Alex's stem cells pass the practice test

In the last blog we talked about how we bank your cells. Today, we will give you an overview of how to make sure that the cell we have are actually pluripotent stem cells, a process we call characterization.

Lauren: We use four methods to characterize your cells. In the image below you can see a summary of these methods. Two methods test the pluripotency of the newly derived cells and the other two methods make sure that the DNA was not damaged or changed during the reprogramming process.

Summary of characterization process

Alex: I know we have talked about pluripotency before, but can you remind me what that is?

Lauren: Pluripotency refers to the ability of the cells to give rise to all of the cell types that make up the human body. The first way we test pluripotency is by staining the cells for certain markers. All cell types have markers on the surface of their cells and within the cells. These markers are specific to each type of cell so that we can distinguish different cells from one another. We use multiple markers that are specific for pluripotency to be sure that the cells are indeed pluripotent.

Alex: Well that seems fairly straightforward. How else do you test for pluripotency?

Lauren: Another way that we test for pluripotency is to spontaneously differentiate these stem cells. The spontaneous differentiation begins by forming embryoid bodies. Embryoid bodies are 3-D cell aggregates as shown in the picture below. The cells are no longer adherent to the bottom of the dish but are floating in the media. Embryoid bodies are meant to mimic the early development of an embryo. This stage primes the stem cells and gets them ready to differentiate into all cell types. We use specific cell markers to confirm that the stem cells can differentiate into various tissue types.

Example of embryoid bodies

Alex: I did not realize that so much work went into these processes. What else needs to be done in order to call my newly made stem cells "induced pluripotent stem cells"?

Lauren: The next two methods of characterization ensure that the DNA within the cells is not damaged and that the virus has not "sneaked into" the DNA by the reprogramming process. The first method is called a karyotype which shows the number and shape of chromosomes (packaged DNA) of the cell. For humans, a normal karyotype consists of 22 pairs of chromosomes and the two sex chromosomes X and/or Y. The image below is an example of how a normal karyotype looks like.

Example of a normal karyotype, 46 XY

Alex: I hope my karyotype comes out normal. What is the next step?

Lauren: To make sure that the genetic material of these reprogrammed cells has not been modified, we measure the virus in the cells which we used to deliver the reprogramming factors.  After 10 to 12 passages, we should not detect the virus anymore. We can detect the virus by an amplification method that will be explain in more detail later.

Alex: Thank you for the overview. I am looking forward to learning more about each of these methods and seeing if my cells pass these tests. It is a bit nerve racking almost like passing a practice driving test.

Lauren: Indeed, I have quite nervous about some of the test results of your cells. In the next few blogs we will go into more detail about each of these methods and show the results in you cells. The first process we will discuss is staining for the pluripotency cell markers.

A Backup for Rainy Days

In the last blog, we talked about the process of picking your stem cell colonies. Now that we have cells that morphologically look like stem cells, we need to create a bank of these cells before we can characterize them to ensure that they are truly induced pluripotent stem cells (iPS cells) derived from your skin biopsy.

Alex: I am always looking forward to hearing about the progress my cells make with your expertise. Can you explain what a “bank” of cells is?

Lauren: A bank of stem cells consists of many vials of your cells frozen and stored in liquid nitrogen for future use. It is like a backup for rainy days. We need these cells for the characterization process and eventually for neuronal differentiation, but we also want to make sure there are enough cells cryopreserved for future experiments. Banking at this stage also allows us to preserve cells that are younger since long-term tissue culture can lead to abnormalities of growth and survival of cells. Lastly, it takes a lot of time and effort to make your iPS cells, a bank of cells will ensure that we do not have to go through this process again.

Alex: That makes a lot of sense. How do you create a bank of my stem cells?

Lauren: All of your stem cell lines that I picked over the last three to four weeks started from one single colony of cells and each stem cell line was ultimately derived from only one cell from your skin biopsy. Now we need to expand this one colony into many more colonies so that we can freeze them and still have some to further expand. Expanding your stem cells or –we like to be short and use jargon “iPS cells”- is similar to expanding skin cells as we talked about in the blog: Alex’s cells growing out of their “clothes”. But there are two main differences between expanding skin cells and expanding iPS cells. The first difference is that iPS cells do not grow as quickly. The second difference is that we will manually passage your iPS cells whereas we used the enzyme trypsin to break apart and dissociate your skin cells.

Expanding and banking pluripotent stem cells

Alex: What does “expanding” actually mean in this context? How do my cells expand?

Lauren: Cells are constantly dividing or multiplying. In the last blog you saw the development of a colony. It takes only one cell that has divided many times to create a colony of cells. In the picture below you can see how your cells divide. Each red circle shows a different time point in the division or mitosis of your cells. The blue in the image is the nucleus of the cell containing the genetic material in form of large DNA structures, called chromosomes, and the green is the rest of the cell. Circle 1 shows a cell that has started the division process and all blue chromosomes align in the middle of the cell, which is called metaphase. Circle 2 shows a cell in which the blue chromosomes starting to pull apart from each other, a stage that is known as telophase. Then in circle 3 we can see two cells that have just completely separated from each other. All of these cells divide many times which helps to expand or increase the number of cells we have so that we can create a cell bank. If you look closely at the image, you can see additional cells dividing.

Stem cell colony with dividing cells

Alex: Fascinating. I can identify more cells dividing. Another question: you also used the term “manually passaging” iPS cells? That sounds tedious.

Lauren:“Manual passaging” is similar to what I did when I was picking your stem cell colonies. As the cells grow, the colonies get larger, when the colonies are large enough I cut them with a needle into smaller pieces by cutting the colonies in squares like a grid, as you can see in the picture below, lift them off the plate with a pipette and transfer them into a new plate. The entire banking process will take me a few weeks, but in the meantime, I will start characterizing your stem cells.

Example of how to manually passage with a needle

Alex: Thank you, Lauren, this needs a lot of diligence and attention to detail. I am glad you are taking good care of my cells.

Lauren:You are very welcome. Next time, I will explain how to characterize your iPS cells.

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

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.

How the “rejuvenation” process of Alex’s skin cells works

Alex: Now that you have grown all of these skin cells what’s next?

Lauren: We are going to turn your skin cells into stem cells. To make stem cells, we are using a technology called cellular reprogramming for which the Nobel Prize in Physiology/Medicine 2012 was awarded to Drs. Sir John Gurdon and Shinya Yamanaka. This ground-breaking discovery broke a central dogma of biology which basically stated that you cannot ‘turn the clock back’ or reverse the fate of a differentiated cell. Cellular differentiation refers to the developmental process of taking a cell from an undifferentiated or pluripotent state, e.g. stem cell, to a specialized state with specific function, e.g. skin, heart, liver.

Alex: How are you going to make stem cells from my skin cells?

Lauren: We are introducing four different proteins delivered by a virus into your skin cells. The proteins force and "remodel" your skin cells and turn them into stem-cell like cells that we call induced pluripotent stem cells or "iPS" cells. A scheme of the process is shown in the image below. iPS cells share similar qualities to embryonic stem cells, they can divide almost indefinitely in the culture dish, that means practically, you can grow them continuously for years. iPS cells also have the characteristic to differentiate into any cell or tissue type of the human body.

Alex: What is your ultimate goal? What do you want to do with my cells?

Birgitt: We want to find out why you got Parkinson’s disease and why your dopaminergic neurons in your brain are dying. We think we can do this in this individualized or personalized approach by making your stem cells differentiate into your dopamine producing nerve cells. Then we can compare them to healthy control cultures and define differences and changes. Once we know why your dopaminergic neurons are susceptible, they can be tested with known or novel compounds that potentially reverse the changes and restore them to their normal function. This is the long-term goal, but we will explain in this blog how we build the cell model from your skin cells.

Alex: You make it sound so simple and easy, but I am sure there is more behind the scenes.

Birgitt: There are definitely additional hurdles and challenges to overcome, but the concept works and the model has obvious advantages -using cells from someone who has the disease- over the traditional cancer cell models that have been used for the last few decades. If you are interested in more details, background and stem cell news, here are some links and blogs.

• http://stemcells.nih.gov/info/basics/pages/basics1.aspx
• http://www.isscr.org/home/resources/learn-about-stem-cells
• http://www.celltherapyblog.com/
• http://californiastemcellreport.blogspot.com/
• http://www.ipscell.com/