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.