The overall goal of this method is to purify endodermal cells generated from human embryonic stem or ES cells. This method can help answer key questions in the stem cell field about endodermal differentiation. The main advantage of this technique is that a pure endoderm cell population can be obtained after the removal of undifferentiated cells.
Before beginning the procedure, coat six well cell culture plates with one milliliter of basement membrane matrix per well. For at least 30 minutes at room temperature. Then, to harvest the human ES cells, aspirate the medium from an 80 to 90%confluent human embryonic stem cell culture with a sterile glass Pasture pipette.
And wash the cells with two milliliters of PBS per well. Shaking the plate and aspirating the saline to remove the dead cells and debris. Next, gently dissociate the cell clusters with one milliliter of enzyme-free passaging solution reagent, at 37 degrees Celsius and 5%carbon dioxide until the cells exhibit clear signs of disruption into smaller clusters.
After about seven minutes, add one milliliter of DMMF12 medium to the wells. And pipette up and down a few times with a one milliliter pipette tip to detach the remaining cell aggregates into a single cell suspension. Using the same pipette, transfer the cells into a centrifugation tube and rinse the wells with one milliliter of fresh DMMF12 medium, pooling the washes in the tube.
After spinning down the cells, re-suspend the pellet in five milliliters of ES cell culture medium supplemented with ROCK inhibitor. Count the cells and seed 1.5:4 times 10 to the fifth cells per well on the basement membrane matrix coated plates in culture medium supplemented with ROCK inhibitor to prevent apoptosis. Incubate the cells for approximately 24 hours.
And then use a sterile, glass Pasteur pipette to replace the medium in each well with 2 milliliters of primitive streak induction medium. After 24 more hours of culture, replace the medium with endoderm induction medium for a 48 hour incubation with daily medium changes. On the last day of culture and at least one hour before harvesting the cells, add fresh ROCK inhibitor to the culture medium.
Then use a sterile, glass Pasteur pipette to aspirate the medium from each of the wells and dissociate the cells with enzyme-free passaging solution reagent as just demonstrated. When a single-cell suspension has been achieved, count the cells and centrifuge them. Make sure that from this point on, all medium and buffers contain the ROCK inhibitor to prevent the cells from dying.
Otherwise, they will not be attached properly after receding. Re-suspend the pellet in 100 microliters of PEB buffer, supplemented with ROCK inhibitor per 1 times 10 to the seventh cells. Then add CXCR4 APC antibody to the cells.
Gently flicking the tube to mix. After 15 minutes at 4 degrees Celsius, wash the cells in one to two milliliters of PEB buffer and use a sterile, glass Pasteur pipette to aspirate the supernatant. Re-suspend the pellet in 80 microliters of PEB buffer per 1 times 10 to the seventh cells and add 20 microliters of anti-APC microbeads to the cells.
Mix the sample with gentle flicking. Then incubate the cells at four degrees Celsius for 15 minutes. At the end of the incubation, wash the cells with one to two milliliters of PEB buffer supplemented with ROCK inhibitor.
And re-suspend the pellet in 500 microliters of fresh PEB buffer plus inhibitor. To isolate the CXCR4+cells by magnetic bead separation, load a medium sized magnetic column onto an appropriately sized magnet and pre-rinse the column with 500 microliters PEB buffer plus ROCK inhibitor. When all of the wash has drained from the top of the column, apply the entire magnetic bead labeled cell volume to the column, collecting the flow through in a conical tube.
Wash the column with three applications of 500 microliters of PEB buffer plus ROCK inhibitor. Then transfer the column from the magnet into a suitable collection tube. And plunge one milliliter of PEB buffer plus ROCK inhibitor through the column to collect the CXCR4+cells.
Optionally, all of the flow through samples can be collected separately and 20 microliter aliquots from each sample can be used to determine the percentage of CXCR4+cells in each alouette by flow cytometry. This procedure can be repeated with the first flow through to collect cells that did not bind to the column. Then pool both allouted CXCR4+samples and centrifuge them.
Finally, re-suspend the pellet in one milliliter of endoderm induction medium supplemented with ROCK inhibitor And seed the cells at the appropriate density in a basement membrane matrix coated cell culture container. Prior to their differentiation, human ES cells express high levels of the pluripotency marker SOX2. Whereas, the definitive endoderm marker, FOXA2 is not detected.
Upon differentiation, the ES cells undergo drastic changes in their gene and protein expression. Indeed, after four days in differentiation medium, SOX17 and FOXA2 are co-expressed uniformly within the nuclei of many of the former ES cells, a hallmark of definitive endoderm commitment. Some cells resist the differentiation process, expressing neither of the two marker proteins.
And in fact, retain their pluripotency marker SOX2 expression. In this representative experiment, on day three of differentiation, nearly 60%of the harvested ES cells expressed CXCR4, the purity of which was enriched to over 85%after magnetic bead sorting of the pluripotent and other unwanted lineage cells. After seeding the purified CXCR4+cells, only a few SOX2+cells are detected, with the majority of the seeded cells expressing FOXA2.
Once mastered, this technique can be completed within two hours, if performed properly. Following this procedure, other like immune histochemistry or QPCR can be performed to answer additional questions about the differentiation process. After watching this video, you should have a good understanding of how to purify endoderm cells by magnetic bead sorting.
