The overall goal of this procedure is to sort human embryonic stem cell subsets by their cell surface antigen binding of the TG 30 and GC TM two monoclonal antibodies. This is accomplished by first dissociating, human embryonic stem cell or HESC cultures into single cell suspensions. In the second step, the HESC are stained with the monoclonal antibodies, TG 30 and GCTM two.
Finally, the cells are separated by fluorescence activated cell sorting and returned to culture. Ultimately, the colony forming ability of the sorted cells can be assessed by flow cytometry and phase contrast microscopy. The main image of this technique over existing methods like visual observation, is that it provides an objective measure of pluripotency on a cell by cell basis for a large number of cells, and this facilitates the separation of the embryonic stem cells from the differentiated cells.
This method can help answer key questions in the pluripotency field, such as how changes in the culture conditions quantitatively affect different human embryonic stem cell and human induced pluripotent stem cell lines. Demonstrating the procedure today will be research scientists from my laboratory, George Zoe Hun Chi and Bay Wang, To make a Hess single cell suspension begin by aspirating the Hess Culture media from two T 75 flasks containing a cultured Hess cell line. Wash each flask with 10 milliliters of DPBS and then incubate each culture with three milliliters of triple E expressed dissociation solution at 37 degrees Celsius and 5%CO2.
After five minutes, bump the side of the flask to dislodge the cells and then confirm detachment under the microscope. Next, add 10 milliliters of cold culture media to each flask using a sterile 10 milliliter pipette. Gently tritrate the dissociated cells several times against the flask wall to achieve a predominantly single cell suspension.
Now pool the hes single cell suspensions through a 70 micron cell strainer into a sterile 50 milliliter polypropylene tube to stain Hess cell surface antigens. First Eloqua about 100, 000 cells in 150 microliters of media into each of six control 1.5 milliliter micro centrifuge tubes. Next, incubate the control tubes and the remaining sort sample with the primary antibodies of interest in final reaction volumes of 200 microliters and about nine milliliters respectively in culture media including the isotype controls.
Place the tubes horizontally on ice and mix them gently for 30 minutes on a rocking platform. After spinning down the cells, discard the supernatant and then wash the sort sample in 20 milliliters of cold culture media and the control samples in 200 microliters of the same. Now incubate the sort sample in a 2.5 milliliter final volume with the appropriate secondary antibodies in cold culture media and the controls in a 200 microliter final volume of the same add freshly prepared PE anti-US CD 90.2 antibody to the appropriate control tube at this time as well.
Once again, gently mix the tubes horizontally on ice for 30 minutes on a rocking platform. After spinning down the cells, again resuspend the sort sample in 20 milliliters of cold culture media and the controls in 200 microliters and wash the cells twice in the media. Resuspend the sort sample in two to three milliliters of cold culture media supplemented with propidium iodide and each of the controls except the unstained cells in 300 microliters of the same.
Then strain each individual cell suspension through a 35 micron lid cell strainer coupled to five milliliter polystyrene round bottom test tubes and centrifuge the tubes to force any residual cell suspension on the strainer lids into the tubes. Then tap the sides of the tubes to resuspend each single cell suspension and place the cells on ice. Set up the flow cytometer for TG 30 GCTM two fluorescence activated cell sorting so that the dissociated Hess cell suspensions are gated as single cells to eliminate potential clumps and doublets.
Next, remove the potential debris with the forward and side scatter gates so that only the intact cells are sorted. Then exclude the non-viable cells based on their propidium iodide expression. Gate out the mouse embryonic fibroblasts or meth feeder cells by negative selection based on their expression of mouse CD 90.2 pe.
Set the TG 30 G CTM two negative gait according to the background autofluorescence for the unstained cells and also the isotype controls. The isolated viable HESC fraction free of meth feeders can then finally be fractionated into TG 30 negative GCT M two negative TG 30 low GCT M two low TG 30 mid GC TM two mid and TG 30 high GCTM two high subpopulations. After setting up the flow cytometer, add one milliliter of meth conditioned media to each of two five milliliter polystyrene round bottom test tubes and place them on ice.
Then recover 400, 000 cells each from the differentiated TG 30 negative GC TM two negative and the pluripotent TG 30 high GCTM two high cell subfractions. After spinning down the collection tubes, resus suspend each cell pellet in one milliliter of pre equilibrated MEF conditioned media. Then seed one pre equilibrated, one third MEF density T 75 flask with the HESC from each fraction in meth conditioned media incubate the cell cultures at 37 degrees Celsius at 5%CO2, refreshing the media daily for approximately two weeks.
Cultures of pluripotent TG 30 high GCTM two high cells show human stem-like colonies after one week and reach about 80%co fluency at nine to 11 days. Cultures of TG 30 negative GC TM two negative cells mostly grow as a lawn of fibroblasts like cells that reaches co fluency at 11 to 13 days. The combined detection of cell surface antigens TG 30 and GC TM two allows the discrimination of a number of cell subsets at the pluripotent stage and at various stages of early HESC differentiation as indicated by their expression of stem cell markers and lineage specific transcription factors in agreement with previous reports, the TG 30 GC TM two negative low, mid, and high subfractions are able to be discriminated within the human embryonic stem cell male one line used for this study here, the percentages of the different gated subpopulations typical of a TG 30 GCTM two flow cytometry analysis are shown in the experiment shown here.
A defined number of TG 30 negative GCT M two negative cells were consecutively cultured post sorting for approximately two weeks, and then resorted to collect only the TG 30 negative GCT M two negative fraction prior to replating. At each passage, the results indicate that the initial TG 30 negative gct M two negative subfraction enriches for TG 30 negative GCT M two negative differentiated cells after consecutive packaging. A minor population of TG 30 high GCT M two high cells is detected at the initial passage, but eventually disappears with further packaging leaving only pluripotent stem cell free samples.
Here the typical morphology of the lawn of differentiated TG 30 negative GCT M two negative cells after 14 days is shown. Note that the sporadic HESC like colonies seen after 14 days intermingled with the TG 30 negative GC TM two negative cells that are most likely generated by the contaminating TG 30 high GCT M two high cells. Note this only occurs rarely and in the initial post facts passage based on previous observations using this assay, it is expected that the collection and culture of TG 30 high GCTM two high cells should enrich for colony forming pluripotent HSCs.
Therefore, consecutive packaging of pluripotent TG 30 high GCTM two high cells after sorting by flow cytometry was also investigated in these representative cultures, the cells were fax sorted using TG 30 and G CT M two, replacing only the TG 30 high G CTM two high cells at each passage. In this way, it was observed that the majority of cells were located in the subfractions associated with pluripotency indicating an enriched population of pluripotent cells that also maintains a capacity to differentiate and regenerate a small percentage of TG 30 negative G CTM two negative cells. Furthermore, consecutive cultures of TG 30 high G CTM two high sorted cells exhibit a large number of HESC likelike colonies post passaging also indicating maintenance of the pluripotency state.
While attempting this procedure, it's important to remember to calibrate fax machine before use every time to ensure the reproducibility of the results Following this procedure. Other methods like microarrays, whole genome sequencing or teratomas can be performed to answer questions such as are these cells truly pluripotent After its development? This technique paved the way for researchers in the field of induced pluripotent stem cells to explore the safety and stability of these cells in comparison with human embryonic stem cells.
After watching this video, you should have a good understanding of how to carefully prepare and stain mixed cultures of human enbr stem cells in order to enrich for periet cells. Don't forget that working with human cells can be extremely hazardous and the precautions such as wearing gloves and working with class two by safety cabinet should always be taken while performing this procedure.
