Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Attaining a high and consistent efficiency in the differentiation process for converting pluripotent stem cells into human beta cells is an notable challenge. This challenge is influenced by the precision and the consistency with which the experimenter follows the protocol. Moreover, the yells of beta cells obtained in each experiment is quite variable, which may result in a reduced functional beta cells yells across multiple experiments.

In the context of islet and beta cell research in diabetes, the availability of cadaveric islet donors is quite limited. Consequently, the use of stem cell differentiated beta cells is offering an unlimited and abundant supply of insulin secreting cells. Beta cell differentiation is a pivotal advancement in our field, offering numerous benefits.

It allows us to explore the development and function of beta cells, leading to a deeper understanding of diabetes causes and beta cell dysfunction. We aim to use this technique for our research focusing on the genetics of diabetes. Our primary objective is to focus on mutation of certain genes that can influence beta cells and islet function.

So this protocol will facilitate the access to human islet derived from pluripotent stem cells with mutations of interest. Before cell passage, coat a six-well plate with cold coating solution and incubate at 37 degrees Celsius with 5%carbon dioxide for one hour. Aspirate the stem cell medium from the six-well plate containing human pluripotent stem cells and add one milliliter of calcium magnesium free DPBS into each well.

After the second wash, add 500 microliters of dissociation solution into each well and incubate for two to five minutes at room temperature. In the meantime, aspirate the coating solution from the six-well plate, and add one milliliter of calcium magnesium free DPBS into each well. Under an inverted microscope, observe the cell dissociation process.

When 80 to 90%of the cells appear to be rounded and adherent, aspirate the dissociation solution from the wells of the six-well plate. Then add one milliliter of stem cell medium containing 10 micromolar ROCK inhibitor into the six-well plate. Add 10 microliters of dissociated cell suspension into a tube, then add an equal volume of Trypan blue.

Mix gently and count the cells using a hemocytometer. After cell counting, add two milliliters of stem cell medium per well, containing 0.8 to one times 10 to the six cells per milliliter and 10 micromolar ROCK inhibitor. Quickly move the plate back and forth, side to side three times.

Place the plate in a 37 degree Celsius 5%carbon dioxide incubator. Quickly move the plate back and forth and side to side 10 times before incubating for two hours. Thaw both the basal medium for days zero and four and supplements on ice.

Warm two milliliters each day one medium and washing medium one, in a 37 degree Celsius water bath for five minutes. Next, aspirate the stem cell medium from the wells of a six-well plate and add two milliliters of washing medium one. After aspirating washing medium one, immediately add two milliliters of day one medium to the side of the well.

Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours. On day 12 of the differentiation process, treat a six-well plate containing 400-micrometer microwells with two milliliters of anti-adherence rinsing solution. Under an inverted microscope, observe the plate for bubbles.

If no bubbles are observed, aspirate the anti-adherence rinsing solution and immediately add two milliliters of washing medium two. Then aspirate pancreatic progenitor medium and add 0.5 milliliters of dissociation buffer per well. Incubate the cells at room temperature for two to five minutes.

When most of the cells are rounded and are still adherent, aspirate the dissociation buffer and immediately add one milliliter of warm cluster medium to each well. Using a P1000 pipette tip, dissociate the cells gently, pipetting up and down, while alternately using a circular motion and moving from the top to the bottom of the well to detach cells evenly throughout the well. Transfer the dissociated cell mixture into a 50-milliliter conical tube.

Add one milliliter of the cluster medium into the six-well plate to collect all remaining cells. Next, aspirate washing medium two from the microwell six-well plate and add dissociated cell suspension. Incubate the cells at 37 degrees Celsius for 24 hours.

The next day, using a P1000 pipette tip, slowly collect clusters from the microwell six-well plate into a 50-milliliter conical tube. Add one milliliter of day 13 medium to collect the remaining clusters and transfer the clusters into the tube. Allow the cells to set naturally under gravity to the bottom of the tube for five minutes.

Carefully aspirate the supernatant from the tube without disturbing the cell clusters. Then, add two milliliters of day 13 medium into the tube. Gently mix by pipetting up and down, avoiding the aspiration of clusters.

Transfer the suspension to a very low adherence six-well plate. Quickly move the plate back and forth, side to side six times. Incubate the cells at 37 degrees Celsius for 48 hours.

Immunofluorescence images of the clusters showed of predominance of insulin producing cells co-expressing the pancreatic beta cell markers. The expression of beta cell signature genes revealed approximately 25%of cells expressing Nkx6.1 and 40%expressing Pdx1. During the glucose stimulated insulin secretion assay, the MEL-1 derived clusters secreted significantly higher levels of insulin in response to high glucose concentrations compared to low glucose concentrations.