The overall goal of the following experiment is to differentiate murine embryonic stem cells into various primitive and definitive hematopoietic lineages. And to characterize all ES progeny, murine hematopoietic cell differentiation is achieved by placing ES cells onto a confluent fluorescently labeled M strawberry OP nine stromal cell layer. Each day of differentiation or co-culture represents the various stages of hematopoietic development at the appropriate harvest time.
Differentiated ES cell progeny can be separated from the M strawberry OP nine stromal cell line by cell sorting. This ES differentiation protocol recapitulates primitive and definitive hematopoiesis as observed in vivo and can be used to characterize hematopoietic defects associated with knockout ES cell lines without the use of embryos. Hello, welcome to the lab of Dr.Judith Gassen at UCLA's Johnson Comprehensive Cancer Center.
My name is he Alicia Poz. The main advantage of the technique I'm going to show you today over existing ESO nine co-culture methods is that this procedure allows for the passage of all day five and day eight cells, as well as the complete separation of all ES derived progeny. From the nine stroma M strawberry OP nine cells are grown in OP nine cell culture medium, which must be changed every two days.
The OP nine cells should be subculture three days prior to when they're needed for co culturing. It is important to note that during maintenance, the OP nine cells should never be maintained at a density above 90%Co fluency first wash the cells two times with PBS, then add PREWARM 0.1%trypsin EDTA for five minutes when the cells have detached. ADD OP nine cell culture medium and transfer the cells to a 50 milliliter conical tube.
Tube pellet cells at 300 G for five minutes at room temperature after centrifugation, discard the supernatant and resuspend cells in fresh M strawberry OP nine cell culture medium plate cells at a minimum density of 10 to the fourth cells centimeter squared on the day the co-culture experiment begins. The OP nine cells that were subculture three days ago should be confluent. The ES cells should be cultured according to the standard protocol for your particular cell line.
Wash ES cells two times with PBS trypsin eyes with 0.25%tryin EDTA and incubate for five minutes at 37 degrees Celsius. Next, add co-culture differentiation media to the ES cells and transfer them to 15 milliliter conical tubes. Pellet the ES cells at 400 G for five to seven minutes at room temperature.
After removing the supernatant Resus suspend ES cells in co-culture differentiation media. Use a 21 gauge blunt end needle to obtain a single cell suspension. Count cells using trian blue and a hemo cytometer.
Now retrieve the flask of M strawberry. OP nine cells from the incubator. Remove media from the OP nine cells and add co-culture differentiation media to the flask plate.
One times 10 to the third ES cells per centimeter square to each flask of M strawberry OP nine cells incubate cells at 37 degrees Celsius, 5%carbon dioxide with humidity. This is day zero and the co-culture should look like this. Three days after the start of the ES M strawberry OP nine CO-culture.
Mostly large flat clusters of ES derived progeny with defined borders are observed and some whirls of cells will have begun to form. Carefully remove medium from the co-culture and replace with fresh differentiation Medium five days after the start of the co-culture. Proper differentiation of ES cells on a confluent layer of M strawberry OP nine cells should result in heman eblasts, which appear as piled up worlds of cells.
The most critical aspect of this procedure is making sure you get proper differentiation on day five of co-culture. At this point, you could see the heman eblast colonies not only microscopically but macroscopically as well. Carefully wash the co-culture two times with PBS and trypsin ice cells with 0.25%tripsin EDTA, wash cells off flask with ES differentiation media and transfer to a tube pellet cells at 400 G for seven minutes at room temperature.
After centrifugation, remove the supernatant and add new media. Then use a 21 gauge blunt ended needle to resuspend cells and to ensure a single cell suspension, count the cells using a hemo cytometer, there should be a 100 to 150 fold increase in ES cell number from day zero reseed co-culture cells onto a new layer of confluent M strawberry OP nine cells on day eight or nine and day 12, count the cells again. Remove the media with the hematopoietic cells and reserve.
Wash the monolayer one time with PBS To remove hematopoietic cells in suspension and reserve, add 0.25%tripsin EDTA to flask after trypsin adherence cells wash cells off the flask with ES differentiation media and add to the hematopoietic cells found in suspension palate cells at 400 G for seven minutes at room temperature. Finally resuspend the cells using a 21 gauge blunt end needle and count the cells. The cells are now ready to be replated or used in downstream assays.
The ES M strawberry OP nine co-culture system can be used to model hematopoietic development outlined. Here is an overview of the co-culture time course days when differentiated ES progeny need to be transferred to new M strawberry OP nine cells are indicated. Proper differentiation of ES cells on a confluent layer of M strawberry OP nine cells will result in the production of flick one positive.
He angio blasts at day five of co-culture Heman osteoblasts are the common mesodermal precursor for the endothelial and hematopoietic lineages and should appear as piled up worlds of cells as shown in this image. As the co-culture proceeds two to four celled clusters should appear at day six or seven and grow into larger cell clusters. By day eight or nine hematopoietic precursors are present by day six while terminally differentiated hematopoietic lineages appear by day 14.
In contrast, a co-culture with improper ESL differentiation will result in ES derived progeny that do not appear in a world pattern at day five. The absence of cells in a world pattern indicates improper GIO blasts formation. A co-culture with improper eblast formation will ultimately result in stunted hematopoietic lineage differentiation and should be discarded with this modified ES OP nine Co-culture protocol.
ES derived progeny are m strawberry negative and can be flow sorted from M strawberry positive. OP nine cells on any day of co-culture for evaluation. Results from a representative experiment are shown here.
Previously published ES OP nine co-culture protocols have focused solely on the passage and evaluation of the non-adherent floating cells. In this modified protocol, all ES derived cells, both adherent and non-adherent cells are passaged and can be evaluated. This leads to increased yields of hematopoietic cells, allows for the identification of rare populations of cells, and provides for complete characterization of all ES progeny, both hematopoietic and non hematopoietic.
