The Good, the Bad and the Ugly

Last time we talked about how we can turn your skin cells into pluripotent stem cells by adding the reprogramming factors to the cells. Now, we will show you how stem cell colonies begin to form and emerge in the culture dish.

Timeline of reprogramming skin cells

Alex: It has been two weeks since you added the booster shot to my skin cells. Are my cells stem cells yet?

Lauren: Almost there. I started to see aggregated cell clumps after 10 days in the culture dish. I am really excited and hope that these clumps will become larger and form stem cell colonies.

In our last conversation, I showed you that the reprogramming took place in 1-well of a 6-well plate. Seven days after I added the reprogramming factors, I transferred your cells into larger dishes each containing about 200,000 cells to provide the cells with enough space to grow and divide. These larger culture dishes also have a layer of feeder cells which give your transforming skin cells structural support and they also release growth factors. I am expecting colonies to start emerging 2-3 weeks after reprogramming.

Alex: 200,000 cells per dish? How do you actually count the cells?

Lauren:In the past, you would count them yourself under the microscope in a Neubauer counting chamber, but we have now a device in the laboratory that does it for us which saves a lot of time.

Alex: Are there any other steps involved while you are waiting for the colonies to emerge?

Lauren: While we wait, I continue feeding your cells with fresh media every day and observe them under the microscope to see if any colonies are starting to appear. Once the colonies are large enough, I will transfer them individually onto new separate plates. This allows me to isolate one colony at a time and expand. A cell culture derived from one colony is termed a clonal cell line or a clone.

Alex: What does it look like when a colony is starting to appear?

Lauren: As you can see in the picture below, potential clumps of cells start developing. I track the cell clumps and observe whether, over time, they start breaking apart and whether they have (or are beginning to show) the morphological characteristics normally associated with stem cells. This colony pictured here is most likely not fully reprogrammed, but we cannot tell for sure at this point.

Partially reprogrammed cells 10 days after reprogramming

Alex: How would a colony that is fully reprogrammed look different?

Lauren: What I noticed is that colonies that were smaller, more compact, and had a "glowing" appearance under the microscope were the ones that were more likely to become stem cell colonies. The picture below is an example of these colonies. As you can see, they are more compact, have distinct borders, and have a cobblestone-like appearance.

Small stem cells colony 15 days after reprogramming

Lauren: Pluripotent stem cell colonies have a unique appearance. What we will see in the culture dish when fully reprogrammed stem cell colonies emerge are round, compact cell aggregates with sharp, distinct borders. The picture below is an example of a “pristine” stem cell colony. A stem cell colony can contain –depending on its size- between 3000-5000 individual cells

An example of an ideal iPSC colony

Alex:I will keep my fingers and toes crossed. Thanks so much for taking me on this journey.

Lauren: My pleasure, Alex. In the next blog, I will show you how I discern the good colonies from the bad ones. The good colonies are actually pretty ugly in the beginning