Subcloning of genotype-confirmed hPSCs clones
Yogendra Verma, Hanqin Li, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs).
General notes:
- Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
Change medium to hPSCs medium + Rock inhibitor one day before subcloning
hPSC medium
| A | B |
|---|---|
| DMEM/F12 | 385 ml |
| Fetal Bovine Serum (FBS) | 75 ml |
| Knockout Serum Replacement | 25 ml |
| L-Glutamine (100X) | 5 ml |
| Penicillin & Streptomycin (100X) | 5 ml |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
| 2-Mercaptoethanol (10,000X) | 50 µl |
| Heat Stable Recombinant Human FGF2 (25ug/ml)* | 80 µl |
*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2. Final volume: 500ml
L-Glutamine (100X)
| A | B |
|---|---|
| L-Glutamine, powder | 14.6 g |
| MilliQ H2O | 500 ml |
2-Mercaptoethanol (10,000X)
| A | B |
|---|---|
| 2-Mercaptoethanol | 0.78 ml |
| MilliQ H2O | 9.22 ml |
Heat Stable Recombinant Human FGF2 (25µg/ml)
| A | B |
|---|---|
| Heat Stable Recombinant Human FGF2 | 500 µg |
| 0.1% BSA | 20 ml |
Final volume: 20ml
Y-27632 (1,000X)
| A | B |
|---|---|
| Y-27632 | 5 mg |
| DMSO | 1.56 ml |
hPSC medium + Rock inhibitor
| A | B |
|---|---|
| hPSCs medium | 500 ml |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500ml
Wash the genotype-confirmed wells with DPBS
Add 25 µl Trypsin to those wells
Incubate 0h 5m 0s 37°C
Add 75 µl hPSCs medium + Rock inhibitor, mix well by pipetting
Transfer dissociated cells to a 15 conical tube
Prepare three MEF wells of a 6-well plate. Seed 1/100, 1/1,1000 and the rest of the cells respectively to those wells
Change medium daily for the high density well starting from day 3. Change medium every 3 days for the low density wells in the first week, then every other day in the second week
Once the high density wells grow to 50-70% confluency, freeze.
For a detailed protocol on freezing hPSCs, refer to "Freezing of hPSCs grown on MEFs" in the "Thawing, Passaging and Freezing of hPSCs on MEFs" collection; dx.doi.org/10.17504/protocols.io.b4msqu6e
For a detailed protocol on freezing feeder-free hPSCs, refer to "Freezing of feeder-free hPSCs" in the "Feeder-free culturing of hPSCs" collection; dx.doi.org/10.17504/protocols.io.b4mcqu2w
When big hPSCs colonies form in the low density wells, change medium to hPSCs medium + Rock inhibitor
The day after, proceed with manual colony picking
For each original clone, prepare six microcentrifuge tubes pre-added with 20 µl Trypsin
Aspirate medium and add 2 ml DMEM/F12
Change medium to DPBS for the well where colonies will be picked
Under the dissecting microscope, use mouth pipet or a fine 10 µl tip to pick one undifferentiated colony which is fully separated from other colonies.
Transfer the colony to one microcentrifuge tube from step 12.
Repeat step 15-16 to pick another five colonies
Incubate the microcentrifuge tubes at 37°C 0h 5m 0s
Add 70 µl hPSCs medium + Rock inhibitor to each tube, pipet to mix
Seed all cells into one well of a 12-well MEF plate
Shake plates to distribute cells evenly
Culture for 7-10 days with medium change daily from day 3
When the subclones grow to 50-70% confluence, passage and prepare crude cell lysis for NGS genotyping
Crude lysis buffer (2x)
| A | B |
|---|---|
| KCl | 100 mM |
| MgCl2 | 4 mM |
| NP-40 | 0.9% |
| Tween-20 | 0.9% |
| Tris | 20 mM |
| Proteinase K (add before use) | 100 µg/ml |
pH: 8
Once genotype confirmed, expand and freeze the one subclone, which shows the best hPSCs morphology and proliferates normally.
Test for mycroplasma, stain for pluripotent markers, and karyotyping
