The Ugly Ducklings

Last time we talked about how partially reprogrammed cells and pristine stem cell colonies look like the Good, the Bad, and the Ugly. Today, we will talk about the challenges of ‘giving birth’ to Alex’s stem cells.

Timeline of reprogramming skin cells

Alex: What exciting things have happened over the last two weeks? Are my stem cells “born”, yet?

Lauren: Well, in the last two weeks, I finally began to see colonies that matched the morphological characteristics of induced pluripotent stem cells, also called iPSCs, we talked about last time. The process reminds me of “hatching eggs”. The hen needs to care and keep the eggs warm before they hatch. Once the first egg starts to crack, all the eggs start to crack and these ugly little chicks come out and eventually turn into cute fluffy little chickens. For the stem cells it is similar, first I need to feed them and check on them every day for changes, then within a couple of days the colonies come together and “pop up” almost overnight. Usually the colonies are hard to see because there are other cells growing on top of them, hiding them and making them look “ugly”. The red circles in the images below show some of these colonies that were a little trickier to see.

Examples of forming iPSC colonies

Alex: There is more to these cultures than meets the eye. It looks like the colonies need a lot of TLC and someone with experience to take care of them.

Lauren: That is very true Alex, it takes a lot of time and work to care for these cells. Since some of the colonies were harder to see I had to “clean them up” before I could transfer them to a new plate. Cleaning the colonies entails carefully scraping off the differentiated cells so that only the stem cells can be transferred. The pictures below show an example of what the colonies look like before and after “cleaning”.

iPSC colony before and after “cleaning"

Alex: Wow, that is precision work. What instrument do you use to clean the cells?

Lauren: I use a fine tip of a pipette and carefully push the differentiated cells aside. If the colony is a true stem cell colony, it seems like you can peel off the layer of differentiated cells. I do this procedure under the microscope in a laminar flow hood.

"Cleaning" cells with fine pipette

Alex: Amazing. How many of my stem cell colonies are being “delivered”?

Lauren: I was not prepared for the amount of colonies that began to show up in the dishes. It felt like an explosion of colonies all at one time. Each day, I was picking between 15 and 20 colonies of your cells. I would cut the colonies into smaller pieces and then transfer them to a different plate. Some of the colonies successfully attached and continued to grow when I replated them and some of them did not. In total I picked 75 colonies and 50 of those colonies attached and continued to grow. Each colony that I picked is now considered its own clone and given an identifying clone number to keep track of them. These iPSC colonies just kept emerging on the original plates, I finally had to make a decision to just stop picking new colonies and focus on taking care of the all the colonies I had already picked. Eventually I will need to narrow down the number of clones to 3 which is more manageable.

Alex: I am truly fascinated by this biology. And it all came from a little piece of my skin that I donated the last time I visited you. What will happen next?

Lauren: In the next blog, I will talk about how we create a master bank of each stem cell clone. I will also give you an introduction on how we characterize each of your stem cell clones for future experiments. 

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