If you’ve seen an old movie about the wild west, you’ve probably seen them. A tanned man with a heavy southern accent jostles a metal dish side to side to find small chunks of glistening yellow metal.
Gold panning is an incredibly satisfying process. I mean, who doesn’t love seeing the dirt slowly seep out of the pan, revealing the gleaming jewels left behind.
While I’m no expert, I too can appreciate the cleverness of being able to separate something so valuable from the rest of the crowd. After all, we can’t identify what we need if it’s blending in with the majority, which happens to be plain rocks in the context of gold panning.
It seems that science appreciates this concept too, with all sorts of diagnostic technologies that help us identify anomalies in our bodies. In fact, this same approach is used in the world of stem cells through a process called fluorescence-activated cell sorting, or FACS for short!
Before we get any further, let’s take a step back and break down what stem cells are.
What even are stem cells? 🔬
If you walk into a factory, every worker you see has a specific task.
The assembly line workers package the products, the managers oversee them, and the technicians fix the machines.
Similarly, the vast majority of our body’s cells each have a specific function, a raison d’etre (that’s french for ‘reason for living’ 💃).
We call these ‘specialized cells’. Each of them is equipped with the necessary toolkit and skillset they need to get their job done. Kind of like how the factory workers would have hardhats, screwdrivers, safety goggles, you name it.
For example, your nerve cells, aka your neurons, have long connections that help them transmit messages throughout our nervous system.
Heart cells, on the other hand, don't have this feature, meaning they will never be able to carry out the tasks a neuron can and vice versa.
Now you might be thinking, “this is good! it’s important for all our cells to have a purpose and the necessary qualities to help them get their job done”.
And you would be absolutely right, it really is a marvel of nature that our body has been able to develop all these helpful properties for our cells to get their job done.
However, what happens if a factory worker gets injured or has to be laid off because they’re getting old? There needs to be an, up until then, unspecialized worker who takes their place to ensure the factory can keep functioning.
And this, my friends, is where stem cells come in. Unlike their specialized buddies, stem cells are much like a recent university graduate. They have no idea what they want to do or who they want to be. This may seem like a bleak description but their indecisiveness is their superpower.
Simply put, stem cells are unspecialized cells that can self-renew.
Stem Cell Uses
Stem cells are like a wild card in Uno. Their purpose can be specified based on the situation and what needs to be done. And because of this, they are the holy grail in treating injury and disease.
The focus of this article is on how we can isolate stem cells using FACS, but let’s briefly look into just a few of the ways stem cells are useful to us..
Stem cell transplants can be used to repopulate healthy cells in regions of the body ravaged by cancer or injury.
Another way stem cells are useful are their potential to aid research. What better way to see how cells divide and differentiate than with an unspecialized stem cell?
Scientists have many plans for what they believe stem cells can be used for in the future. Diseased heart? Replace the damaged tissue with stem cells that will develop into healthy heart cells. Another goal is to grow functioning organs from stem cells that match the patient’s immune system to prevent complications.
Unfortunately, there are many barriers preventing these goals from being a reality today. One of the most obvious ones being how we isolate stem cells from their specialized counterparts.
Stem cells categorized by potency
To get to understand how we isolate stem cells, let’s first understand the concept of potency.
What does potency mean, you ask?
Well, potency describes the number of different fates the stem cell can have.
The higher the potency, the more types of cells that the stem cell can differentiate into.
The following list categorizes stem cells based on their potency, going from highest to lowest.
Some stem cells can differentiate into any type of cell in the body. These are called omnipotent stem cells.
Cells that can do this are found when the zygote first starts to divide in the uterus.
This type can differentiate into many, but not quite all types. They can differentiate into any of the three germ layers: ectoderm, endoderm, and mesoderm. These germ layers later specialize into all the tissues and organs.
Adult lymphoid or embryonic stem cells are good examples.
Stem cells that can differentiate into cells from a closely related family are multipotent.
An example of this type are mesenchymal stem cells.
These type of stem cells can differentiate into different types of cells, however, they have lower potency than the previously mentioned types.
An example would be myeloid stem cells.
Unipotent stem cells only produce cells of their own type. However, they still stem cells because they can renew themselves.
Examples include adult muscle stem cells.
Stem cells categorized by source
Now that we know how to classify stem cells based on their potency, let’s look at where stem cells can be found in the body. Potency and source are deeply related, with certain areas having cells with higher potency than others.
Here are some sources of stem cells:
Adult stem cells
- Small amounts in the majority of adult tissues (fat, bone marrow, etc.)
- Replace existing cells in organs once they get damaged or die
- Less potent than embryonic stem cells
Embryonic stem cells
- Come from the inner cell mass (the part that will develop into the human body) of a blastocyst that is 4–5 days old
Note: Blastocysts are a hollow ball of cells, an early stage in the development of mammals
Mesenchymal stem cells (MSCs)
- Found in connective tissue, most commonly bone marrow
- Can be used to create new tissues such as bone, fat, and cartilage
Induced pluripotent stem cells (iPS)
- Adult stem cells that have been reprogrammed to have similar potency to embryonic stem cells
- Can regenerate and specialize into all cell types of the body (except for cells in extra-embryonic tissues such as the placenta)
Why do we need to isolate stem cells?
Let’s get one thing clear. Stem cells are hard to come by.
Even in the most researched stem cells, such as hematopoietic ones, the chances of finding one in a random sampling are incredibly low. In fact, only about 1 in 15,000 bone marrow cells is thought to be a stem cell.
Research and treatment regarding stem cells require isolating populations of them. If the mixture is full of other types of cells that are differentiated, the treatment would be pointless and research findings would be irrelevant.
This is why we need a precise and efficient method of separating stem cells from the rest. Enter fluorescence-activated cell sorting.
A step-by-step guide to FACS
Fluorescence-activated cell sorting, or FACS for short, is a process that identifies and separates target cells from the rest of a population by tagging the target cells with a ‘fluorescent tag’.
The process has eight steps, so let’s break them down.
Step 1: Identify the unique cell surface receptors 🧫
All cells have cell surface receptors. These are proteins attached to the cell’s membrane that dictate how it interacts with the environment. Based on their function and other properties, cells will have different surface receptors. This applies to stem cells as well.
Step 2: Identify the unique antibodies 🔍
Antibodies are Y-shaped proteins that bind to antigens in order to prevent them from harming the body. They do this because they are similarly structured at the tip of their ‘Y’ shape to their corresponding protein.
Our immune system creates antibodies in order to fight off harmful intruders to our body. Since cell surface receptors can cause a reaction from the immune system, they are considered antigens.
Each antibody is like a key that can only open (or bind to, in this case) one lock: their fated protein.
Step 3: Tag your target antibodies 🏷️
By tagging the antibodies with a fluorophore, a type of fluorescent-chemical compound, we can ensure that target cells emit fluorescence since the antibodies will attach to their cell surface receptors.
This process is called immunofluorescence.
Once tagged with the fluorophore, they are considered conjugated antibodies.
There are two ways to conjugate the antibodies:
Direct — conjugate the primary antibody with the fluorophore
Indirect — conjugate a secondary antibody with the fluorophore
Step 4: Stain your target cells 🎨
Mix the fluorescent-conjugated antibodies with the cell population so they can bind to their target antigens.
5. Pass cells through the laser beam 💡
The single-file line of cells passes through a laser beam one by one. This laser beam gives three important data points about the cell
a) Side scatter —how complex is the cell?
Laser beams are made of photons, the tiny packages of light. When a photon strikes an organelle such as the nucleus or endoplasmic reticulum in a cell, it will be reflected at a larger angle than if it didn’t.
Side scatter collects this information to indicate how complex a cell is!
b) Forward Scatter — what’s the cell’s size?
The majority of photons from the laser beam will pass through the cell, however, their angle of movement will change depending on where they hit the cell membrane.
This angular change causes the photons to end up deviated from their path as they go further away from the cell. The more they deviate, the bigger the cell.
This measurement of deviation is called forward scatter!
c) Fluorescence detectors — does the cell express the protein?
The laser beam excites the fluorophore as it passes through it, so cells that express the target antibody would emit photons at a higher wavelength.
The photons from the fluorophore release are funnelled through filters, mirrors, and photo multiplying tubes for detection.
Fluorescent detectors measure the wavelengths of the photons emitted by each cell and can categorize the fluorescence (a.k.a. whether the cell has the target protein) based on this data!
6. Create a single file line of cells 🦠
A vibrating mechanism jostles the cells at a certain frequency so that they all separate into ‘droplets’ of sorts and only one cell passes through the mechanism at a time.
This ensures each cell will end up in the relevant collection tube.
7. Assigning an electrical charge to each cell ⚡
Based on the fluorescence measurement from step 5, each cell will be electrically charged by an electric charging ring.
How is it so fast? Remember, photons = light and light is very, very speedy.
Fluorescence indicating the presence of the target cell surface protein will be given a positive charge while fluorescence indicating the absence of the protein will be given a negative charge.
If we are looking to collect two types of cells, there can be a third option: cells that express neither of the target cell surface proteins will remain neutrally charged and end up in the waste collection tube.
8. Cells separated into corresponding collection tubes 🧪
Electrostatic deflector plates, which modify the path of a beam of the cells based on their electrical charge by using the electrical field.
This ensures that cells expressing high fluorescence end up in one tube and cells that don’t end up in another.
Important note: all of the detectors such as the FSC, SSC, and fluorescence detectors are connected to a computer that collects the data so that scientists can analyze it after the process is complete!
What results do we drive from FACS?
We now have two groups of cells. Those that do express the cell surface protein we are targeting and those that don’t.
By doing this while targeting proteins specific to stem cells, we now have a population of stem cells that we can use in experiments and clinically!
Using the information we got from FSC (forward scatter), the computer can now generate graphs that tell us about the size of the cells in our population.
Using the information we got from SSC (side scatter), the computer can generate graphs that tell us about the complexity of the cells.
Combining FSC and SSC, we can get valuable graphs like this one
This graph combines data about:
- forward scatter, telling us about the size, on the x-axis
- side scatter, telling us about complexity, on the y-axis.
Since the size and complexity of cells are two important data points, by plotting the cells we sorted onto this graph, we can tell how many cells of a certain type were present in our population.
In this situation, we can see that the larger cells with high complexity are called macrophages and that the smaller cells with low complexity are called erythrocytes.
Benefits of FACS
Speed is of the essence
Current FACS machines can sort and analyze thousands of cells per second.
There are 5 million cells in a single pinhead-sized drop of blood so speedy sorting is necessary if we want any progress.
Sorting cells based on one factor alone, such as size, is risky. By comparing size, granularity, and the presence of certain cell surface proteins, FACS separates cells by taking multiple factors into account.
Once the scientists adjust the settings of the laser beam, detectors, and type of antibodies used to stain the target cells, they no longer have to be involved during the process of sorting the cells.
This is important because highly labour-intensive methods take up valuable hours of scientist’s time. Hours that could be spent on actually doing experiments with the stem cells!
Everything from the cells you’re targeting, the number of proteins you’re looking for (you can target more than one), and the antibodies and fluorophores you use can be customized depending on the need for the experiment.
As long as you adjust the machinery such as the laser beam so that you are able to get the results you wish, FACS can be used to sort and analyze practically any type of cell.
How is FACS relevant to stem cells?
As we already learned, stem cells are hard to come by. Even if we take cell populations from areas of the body that contain stem cells, there will be plenty of contamination from other types of cells.
Let’s look at a real-life example in the Stanford Institute for Stem Cell Biology and Regenerative Medicine.
At these labs, important work is being done to separate cancer stem cells from human samples. Time is of the essence in this situation and the data gained from samples can become useless if the separation isn’t done in time.
According to the institute themselves, “ FACS allows rapid and accurate characterization of stem cell populations as well as isolation of rare stem cells or differentiated cells from contaminating cell populations”.
“ FACS allows rapid and accurate characterization of stem cell populations as well as isolation of rare stem cells or differentiated cells from contaminating cell populations”.
For researchers like the ones at the Stanford lab, FACS is the only way they can separate cell populations accurately and efficiently.
After all, how would an experiment on blood stem cells be beneficial if half the sample was contaminated with regular blood cells, and another 30% was just plasma?
- Stem cells are cells that are unspecialized and can self-renew. This makes them incredibly useful in treating diseases.
- Fluorescence-activated cell sorting (FACS) is a method that can be used to separate stem cells from other cell populations.
- It does this by tagging the stem cells with a fluorescent antibody so that it releases light at a different wavelength when passed through a laser.
- This method is important for scientists to isolate stem cells for research and clinical trials.
Our bodies are like tapestries with thousands of different strings weaving together to create one magnificent final product.
In order to treat diseases, we need a way for healthy cells of the afflicted region to grow and replaced damaged ones. Stem cells offer us a solution to this problem, however, we can’t make the most of their potential to cure if we can’t isolate them from the specialized cells.
Like gold panning, FACS gives us a way of separating the valuable from the rest. By understanding methods like FACS better and building upon them, the process of getting stem cells from labs to hospitals becomes infinitely faster.
FACS may not be the treatment for hundreds of diseases, but it’s a vital part of the process that allows stem cell therapies such as hematopoietic stem cell transplantation to exist.