ヒト膵臓の開発にリネージュの決定要因を問い合わせるためのゲノム編集とhPSCsの指向性分化

This protocol demonstrates how to establish the iCRISPR platform for rapid genome editing in hPSCs, and how to incorporate genome-edited lines with hPSC-directed pancreatic differentiation to study human pancreatic development and disease. These methods can help answer key questions in the human pancreatic development field, such as a genetic control of normal pancreatic development, and the molecular basis of diabetes. The main advantage of these techniques is they allow us to efficiently modify genes of interest in hPSCs and to use these modified cell lines for studies of human pancreatic development and disease in vitro.

Culture hPSCs in chemically defined and feeder-free conditions on truncated human vitronectin-coated dishes. The day before electroporation, add 10 micromolar ROCK inhibitor during the media change. On the day of electroporation, prepare vitronectin-coated 10-centimeter dishes in advance.

Then remove culture medium from the cells, wash once with PBS without calcium and magnesium, then treat the cells with 1X dissociation reagent at 37 degrees Celsius for approximately three minutes. Next, aspirate the dissociation reagent before the cells have detached and add 10.5 milliliters of complete medium. Use gentle pipetting to form a single-cell suspension before transferring to centrifuge tubes.

Perform a cell count on 500 microliters of cell suspension using an automated cell counter. Then pellet the hPSCs at 200 times G for five minutes. Resuspend the cells in cold PBS at 12.5 times 10 to the sixth cells per milliliter, and aliquot into 800-microliter volumes.

Add the plasmids into each 800-microliter hPSC suspension and mix well. Transfer the mixture to a 0.4 centimeter electroporation cuvette, and keep on ice for approximately five minutes. Electroporate the cells using an electroporation system at 250 volts and 500 microfarads.

It's critical to use healthy and proliferating hPSCs for genome editing. After electroporation, transfer the cells to a 15 milliliter conical tube with five milliliters of pre-warmed, complete medium, and then pellet the cells. Resuspend the cells in 10 milliliters of complete medium with 10 micromolar ROCK inhibitor, and plate one times 10 to the sixth, 2.5 times 10 to the sixth, and five times 10 to the sixth cells from each electroporation onto vitronectin-coated dishes, so at least one of the plates will have sufficient single-cell colonies for picking.

Culture the cells at 37 degrees Celsius. On day two, start neomycin selection by adding medium containing 500 micrograms per milliliter G418 sulfate. On day six, start puromycin selection by adding medium containing one microgram per milliliter puromycin dihydrochloride.

On days 10 to 12, the hPSC single-cell colonies will reach one to two millimeters in diameter. At this point, pick 12 to 24 colonies under a stereo microscope. Mechanically disaggregate the hPSC colonies into small pieces using a 200-microliter pipette tip, and transfer the cells directly into vitronectin-coated 24-well plates.

24 hours before guide RNA transfection, treat iCas9 hPSCs with two micrograms per milliliter of doxycycline. On the day of transfection, prepare vitronectin-coated 24-well plates and dissociate the iCas9 cells into single cells using 1X dissociation reagent as before. Next, pellet the cells at 200 times G for five minutes and resuspend the cells at 0.5 times 10 to the sixth cells per milliliter in complete medium supplemented with two micrograms per milliliter doxycycline and 10 micromolar ROCK inhibitor.

Plate 500 microliters of the resuspended cells into individual wells of the 24-well plate. For each guide RNA, make the following transfection mixtures. Mix A, 50 microliters of reduced serum medium plus one microliter of guide RNA.

Mix B, 50 microliters of reduced serum medium plus three microliters of transfection reagent. Combine mix A and B to make a 100-microliter mixture and incubate for five minutes at room temperature. Add 50 microliters of the mixture to cells in the duplicate wells of the 24-well plate and mix well.

After four days, extract genomic DNA, PCR amplify the target regions flanking the guide RNA targeting sequences, and estimate the editing efficiency using T7E1 digestion. Begin differentiation of hPSC lines when cells have reached 80%confluency two days after seeding. Add differentiation day zero medium and then change the media daily using the formulas shown here.

On day 10, examine the later pancreatic progenitor PP2 markers, PDX1, and NKX6.1 by immunofluorescent staining a subset of the cells. Then proceed to air-liquid interface culture by first treating the PP2 cells with 10 micromolar ROCK inhibitor for four hours. Once the incubation time has elapsed, dissociate cells as before.

Minimize the duration of dissociation reagent treatment to maximize cell survival for seeding at the air-liquid interface stage of differentiation. Aspirate the dissociation reagent before the cells have detached. Then add 10 milliliters of boo-lah medium to the plate and disperse the PP2 cells into single cells by gently pipetting up and down.

Collect the single-cell suspension. Count the cell number, and pellet the cells. After resuspending the cell pellet in S5 differentiation medium, spot five to 10 microliters of cells per spot on a transwell insert filter.

Add S5 medium to the bottom of each transwell insert and incubate with media changes until pancreatic endocrine markers are detectable by immunofluorescent staining. This figure shows iCRISPR-mediated non-homologous end joining in hPSCs. Gene knockout is highly efficient, as no selection process is involved, and 20%to 50%biallelic mutants can be easily achieved.

For efficient and precise genetic alterations, guide RNAs are cotransfected with a single-stranded DNA donor carrying the specific sequence alteration, shown in red. Often, to prevent re-cutting in modified alleles, it is recommended to include a silent mutation in the PAM sequence, shown here in green. With this system, approximately 10%of clones carrying the desired homology-directed repair-mediated genome modification, without additional alterations in both alleles, can be achieved.

Once mastered, it takes less than two months to establish a new iCas9 hPSC line. After that, a variety of hPSC mutant colony lines can be generated within one to two months. Following generation of hPSC genetic mutant lines, in vitro stepwise differentiation of the mutant cell lines into pancreatic beta-like cells allows us to study the specific developmental steps affected by these mutations, which'will permit better understanding of underlying disease mechanisms.