利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

The overall goal of this procedure is to generate footprint-free induced pluripotent stem cells or IPSCs from human pancreatic cells in feeder-free conditions, then to edit the cells using CRISPR-Cas9 ribonucleoproteins and finally to characterize the modified single cell clones. This method can help answer key questions in the human stem cell genome editing field such as generation of footprint-free IPSCs and editing with CRISPR-Cas9 ribonucleoproteins. The main advantage of this technique is that clonal lines of edited IPSCs can be generated with high reliability and there is no evidence of mosaicism.

After coating a six well plate with cold collagen, plate early passage human primary pancreatic cells in Prigrow III medium on day 2 to achieve approximately 2.5 x 10 to the 5 cells or at least 60%confluency per well on the day of transduction or day zero. On the day of transduction after harvesting and counting the cells according to the text protocol, thaw on ice one set of Sendai vector tubes and carefully add the calculated volumes of each tube to 1 mL of prewarmed Prigrow III medium. Gently pipette to mix the solution.

Aspirate the Prigrow III from the cells and slowly add the reprogramming virus mixture to the wells. Then incubate the cells in a 37 degree Celsius incubator overnight. Twenty-four hours after transduction, carefully discard the virus mixture on the cells and replace it with fresh Prigrow III medium.

On day six, add 1 mL of cell detachment solution to the cells and incubate them for 10 minutes. Then, using Prigrow III with rock inhibitor, plate all the cells on dishes of MEFs prepared the day before. Incubate the cells at 37 degrees Celsius overnight.

Observe the plates regularly for the emergence of cell clumps or colonies indicative of reprogrammed cells which will form clonal aggregates with a cobblestone morphology and a big nucleus and nucleoli. Mark the probable IPSC colonies and regularly check them for growth. Almost four weeks following transduction after preparing 24 well MEF plates, prepare matrix membrane by aliquoting it based on the dilution factor on the certificate of analysis.

Pick 24-48 colonies and transfer them onto matrix membrane coated 24 well plates with mTeSR1. Incubate the plates for 24 hours and change the medium every day. Refer to the text protocol for additional details.

Add 500 mcL of dispase to detach the robust colonies plated on the matrix membrane. After incubating the cells for 20 minutes, plate them again on matrix membrane coated 12 well plates. Expand and characterize the clones using alkaline phosphatase staining, immuno staining and FACS analysis according to the text protocol.

To transfect HIPSCs after synthesizing SGRNAs according to the text protocol, prepare nucleofaction master mix for each sample by combining 0.5 mcg of Cas9 protein and 0.5 mcg of each SGRNA in a 22 mcL reaction volume. After aspirating the medium containing rock inhibitor, use 2 mL of RTPBS to wash each well of ISPCs. Then aspirate the PBS and add 1 mL of cell detachment solution.

Incubate the plate at 37 degrees Celsius for 10 minutes. Resuspend the cells in 3 mL of mTeSR1 medium and gently pipette up and down to generate a single cell suspension. Transfer the dissociated cells to a 15 mL centrifuge tube containing 5 mL of mTeSR1 medium.

To the nucleofection master mix, add 16.4 mcL of P3 primary cell supplement and 3.6 mcL of supplement one from the Nucleofector kit. After counting and spinning the cells, resuspend each unit of 0.5 x 10 to the 6th cells in 22 mcL of the previously prepared transfection master mix. Quickly transfer the cells into the central chamber of one well of a Nucleocuvette strip.

Then, place the strip into a Nucleofector device and nucleofect the cells using program CB150. Following nucleofection, quickly add 80 mcL of prewarmed mTeSR1 medium containing 10 micromolar rock inhibitor to each well of the nucleofected cells. Gently mix by pipetting up and down.

Gently transfer the cells from the strip to wells of the matrix membrane precoated 12 well plate containing mTeSR1 medium with rock inhibitor. On the second day of incubation after preparing MEF plates, replace the medium on the HIPSCs from mTeSR1 to mTeSR1 supplemented with 1XSMC4 for at least two hours before single cell sorting. Aspirate the medium from HIPSCs and use PBS to gently wash the cells.

Then add 500 mcL of cell detachment solution into each well and incubate the cells at 37 degrees Celsius for 10 minutes. Generate a single cell suspension by adding 1 mL of mTeSR1 to each well and gently pipette up and down several times. Sort single cells into individual wells of a previously prepared 96 well plate.

Four days after sorting, colony formation should be apparent. At this point, replace the culture medium with HESC medium supplemented with 1X SMC4. After extracting and expanding the target DNA according the text protocol, use a fragment analyzer kit to run the PCR amplified target genomic region of all clones through the gel dye mix per the manufacturer's instructions.

Confirm pluripotent IPSC clones according to the text protocol. Before Sendai virus transduction, pancreatic cells show typical spindle-like morphology. Small tight colonies appear on MEFs by day 12 resembling human pluripotent cell colonies.

Normally, the fully reprogrammed human IPSC colonies have very clear boundaries and can be picked up by days 23 to 30. A dissociated colony at day 23 is shown here. Presented here is a typical phase contrast image of an IPSC colony after picking and culture in feeder-free conditions after Sendai virus reprogramming of pancreatic cells.

Alkaline phosphatase stained red IPSC colonies are on the right. IPSCs were confirmed by immuno staining for the pluripotency markers OCT4 and NANOG as seen in these panels. In this experiment, IPSCs were stained with cell surface markers and FACS analysis was carried out on a single cell suspension.

The green peak represents pancreatic IPSCs, and the purple peak contains IPSC negative cells. While attempting this procedure, it is important to remember to maintain aseptic or sterile conditions for cell sorting and cell culture. Following this procedure, other methods like gene targeting in IPSCs and ESCs can be performed, and additional questions can be answered by performing precise genetic modifications.

After watching this video, you should have a good understanding of how to reliably generate footprint-free and feeder-free IPSCs and perform genome editing biallelically.