Clonal Expansion of
Single Pluripotent Stem Cells
Pluripotent stem cells are known for the need to grow as colonies in order to prevent differentiation. This adds complexity to the isolation/expansion of clonal populations following research techniques such as gene editing. Have you ever wished you could isolate and expand clonal lines of pluripotent stem cells via limiting dilution starting from a single cell? Now you can.
Below is an example of expansion of induced pluripotent stem cells in PluriQ™ G9™ Cloning Medium starting from a single cell.
PluriQ™ G9™ Cloning Medium (100 mL) supports single cell plating and clonal expansion of human iPS or ES cells while maintaining pluripotency. From cells in culture, dissociate to single cells, set up a serial dilution to get to 10 cells/mL in PluriQ™ G9™ Cloning Medium and plate 100 µl such that the majority of cell containing wells will have a single cell. Feed daily with PluriQ™ G9™ Cloning Medium according to the protocol and in 2 weeks the cells will have expanded such that the clonal line can be split to create duplicate plates for freezing and screening or to move into a larger plate for further expansion. By conditioning our PluriQ™ G9™ Maintenance Medium on proprietary human stromal cells, the complete PluriQ™ G9™ Cloning Medium supports single cell survival and expansion without the need for additional supplements. Clonal human ES and iPS cells can subsequently be expanded in our regular PluriQ™ G9™ Maintenance Medium on G9™ VTN for further characterization and experimentation in continuous feeder-free, xeno-free conditions. PluriQ™ G9™ Cloning Medium was optimized for use with human Pluripotent Stem Cell Lines (PSCs) grown in PluriQ™ G9™ Maintenance Medium, but it is also compatible with starting cultures grown in other media such as mTeSR1™ and Essential 8™. Colony formation after depositing single cells by flow cytometry directly into the G9 Cloning Medium has also been successful.
Figure 1: Chart showing the percentage of wells with no colonies, a single colony or multiple colonies after plating 100 µl at 10 cell/ml of human ES or iPS cells in PluriQ™ G9™ Cloning Medium, as described, per well of a 96-well plate. Standard error bars were calculated for the total number of wells with colonies out of 32 wells from 3 independent experiments. Cells were grown for 2 weeks as described in the protocol.
When used in conjunction with our PluriQ™ G9™ Gene Editing System, where high-efficiency CRISPR/Cas9 gene editing is readily achievable, clonal lines can easily be isolated without flow-sorting, manual isolation or chemical selection. Human ES and iPS cells typically die or differentiate, when enzymatically dissociated and plated as individual cells in most media. Up to 40% of human ES and iPS cells will survive and form clonogenic lines, when dissociated using G9™ Versene Solution (1X) and plated at an average of 1 cell per well on G9™ VTN (vitronectin) in PluriQ™ G9™ Cloning Medium, while maintaining pluripotency.
Figure 2. Human iPSC line NCRM-1 subclone 485 after transfection with EditPro™ Stem, expanded from a single cell in G9 Cloning Medium on Vitronectin to a T75 flask in 4 passages, maintaining Oct4 expression a marker of pluripotency.
- Supports pluripotent stem cell expansion from a single cell
- Maintains pluirpotency
- Ready to use, just thaw.
- Xeno-free, feeder-free.
- Compatible coming from PluriQ™ G9™ Medium and other popular commercial media and plating on G9™ VTN (vitronectin) as well as other common matrices.
Download PluriQ™ G9™ Cloning Medium Protocol
- Kehler, J., Greco, M., Martino, V., Pachiappan, M., Yokoe, H., Chen, A., Yang, M., Jessee, J., Gotte, M., Milanesi, L., Albertini, A., Bellipanni, G., Zucchi, I., Reinbold, R. A. and Giordano, A. (2016), RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy. J. Cell. Physiol. 2016 Sep 15. doi: 10.1002/jcp.25597. [Epub ahead of print]
|Recommended Use||Expansion of human ESC and iPSCs from single cells to establish clonal lines.|
|Storage||Keep at -20°C for long term storage. It is stable for up to 2 weeks at 4°C. Do not subject to repeated freezing and thawing.|
|Quality Testing||Mycoplasma, Sterility, Cloning Efficiency|
|Shipping||Frozen on dry ice.|
PluriQ™ G9™ Cloning Medium
- Product Code: GSM–9020
- Availability: In Stock