ゲノム編集したヒト多能性幹細胞株の樹立：分離へのターゲティングから

The overall goal of this procedure is to genetically engineer human pluripotent stem cells using modern genome editing technology. Though this procedure can provide insight into stem cell biology, it can also be applied to facilitate human disease modeling in a genetically defined setting. Generally, individuals new to this method will need to practice isolating colonies before becoming proficient.

Visual demonstration of this method is essential as identification and picking colonies can be difficult. Begin by culturing human pluripotent stem cells in hESC media on a six-well plate containing mitomycin C inactivated mouse embryonic fibroblasts, or MEF feeder cells grown on gelatin. Each subsequent day after plating, until the hPSCs reach 50%confluency, use a glass pipette and vacuum to remove the entire volume of media.

Replace with three milliliters of warm hESC media per well. One day before targeting, remove the hESC media and add fresh pre-warmed hESC media supplemented with 10 micromolar Y-27632. Also, prepare one to two six-well plates of drug resistant MEF feeder cells from DR4 mice.

On the day of targeting, prepare the transvection solutions by pipetting five micrograms of Zinc finger nuclease expression plasmid 1 and 2, TALON 1 and 2 expression plasmid, or 15 micrograms of the CRISPR cas9 px330 encoding plasmid into a one point five milliliter tube. Add 30 micrograms of the repaired donor plasmid, followed by enough 1x phosphate buffered saline to bring the volume to 300 microliters. Inspect the cells under a microscope to ensure 50%confluency.

Next, use a glass pipette and vacuum to remove the media from the plate of hPFCs, then wash the cells with 2 milliliters of warm 1x PBS. After aspirating the PBS, add zero point five milliters of 0.25%Trypsin-EDTA solution directly onto the cells. Place in the tissue culture incubator for approximately 10 minutes, or until the feeder layer begins to lift off the plate.

Following the incubation, add two milliliters of warm ES wash media to each well to stop the Trypsin reaction. Collect the cells from each well, ensuring that the feeder cells come off as a sheet. Pipette the contents of each well into a single 50 milliliter conical tube, combining all wells and triturate the cells using a 10 milliliter serological pipette.

Add ES wash media to bring the cell suspension to 40 milliliters. Wait one to two minutes to allow large feeder chunks to settle at the bottom of the tube, then remove the supernatant, using a serological pipette, and deposit into a fresh 50 milliliter conical tube. After centrifuging the cell suspension for five minutes at 190 times g, aspirate the supernatant without disturbing the cell palette.

Resuspend the cells in 500 microliters of 1x pbs. Combine the resuspended cells with the plasma transection solution prepared earlier. Pipette the cells and transfection mixture into a four millimeter electroporation cuvette, and place on ice for three to five minutes.

Set the parameters for the exponential program on the electroporation system to 250 volts, 500 microfarads, infinite resistance, and four millimeter cuvette size. Electroporate the cells, and then place the cuvette back on ice for three minutes. Resuspend the electroporated cells in 18 milliliters of warm hESC media, supplemented with 10 micromolar Y-27632.

Inspect the DR4 MEFs under a microscope. Plate three milliliters of the single cell suspension into each well of a six-well plate containing DR4 feeder cells, and return to the incubator. On day three after plating, replace the media on the cells hESC media without Y-27632.

On day four, replace the unsupplemented media with media containing the appropriate selection antibiotic. Puromycin is used here. On the day before colony picking, prepare one 12-well plate of MEF feeder cells for each colony to be picked.

On day 12 after plating, examine the plate under a dissection microscope. Identify colonies of 800 to 1, 200 microns in diameter that are ready to be picked. Ensure that those colonies do not contain cells that are beginning to differentiate, such as the one shown here.

Also, ensure that the cells are expressing GFP. On the day of picking, inspect MEFs under the microscope to ensure that they are healthy. Remove all media from the MEF feeder cell plates, and replace with one milliliter of hESC media.

Also, change the hESC media on the six-well hPSC plates that are going to be picked. Next, pull glass pipettes for picking colonies. To pick colonies, first place the plate hPFCs onto the stage of the dissection microscope in the tissue culture hood, assemble the picking device by placing the glass pipette end into the suction bulb.

Identify the colony to be picked, and place the tip of the pulled glass pipette over the colony. Then, compress the bulb of the picking device to gently excise and cut and individual colony into 10 to 20 equally sized pieces. Next, draw the excised pieces of colony into the pipette by releasing the bulb.

Try to take as little media as possible while transferring. Compress the bulb to transfer the now broken colony directly into one well of a 12-well MEF feeder cell plate. Label each well to allow unique identification of single cell derived clones.

Change the glass pipette and repeat the procedure for the next colony. Return the plates to the incubator, and gently rock the plates to disperse the cells in the well. The next day, remove the full volume of media, and replace with one point five milliliters of warm hESC media.

Repeat for 10 to 12 days until the cells are 50%confluent. After 10 to 12 days, inspect the hESCs under the scope. Pick one to two colonies from each well, and transfer to new 12-well MEF feeder cell plates to generate a replica plate.

Extract DNA from the remaining colonies in each well of the original plates for genotyping by PCR or southern blot. Weber Three human embryonic stem cells were targeted at the AAVS1 locus using site-specific nucleases and a repair template to introduce an EFFP reporter and a puromycin resistance cassette. These representative images show colonies edited with zinc finger nucleases, TALENs, and CRISPR/Cas9.

These representative PCR genotyping results show untargeted, heterozygous, and homozygous targeted clones using zinc finger nucleases, TALENs, and CRISPR/Cas9 along side a wild type control. This table shows the PCR verified integrations at the AAVS1 locus for each site-specific nuclease in this experiment, compared to Southern Blot verified proper single integrations in previous experiments. CRISPR-Cas9 yielded the most correctly targeted clones, while the TALEN platform had the most homozygously targeted clones.

Once mastered, this technique takes about three to five hours of hands-on time, and you can get edited cells within about three weeks of starting experiments. It's really important with this technique to work safely, and use sterile technique at all times. Following the genetic manipulation, you can see how it affects other types of cells by using stem cell differentiation into other cell types, such as neurons.

The development of this protocol paved the way for researchers to use genome editing in human pluripotent stem cells to establish in vitro disease models. After watching this video, you should have a good understanding of how to genome edit in human pluripotent stem cells.