A booster in your coffee to rejuvenate

In the last blog, we showed and described how to prepare Alex’ skin cells for the reprogramming procedure (to turn skin cells into stem cells). Now, we will show how we are actually turning the skin cells into induced pluripotent stem cells (iPS cells).

Nuclear reprogramming timeline

Lauren:  A couple days ago I transferred your fibroblasts to a 6-well plate in which I will reprogram them. On the picture below you can see one of our tissue culture microscopes with a 6-well plate on the stage.

Fibroblasts in a 6-well plate under microscope

Alex:  It is really exciting to witness the whole process of coaxing my skin cells into stem cells. Tell me more about how you are doing this procedure.

Lauren:  Below is a picture of how your skin cells looked before I started the nuclear reprogramming.

Confluent fibroblast well before reprogramming

The reprogramming system containing four different proteins that are delivered by a virus system into your cells. The proteins delivered by the virus force and "remodel" your skin cells and turn them into iPS cells. The four viral factors are like a booster to your coffee to rejuvenate you. Now, since we are working with human cells and viruses, we need to protect ourselves and wear the appropriate personal protective equipment, like gloves, lab coat and face protection. On the other hand, to avoid contamination of your cells, every step of handling your cells needs to be performed in a biological safety cabinet.

Alex: Coffee sounds great, anywhere but Starbucks. I understand, you added the virus and you need to be careful that you don’t introduce other ‘bugs’ that could chew up my cells. Now I am curious what happens next?

Two days after reprogramming

Lauren:  Here is an image I have taken two days after I added the reprogramming protein factors. You can see how the morphology of the cells has drastically changed. Originally the skin cells were long and stretched out on the plate. With the addition of the reprogramming factors most of the cells have become smaller and more compact, they are not as stretched out as the original skin cells. It seems like the experiment is working and the skin cells start reprogramming into stem cells. Now we wait, change media every other day and watch how these reprogramming factors change the appearance or morphology of your skin cells in the next few days.

Alex: Great, where can we have a coffee?

Lauren: Let's go to Philz for coffee!

In the next blog, I will show you how the stem cell colonies start emerging from the culture and how ‘good looking’ colonies can be identified.

Alex’s cells are alive...and in perfect hibernation

Alex: You told me in another blog that my skin cells have been hibernating. That reminds me of Han Solo in Star Wars when he was trapped in carbonite.

Lauren: That is a great analogy, Alex. Currently, your skin cells are being kept in a tank filled with liquid nitrogen that cryopreserves your cells at -190°C. Your cells can be stored in liquid nitrogen for many years if not decades.

Alex: How long will it take until my skin cells become stem cells?

Lauren: It will take about a month to turn your skin cells into stem cells. Here is a timeline and

Nuclear reprogramming timeline

Between Day -7 to Day 0 your cells recover from the freezing. Then I will add the reprogramming viruses that will express four specific proteins. Between days 0 to 15, I maintain your cells by feeding them or changing the media every day and monitoring the cells under the microscope to determine when colonies emerge. By day 21, some stem cell colonies are large enough to be transferred to individual smaller wells to grow and further expand. I will go into more detail about each of the steps as we get to them in subsequent blogs.

Alex: I am really curious to learn more about the details of the reprogramming process. It still sounds like the Force to me.

Lauren: We actually now understand some of the underlying biology of nuclear reprogramming, it is not pure magic. Practically, in the first week I prepare the cells for the reprogramming process. I remove a vial of your skin cells from the liquid nitrogen tank as you can see on the image below. One cryovial contains about a million cells in one milliliter, and I transfer the cells with a pipette to a round 10 cm dish after removing the hibernation media and then supplementing the cells with enough fresh media.

Removing a cryovial from the liquid nitrogen tank

Culture dish under microscope

On the image below you can see your cells and how they look under the microscope the day after I plated them.

Skin cell 24 hours after plating

Alex: I am curious, since I have Parkinson’s disease, do my cells look different from cells of people without Parkinson's disease?

Lauren: At this stage, we do not notice profound differences between your skin cells and skin cells from donor controls. This is not surprising since Parkinson’s disease is not a skin disease, but a neurological one. The disease particularly affects the substantia nigra located in the brain stem. Also, all cultured cells are grown under the best possible conditions. For example, besides amino acids and serum the cell culture media also contains a lot of sugar, in fact 4.5 g/L glucose, like Orangina (although a serving of Orangina is about 21g of sugars). The growth rate, size and shape of your cells are comparable to cells from people without Parkinson's disease. Here is an example of your cells after five days. The space between the cells compared to the image above is filled with cells.

Skin cell 5 days after plating

As you can see in the image the cells are very tightly packed together, they are running out of room to grow. This is called a confluent plate. In order for the cells to stay healthy I need to remove them from this plate and divide them into other plates. To prepare for reprogramming, I will treat the cells with trypsin and after they are lifted off the plate, I transfer them into a 6-well plate (diameter of 35 mm). This preparation begins 2 days prior to the start of reprogramming.

Alex: Great. I look forward to learning more about the process of rejuvenation of my cells.