Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

The overall goal of this procedure is to generate a purified population of human induced pluripotent stem cell derived cardiomyocytes. First, human induced pluripotent stem cells are cultured in a cell monolayer until they are about 85%confluent. A small molecule compound that activates the wind cell signaling pathway is then added and the cells are incubated for two days to induce the formation of mesoderm.

Next, a small molecule compound that inhibits the wind cell signaling pathway is added to the cells to specify cardiac mesoderm differentiation. The cells are then incubated for two more days. By day seven, the differentiated cardiomyocytes beat the heterogeneous differentiated cell population is then incubated in a low glucose cell culture medium to selectively purify the cardiomyocytes.

Finally, the cells are visually inspected under a light microscope and analyzed by flow cytometry to confirm the production of a highly purified population of beating cardiomyocytes. The main advantage of this technique over other cardiomyocyte differentiation protocol that utilizes small molecule is that it employs an additional glucose deprivation step. This enable us to purify cardiomyocytes, which are able to utilize sources of cellular energy other than glucose.

Non cardiomyocytes can be selectively eliminated from this heterogeneous population of differentiated cells. Cardiomyocytes can then be utilized for downstream purposes, such as in vitro disease modeling or high throughput drug discovery. Assays Prepare for this experiment by Preco six Well plates with extracellular matrix solution or ECMS and thawing, a vial of frozen human-induced pluripotent stem cells, or HI PSCs as described in the accompanying text.

After centrifusion Resus, suspend the thawed cells in E eight with rock inhibitor. Then transfer the contents of the vial to one well of a six well plate pre-coded with ECMS. Place the cells in an incubator at 37 degrees Celsius with 5%carbon dioxide.

After 24 hours, the cells will have adhered. Replace the medium with E eight medium without rock inhibitor. Continue to culture the cells and replace the E eight medium daily.

When the HI PSCs reach 75 to 80%co fluency, it is time to passage them. Aspirate the culture medium quickly. Wash wells with one milliliter of sterile room temperature PBS aspirate the PBS.

Then add 500 microliters of 0.5 millimolar EDTA or another cell detachment solution. Incubate for one to seven minutes at room temperature using a microscope monitor, cell detachment when visible curling or thickening of the colonies around the edges is evident, aspirate the cell detachment solution. Then add one milliliter of E eight medium supplemented with 10 micromolar rock inhibitor using a P 1000 repeatedly pipette up and down to completely detach the H-I-P-S-C colonies from the plate, forming five to 10 cell clumps.

Then transfer the suspension to a 15 milliliter conical tube to further disperse the large cell. Clumps, pipette up and down until smaller clumps have formed as shown here. Then dilute the cells one to 12 by adding 11 milliliters of E eight medium with rock inhibitor to the one milliliter cell suspension.

Next, aspirate the ECMS from the pre-coded plates. Plates, and add two milliliters of resuspended cells to each well approximately 100, 000 cells per well of a six well is ideal aim to dispense the cells evenly around the well to avoid clustering of cells in the center of the well. Place the plate in the incubator for 24 hours.

The next day, replace the medium with E eight that does not contain rock inhibitor. Change the medium every 24 hours until cells become 80%confluent. Typically, the cells are ready to passage again within three to six days after dissociation with EDTA or one Xcel detachment solution seed, approximately 100, 000 human I PSCs on ECMS coated six well culture plates for differentiation.

Following the same steps used for HI PSCs when the cells reach 85%co fluency change to R-P-M-I-B 27, medium without insulin, but with six micromolar of the GSK three beta inhibitor. CHIR 9 9 0 2 1. Incubate the cells for 48 hours each day, examine the cells under a microscope.

Substantial amounts of cell death may be observed after 24 hours of CHIR treatment. This is normal and commonly seen after two days of treatment. The HIPS sees increase in size and number and continue to differentiate towards a mesodermal fate.

Replace the CHIR containing culture medium with R-P-M-I-B 27 insulin medium, and incubate until day three. On the third day, change the medium to R-P-M-I-B 27 without insulin. With five micromolar wind inhibitor IWR one and incubate the cells for 48 hours.

On the fifth day, examine the cells under a microscope. The mesodermal cells should be directed towards the cardiac lineage and begin coalescing and forming characteristic branch like structures. Change the medium back to R-P-M-I-B 27 without insulin and incubate the cells for 48 hours.

On the seventh day. Replace the medium with R-P-M-I-B 27 containing insulin every three days thereafter, replace with the same medium between the eighth and 10th day. Spontaneous beating of cardiomyocytes will be seen after 10 days of differentiation.

More than 50%of cell areas are beating. However, beating cell sheets may be intermingled with non cardiomyocytes on the 10th day branch like structures will be highly pronounced. Terminally differentiated cardiomyocytes will not proliferate beyond this point.

At day 10. Post differentiation, change the medium in each well of the six well plate to two milliliters of low glucose medium and maintain the cells in this medium for three days until day 13. At day 13, return the cells to R-P-M-I-B 27 medium with insulin or optionally to reflate the cells, which facilitates the dissociation of non cardiomyocytes During glucose starvation.

Aspirate the medium and wash once with PBS. Add 500 microliters of cell dissociation enzyme and incubate the cells for five minutes. After five minutes of enzyme treatment, use a P 1000 to manually disperse the cardiomyocytes into a single cell suspension By pipetting up and down up to 30 times after the cells are dissociated, transfer them to a 15 milliliter conical tube.

Filled with five milliliters of R-P-M-I-B 27 medium with insulin to dilute out the cell dissociation enzyme centrifuge for four minutes at 200 Gs following the spin aspirate and discard the supernatant. Then resuspend the cells in two milliliters of R-P-M-I-B 27 medium and seed them onto a new ECMS coated six well plate. Typically cell survival is increased with confluence for optimal cell survival.

Reflate 2 million cells per well in a six well dish For cells, not replated. On the 14th day, change the medium back to two milliliters of low glucose medium for a second glucose deprivation cycle culture. The cells in this low glucose state for three more days.

Most of the non cardiomyocytes will die on the 17th day. Change the medium to two milliliters of R-P-M-I-B 27 with insulin. The remaining cells will be highly purified.Cardiomyocytes.

These cells can be used for gene expression analysis, drug screening, metabolic analysis, and various other downstream assays to examine cardiomyocyte purity following differentiation and purification via glucose starvation method cells with immuno staining against cardiomyocyte marker, cardiac tropin and T CTN T were characterized. Immunofluorescence microscopy of an un purified population of differentiated cells revealed that although many cells were CTN T positive shown in green, many cells lacked CTN T expression suggesting the presence of non cardiomyocytes at day 13. In contrast, after a three day glucose starvation beginning at day 10, almost all cells were CTNT positive at day 13, indicating successful purification of the cardiomyocytes following glucose starvation.

These data were further corroborated using flow cytometry analysis. As indicated in red, about 50%of the cells that did not undergo glucose starvation were tn NT two positive. While 90%of the glucose starved cells were tn NT two positive.

After watching this video, you should have a good understanding of how to differentiate human induced pluripotent stem cells into highly purified populations of cardiomyocytes. These purified cells can be produced in a high throughput fashion for a variety of downstream in vitro applications. When culturing pluripotent stem cells, remember that they should not be allowed to grow to a hundred percent co fluency prior to messaging.

Some stem cell lines differentiate best when the initial pluripotent stem cell co fluency is less than 85%Finally, some stem cell lines may respond more rapidly or severely to the glucose deprivation step. Always monitor these parameters before working with new stem cell lines.