The overall goal of this procedure is to efficiently generate NKX6.1 expressing pancreatic progenitors from human pluripotent stem cells in vitro by differentiating the cells through four key stages of pancreatic development. By differentiating human pluripotent stem cells through four key stages of pancreatic devleopment, this protocol provides a model to study human pancreas development in vitro. The main advantage of this technique over existing protocols is that we are able to generate high percentages of NKX6.1 positive pancreatic progenitor cells using a variety of human pluripotent stem cell lines.

Given that pancreatic progenitors have the potential to generate insulin producing beta like cells, this technique will give us the opportunity to develop cells for diabetes treatment, for drug discovery and for disease modeling. On day zero, begin differentiation by replacing the KnockOut serum replacement media with day zero differentiation media. Then one days one and two, gently shake the plate to remove any dead cells from the monolayer before aspirating the media.

Afterward, replace it with day one to two differentiation media. On day three, harvest the cells for flow cytometry. If the cells express more than 90%of CXCR4 and CD117, the efficiency has been reached.

Then gently shake the plate to remove any dead cells from the monolayer prior to aspiration and replace it with day three to five differentiation media. It is important to ensure efficient induction of the definitive under derm by day three by flow cytometry. If the percentage of cells expressing both CXCR4 and CD117 is lower than 90%differentiation to pancreatic progenitors will likely be compromised.

On days six and seven, replace the medium with day six to seven differentiation media. On days eight, 10 and 12, replace the medium with day eight to 12 differentiation media. Then on day 13, harvest the cells for flow cytometry to determine PDX1 and NKX6.1 expression.

To harvest the cells for flow cytometry analysis, disassociate the cells with commercial Trypsin solution according to the manufacturer's protocol and incubate the cells at 37 degrees Celsius for three minutes. After three minutes, remove the Trypsin solution and re-suspend the single cells in 1, 000 microliters of fax buffer with 30 microliters of DNA One. Then filter the cells using a 35 micrometer nylon mesh cell strainer and transfer them to a micro centrifuge tube.

Centrifuge the cells at 455 times g for five minutes before preparing the cells for live or fixed staining. Now re-suspend the cells in 1, 000 microliters of fax buffer. Transfer 200 microliters into two wells of a 96 well plate for both unstained and stained samples.

Afterward, centrifuge the plate at 931 times g for two minutes, afterward remove the supernatant by inverting the plate, stain the cells with primary conjugated antibody in a final volume of 100 microliters for 30 minutes at room temperature and protect them from light. Subsequently, centrifuge the plate at 931 times g for two minutes, after two minutes remove the supernatant by inverting the plate. Following that, re-suspend the samples in 100 microliters of fax buffer.

Then centrifuge the plate 931 times g for two minutes. And remove the supernatant by inverting the plate. Next re-suspend the samples in 300 to 500 microliters of fax buffer, transfer them to the one milliliter micro test tubes or the five milliliter round bottom tubes and run the samples on flow cytometor.

If the samples cannot be run immediately, store them at four degrees Celsius and protect it from light. In this procedure, re-suspend the cell pellet in commercial fixation and permeable Isation solution for 24 hours at four degrees Celsius. After 24 hours, centrifuge the sample at 455 times g for five minutes, then re-suspend it in 400 microliters of commercial perm wash solution.

Next, transfer 200 microliters of the sample in two wells of a 96 well plate for IGG control and PDX1, NKX6.1 staining, centrifuge the plate at 931 times g for two minutes. Then re-suspend the cell pellets in 100 microliters of commercial perm wash solution containing anti-PDX1 and anti-NKX61 primary or isotope control antibodies. Incubate them overnight at four degrees Celsius.

The next day, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate and re-suspend the samples in 100 microliters of the commercial perm wash solution. Next, centrifuge the plate at 931 times g for two minutes. Remove the supernatant by inverting the plate.

Then re-suspend the samples in 100 microliters of perm wash solution containing secondary antibody at room temperature for one hour and protect it from light. After an house, centrifuge the plate 931 times g for two minutes, then remove the supernatant by inverting the plate, following that, re-suspend the samples in 100 microliters of commercial perm wash solution, centrifuge the plate at 931 times g for two minutes, remove the supernatant by inverting the plate. Then, re-suspend the sample in 100 microliters of commercial perm wash solution and centrifuge the plate at 931 times g for two minutes again before removing the supernatant.

Next, re-suspend the samples in 300 to 500 microliters of fax buffer and transfer them into the separate one milliliter test tubes or five milliliter round bottom tubes for running flow cytometry. Shown here is a sematic of the induction of pancreatic progenitor from human embryonic stem cells which indicates day of differentiation, develop mental stage, base media and the cytokines added at every stage. And here are the phase contrast images at the key stages of pancreatic differentiation.

H1 cells were differentiated according to the four stage differentiation protocol depicted. With undifferentiated human embryonic stem cells at day zero, definitive endo derm at stage one, posterior four gut at stage two, pancreatic endo derm at stage three and NKX6.1 positive endo derm pancreatic progenitors at stage four. This figure shows the day three representative flow cytometry plots for CXCR4 PE and CD117 APC antibody staining.

Here is the unstained sample and here is the stained sample. And this figure shows day 13 representative flow cytometry plots for stage four derived pancreatic progenitor cells expressing NKX61 and PDX1. This is the IGG control and this is the stained sample.

Once mastered, this technique can be done in 13 days. However this may vary depending on the cell line used. While attempting this procedure, it is important to test all agents due to batch to batch variability.

After following the procedure, the NKX6.1 positive pancreatic progenitor cells can be further differentiated to insulin producing beta like cells using other published protocols. This provides a platform for studying beta cell maturation and development in vitro. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells to pancreatic progenitors by taking the cells through four key stages of development.