Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

The overall goal of this procedure is to generate human or mouse cardiac progenitor cells from embryonic stem cells. This is accomplished by first culture in high quality embryonic stem cells or e ESCs or mouse embryonic feeder cells or myths. In the second step, the cells are transferred to gelatin coated petri dishes to facilitate the formation of the first embryo bodies.

Next, the cells are associated and growth factors are added to the cultures to induce the second form embryo bodies. In the final step, the embryo bodies are dissociated again and the resulting cells are treated with additional growth factors. Ultimately, the cardiac progeny cells can be further cultured for cardiomyocyte and smooth muscle cell differentiation.

While attempting this procedure, it is important to test the differentiation efficiency of each cell line to allow the selection of the cells with the best cardiogenic potential. Now, Dr.Inland Lamb, a postdoc fellow in laboratory will demonstrate the procedure To induce cardiac progenitor cell differentiation from mouse embryo bodies Begin by culturing mouse e ESCs, or MES in ESC medium until the cells are 90%cofluent. Next, wash the cells in DPBS and then detach them with 0.4 milliliters of 0.25%tripsin at 37 degrees Celsius, and 5%carbon dioxide.

After five minutes, add one milliliter of prewarm medium to halt the enzymatic reaction. Spin down the cells for five minutes at 200 times G and room temperature, and then resuspend the cells in one milliliter of ESC medium. After counting plate the cells onto gelatin coated plates at a density of three times 10 to the four cells per centimeter squared at 37 degrees Celsius and 5%carbon dioxide changing the medium every day.

When the cells have reached co fluency, detach them with trypsin as just demonstrated. Then culture one times 10 to the six ESCs in one milliliter of differentiation. Medium in a low detachment Petri dish at 37 degrees Celsius for the first embryo body aggregation.

Two days after embryo body formation, transfer the embryo bodies into 50 milliliter tubes and centrifuge. The cells rinse the pellets with 10 milliliters of DPBS without calcium and magnesium, and then desegregate the embryo bodies with one milliliter of trypsin at 37 degrees Celsius. After five minutes, stop the reaction with 4.5 milliliters of differentiation.

Medium pipetting up and down to dissociate the embryo bodies into a single cell suspension. Then after another centrifugation resuspend the cells in one milliliter of medium and count them dilute two times 10, the six cells to one point 10 to the five cells per milliliter in differentiation medium supplemented with VEGF BMP four and active in a then culture. The cells in a Petri dish at 37 degrees Celsius for the second embryo body formation.

40 hours after the second embryo body forms transfer the embryo bodies into 50 milliliter tubes and then dissociate the cells as just demonstrated. Next, re suspend the cells in EmPro 34, medium with VEGF B, FG F and FGF 10 and culture The cells at two times 10 to the five centimeter squared in gelatin coated plates at 37 degrees Celsius after approximately 32 hours, dissociate the embryo bodies into a single cell suspension as demonstrated, and then resuspend the final pellet in 2%F-B-S-P-B-S. Note that culture time for the highest yield of fluorescently labeled cells needs to be determined individually for each cell line.

Run undifferentiated ESCs on a fax machine as a negative control, adjust the forward and side scatters to select the desired population using the forward versus side scatter and forward scatter height versus width to exclude any debris and doublets. Then within the selected subpopulation, draw a gate for A YFP positive histogram to eliminate any autofluorescence by the cells and collect the YFP positive CPCs to induce cardiac progenitor cells from human embryo bodies. Begin by using a cell scrapper to detach a human ESC monolayer and then carefully pipette up and down to break the colonies into small clumps.

Next, culture, the clumps in one to three dilution on coated plates in MTS and medium for at least two passages. When the cells reach 90%co fluency, aspirate the medium and rinse the plate with DPBS. Then incubate the human E ESCs in 0.5 milligrams per milliliter DIS displays at 37 degrees Celsius for 20 minutes.

After rinsing the cultures twice with DPBS, use a cell lifter to remove the cells and then resuspend the cell clumps in differentiation medium, transfer the clumps into six well ultralow attachment plates for embryo body formation and collect the embryo bodies no later than day nine of differentiation following dissociation with trypsin as just demonstrated, dissociate the embryo bodies further into single cells by duration. Then after centrifugation, re suspend the cells in 2%F-B-S-D-P-B-S and filter them through a 40 micrometer cell strainer. Now use the same gating strategy as just demonstrated for the mouse cells to sort the human RFP positive cardiac progenitor cells.

Finally, culture the cardiac progenitor cells onto gelatin coated plates in differentiation medium for seven to nine days until cardiomyocyte and smooth muscle cells are observed. ESCs are routinely maintained on me feeders, which are removed before differentiation upon aggregation of the ESCs into embryo bodies. The second formation of the embryo bodies is treated with BMP four and active in a, to enhance the differentiation of the mesoderm in the mouse cell lines, the ESCs differentiate into cardiac lineage cells as identified by their fluorescent protein expression.

Typically 80 to 90%cardiac progenitor cells are obtained from the mouse ESC differentiation protocol and 0.5 to 5%cardiac progenitor cells are obtained from the human ESC differentiation protocol. After fax purification, further culture facilitates differentiation of the ESCs into cardiomyocytes and smooth muscle cells. After watching this video, you should have a good understanding of how to derive Embry cardiac progenitor cells from both mouse and human embryonic stem cells.

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