Science Buddies: "Ask an Expert"

Hi,

I understand the theory and reasoning behind direct reprogramming, but I am confused about one thing: how would this be done in a lab (specifics)? How do experimenters "force" mature cells to express certain transcription factors associated with a nother type of cell? If anyone can describe the specific process that could be done in a lab, I would really appreciate it. Also, if anyone can explain how chromatin remodeling agents are involved in direct reprogramming (again, specifics) that would be great.

Thanks!

Typically when scientists are altering cell fate they are either going from a stem cell or pluripotent state to a particular cell type through differentiation. This can be accomplished by adding small molecules (growth factors, etc) to the cell culture media and culturing the cells on a matrix that would promote differentiation down a particular lineage. Specific transcription factors can also be added to the media by either DNA/RNA transfections or with protein transduction (Depending on if the transcription factors can be expressed in bacterial systems and further purified). This cell-specific differentiation is not considered reprogramming, however.

If you want to reprogram a differentiated cell, you would be trying to de-differentiate it back to a pluripotent state similar to a stem cell. Once you get to this point you can then go on to differentiate it as mentioned above to a cell type of choice (regardless of starting cell type). Similarly to differentiating stem cells as above, you can introduce small molecules (growth factors, signaling cytokines, morphogens, etc) to the media which would promote the endogenous transcription of genes involved in pluripotency. Another method is again by introducing exogenous DNA or RNA through transfections. Most recent, and I believe the best method, is to introduce proteins to the cells in culture (ex. Oct4, Sox2, etc). This allows the transcription factors to immediately be used in DNA binding to promote chromatin remodeling, which brings us to your last question. As I mentioned before, you can add small molecules or proteins to your cell culture to induce a response in your cells. Similarly, chromatin remodeling agents such as deacetylases such as HDACs can be added to your culture medium as well. Chromatin status is imperative for reprogramming and the main pluripotency genes are involved in the regulation of particular chromatin states, along with other factors, by binding to specific motifs on DNA.

The cool thing about cellular reprogramming is that there are many ways to accomplish the same thing. Everyone is trying to find the "best" and the "most efficient" way by mixing up the composition of what they introduce to the cell to induce reprogramming. If you are looking for an exact protocol, I would suggest figuring out which method (small molecule, DNA, or protein) you want to use to reprogram and then do a literature search on that specifically. You will find a huge amount of literature with all different types of cocktails to choose from.

I hope this helps.
Kelsey

Thank you so much! I really appreciate it, and I now understand the process.