation of T-cell development using cell lines without the use of animals. Begin by diluting 50, 000 mouse ESCs in 10 milliliters of OP nine media per plate.

Then after removing the old media from the OP nine cultures plate, the ESC suspension, taking care to distribute the cells evenly and incubate the cells at 37 degrees Celsius on day five. When 80 to 90%of the colonies display visible mesoderm like morphology, aspirate the media from the co-culture dishes and wash the cells in four milliliters of PBS with swirling. Next, detach the cells with four milliliters of point 25%trypsin at 37 degrees Celsius after approximately five minutes, disrupt the cell monolayer with vigorous pipetting, taking care not to introduce excessive air bubbles until a homogenous mostly single cell suspension is achieved.

Then mix the cells with four milliliters of complete OP nine media and prelate them into a new empty 10 centimeter dish. After 30 minutes, filter the non-adherent non-op nine cells through a 40 micrometer strainer into a 50 milliliter conical tube. Then wash the dish gently with six milliliters of PBS and pass it through the same strainer.

Leaving the attached OP nine cells behind. Spin down the collected cells for five minutes at 400 Gs and four degrees Celsius and resuspend the pellet in three milliliters of complete OP nine media. After counting the cells, remove the media from optimally confluent OP nine cultures and seed 500, 000 cells onto the OP nine plates in a 10 milliliter final volume.

For each analysis time point, then supplement each co-culture with five nanograms per milliliter of human recombinant fit three ligand and place the plates in the incubator. Observe the co cultures under the microscope on day eight. Shiny clusters of hematopoietic progenitor cells or HPCs should be loosely attached to the op nine monolayer.

Collect as many of these cells as possible by washing the OP nine monolayer with the pipette and the existing media on the dish. Taking care not to overly disrupt the monolayer. To prevent foaming, always have at least one milliliter of the media in the tip of the pipette.

Filter the collected cells through a 40 micrometer strainer and then rinse the plate with eight milliliters of PBS. Confirm under the microscope that all the HPCs were collected and then filter the PBS wash into the tube containing the cells from the same plate. After spinning down the cells resus, suspend the pellet in OP nine media supplemented with freshly prepared fit.

Three ligand and IL seven containing master mix and incubate the collected cells from each plate in individual wells of a six well plate coated with the appropriate cells. On day 10, gently pull the media from each well of the six well plate containing the same ESC clone feeder cell type combination. And then add two milliliters of fresh OP nine media master mix to each well of the plate.

Sent a the collected media and resuspend each pellet in one milliliter of fresh OP nine media master mix per well. Then distribute one milliliter of the cells back into each well and return the dishes back to the incubator on day 12 vigorously pipette the existing media in each well to disaggregate all the cells, including the monolayer until a state resembling a single cell suspension is achieved. Then strain and wash the contents of each well as just demonstrated.

Combining the cells from multiple wells containing the same ESC clone feeder cell type combination. Finally place aliquots of the cell suspension for any further analysis to be carried out that day on ice. And then spin down the rest of the cells for receding.

On day 12, flow cytometry analysis of live gated cells differentiated on the OP nine monolayer will reveal erythroid and monocytic lineage cells and the emergence of the early CD four CDH double negative or DN.Stage T lineage cells refer to as the DN one and DN two populations on the OP nine DL one monolayer by day 16. There is a significant increase in the amount of small round shiny cells in the cultures. On OP nine DL ones T lineage differentiation products progressed to the DN three and DN four stages hematopoietic progenitor cells seated on OP nine cells yield CD 19 positive B cells by day 16.

By day 20 of the OP nine DL one mouse ESC co-culture, large amounts of CD four, CD eight, double positive T cells and CD eight single positive T cells will be present. After watching this video, you should have a good understanding of the three types of cell harvesting and transfer steps required to derive T cells and other hematopoietic cell types from mass embryonic stem cells, as well as what the co-culture should look like under the microscope at each of these steps. After its introduction, this technique paved new ways for investigators in the field of immunology to study the role of genes in hematopoiesis, the microenvironmental cues affecting T-cell development and the regulation of T-cell genes.

Questions that were formally only approachable using whole mouse models.