This optimized protocol enhanced the generation of pancreatic beta cell precursors from human pluripotent stem cells, which can be used for cell therapy for diabetes and investigating molecular mechanisms underlining diabetes biogenesis. This is a feasible technique as it is used for 2D monolayer cultures. It is easy to apply, and is consistent across multiple control and patient-derived pluripotent stem cell lines.
These beta cell precursors when transplanted in a diabetic patient can mature into insulin secreting beta cells, and can potentially reverse hyperglycemia. Therefore, the increased percentages of pancreatic progenitors generated using our protocol can be used for cell therapy for diabetes. This enhanced protocol can be used for studying early human pancreatic development in healthy individuals as well as in diabetic patients, which is challenging due to a shortage of tissue samples.
When the human pluripotent stem cell or hPSC colonies have reached 70 to 80%confluence, wash them twice with warm PBS inside the tissue culture hood. Then aspirate the buffer using a portable vacuum aspirator. Next, add stage one differentiation medium containing CHIR-99021 to the colonies, and incubate the plate at 37 degrees Celsius for 24 hours.
The next day, replace the spent media with stage one differentiation medium without CHIR-99021. After aspirating the spent medium and washing the definitive endoderm cells twice with warm PBS, swirl the plate to remove any cell debris. Then fix the cells by adding 4%paraformaldehyde, and place the plate on a two-dimensional shaker.
After fixation, wash the cells with TRIS-buffered saline containing 0.5%Tween, and place the plate on the shaker. Then permeabilize the fixed cells by adding a generous amount of PBS containing 0.5%Triton X-100 and place the plate back on the shaker. Next, add freshly prepared blocking buffer to the permeabilized cells, and incubate the plate for one hour on the shaker.
Then add diluted primary antibodies to the blocked cells, and place the plate on the shaker at four degrees Celsius with gentle shaking. The following day after aspirating the primary antibody solution, wash the wells three times with TBST for 10 minutes each on the shaker. Next, add the secondary antibodies.
Cover the plate with aluminum foil to protect from light, and place the plate on the shaker at room temperature for one hour. Then aspirate the secondary antibody solution, and wash the stained wells with TBST on the shaker for 10 minutes, keeping the plate covered with foil. Next, to stain the nuclei, add Hoechst solution to the wells and place the plate on the shaker.
After two to three minutes, aspirate the Hoechst solution and rinse the wells twice with PBS. Finally, add PBS to the stained cells, and image them using an inverted fluorescence microscope in the dark. On day one of stage two, dissociate the hPSC derived endodermal cells by first washing them with warm PBS, and then adding one milliliter of warm enzyme solution per well of a six well plate.
Place the plate at 37 degrees Celsius and 5%carbon dioxide for three to five minutes or until the cells begin to detach from one another. Dissociate the detached sheets or monolayer of cells in the wells, and collect the detached cells together in a 15 milliliter polypropylene tube using basal stage half media. Then spin the cells down, discard the supernatant, and resuspend the pellet using one milliliter of sterile PBS.
After counting the cells using an automated counter, spin the cells again, and discard the supernatant. Then resuspend the pellet in the appropriate volume of stage two differentiation medium containing a Rock inhibitor. Plate the resuspended cells on one to 50 membrane matrix coated plates and incubate them at 37 degrees Celsius and 5%carbon dioxide.
After 24 hours, replace the media with stage two differentiation medium without the Rock inhibitor. At the end of stage four, detach cells as demonstrated earlier, and collect them in a 15 milliliter tube. Then spin the cells, discard the supernatant, wash the cells with PBS, and dissociate them into single cells.
After counting the cells using an automated cell counter, spin the cells again and resuspend the pellet in 200 microliters of chilled PBS. Next, add two milliliters of chilled 80%ethanol drop wise with the tube on a vortex set low to medium speed. Close the cap tightly, and place the tube slightly tilted on the shaker at four degrees Celsius overnight.
The next day, spin the cells and wash the pellet with PBS to dissociate any clumps of fixed cells. Then block the fixed cells for at least one hour at room temperature or four degrees Celsius overnight on the shaker. Next, to stain for the pancreatic progenitor markers, distribute 200, 000 cells per condition including appropriate isotype controls, unstained, and secondary antibody controls in a 96 well V bottom plate.
Then spin the plate briefly, and flip the plate with a swift motion to discard the supernatant without losing the pellets. Next, incubate the cells in primary antibodies for at least two hours at room temperature on a shaker with gentle shaking. Then wash the stained cells three times with TBST by pipetting up and down in the wells.
Centrifuge the cells again, discard the supernatant, and add secondary antibodies to the cells. After a 30 minute incubation at room temperature, wash the stained cells twice with TBST. Then spin the plate, collect the stained cells in 100 microliters of PBS in light protected fax tubes, and run the samples on a flow cytometry machine.
Compared to the non-optimized method, the optimized protocol enhanced pancreatic progenitor differentiation efficiency by upregulating PDX 1 and NKX 6.1 co-expression. In particular, the disassociation of endodermal cells and their replating on fresh membrane matrix, along with a longer duration of stage three, enhanced NKX 6.1 expression in hPSC derived pancreatic progenitors compared to the non-dissociated protocol. Pancreatic progenitors generated using the optimized protocol also increased numbers of SOX9 positive cells compared to the non-dissociated method.
Further, the optimized method also generated a higher proportion of proliferative NKX 6.1 positive cells that co-expressed the proliferation marker Ki67. In order to enhance the efficiency, it is crucial to dissociate the HbA1 derived endoderm and then extend the duration of stage three treatment with FGF amyloid signaling and Hedgehog inhibition. Scalable generation of pancreatic progenitors using this enhanced protocol has facilitated studies on improving efficiency and maturation of human pluripotent stem cell derived pancreatic beta cells and allowed modeling of different type of diabetes.
