Stem Cells Explained, Part 3: How Do We Know We Have a Stem Cell?

First, before I start, I want to direct you to Euro Stem Cell's wonderful resource on all things stem cell. Their site goes into a lot more detail about the different stem cell types and the specific therapeutic applications of stem cells, but still in very understandable terms. If there are specific therapeutic potentials or stem cell types you're interested in, this is a great place to look them up. (That said, I'm also happy to take topic requests, so if you have any specific questions, please leave them in the comments and I'll do my best to answer them!)

Today's topic of stem cell discussion is how we specifically define and identify stem cells. As I've already discussed, stem cells are vaguely defined as the cells which give root to all other cells, including themselves. This is a useful starting definition, but unfortunately the cells in the body aren't quite that clear cut. This is because cells specialize gradually, and it can be hard to tell them apart. 

As an example, imagine that standing before me are two fairly typical university students. One is in first year and one is in second year, but I haven't been told which is which. Both students probably look fairly similar - they are in their late teens, have textbooks rather than journal articles in their backpacks, and party a lot on the weekends. (Yes, I realize I am stereotyping like crazy here, but bear with me.) Therefore, I am not going to be able to determine their respective level of specialization just by looking at them.

Analogously, in biology, we assess many cell types by what they look like on the outside; this is called phenotypic identification. Sometimes this is done visually, using a microscope, and sometimes it is done by detecting proteins that the cell has on its surface. This is very helpful if I am trying to identify a specific cell type that has specific proteins on the outside of the cell which serve a functional purpose. (For example, dendritic cells, a type of immune cell, has specific proteins on its surface to help it capture bits of invaders and show them to other immune cells (here, this is what the bad guy looks like!) We can, in turn, detect these proteins and say "oh look, a dendritic cell.") However, the problem is that many of the immature, unspecialized cells, like stem cells and their near relatives, all tend to have pretty similar markers and appearances. This makes it very hard to tell the true stem cells apart from all the rest.

Going back to my example, how can I determine which of my students is actually in first year, versus in second year, if I can't tell just by looking at them? What if, instead, I gave them a test? I would expect that if I gave the students a test which covered material taught in second year classes, the second year student would score a lot higher. This is called functional identification, because we are classifying the student based on what they can do, rather than what they look like. 

This is precisely what scientists have to do to identify stem cells. As mentioned before, stem cells are the root of everything in their system, and continue to maintain that system over time. This involves two key properties which can be functionally tested: differentiation and self-renewal. Scientists refer to these (and other) functional tests by the term assay. This is both a noun ("running an assay") and a verb ("assaying for a specific property").

Differentiation Assays

A pipettor (in this case, Nichiryo brand) 

is used by molecular biologists to measure 

and transport very small amounts of liquid.

Differentiation is the process by which cells become specialized to do a very specific job. Going back to our university student analogy, an immature/undifferentiated cell would be a high school graduate starting on their professional career. Leaving high school, the student has a broad, general basis in most subjects, but not a ton of expertise in any one given area. This is like our metaphorical stem cell. Now, let's say our student decides to go into biology. Over the next many years of education, s/he will gain a lot of new, specialized knowledge about biology, and will probably develop some new physical attributes as well (like the proper thumb muscles to use a pipettor). But s/he will likely also lose much of the knowledge s/he had leaving high school, both academic (I know that in the years since I've taken literature, I've forgotten the meaning of technical words like synecdoche), and physical (long hours in class and in the lab can have a negative effect on physical conditioning). The student has been shaped to his/her new career, and it would now be much harder for him/her to go back and enter a different field of specialization, such as literature or athletics. This is the consequence of differentiation in the body as well: to become very, very focused on one specific task, the cell will shut down the other genetic information which isn't relevant to its current task.

Therefore, differentiation is the process by which the cell commits, usually irrevocably, to one very specific career path in life. As these cells get more and more specialized, the daughter cells they produce will also be restricted to their area of specialization. This makes sense: a biology professor can effectively teach new biology students, but is unlikely to be a good teacher to new fine arts students.

Because the stem cell is, by definition, the root of the whole system, the single stem cell must be able to differentiate into every type of cell in that system. In the lab, this is tested by isolating individual cells, and either transplanting them into a host recipient (often a mouse), or else growing that cell in the lab, and showing that it can become all the relevant kinds of specialized cells. Because cells specialize gradually over time, and commit to general types before sub-specializing, we can usually use proxies so that we aren't testing for every specific kind of cell. Going back once again to our student metaphor, we might test to ensure that the student has the ability to become a humanities student, a social scientist, a physical scientist, or an artist, without having to specifically test for both sociology and anthropology. As mentioned before, cells with specific functions are much easier for us to detect just by appearance, which makes it easier to read out this assay.

The key for testing differentiation is to make sure that all the cells that we detect in our differentiation assay descended from the same single starting cell: this is called clonality. Otherwise, it is possible that we could see all the different cell types because we started with two slightly more specialized cells, neither of which was actually a stem cell (e.g. a first year in Arts and a first year in Sciences could together produce most of the possible university majors, without either being our metaphorical high school stem cell).

Self-Renewal Assays 

However, the ability to differentiate into all cell types is only one part of what makes a stem cell. To explain this second property, we need to revisit the process by which new cells are produced. New cells are produced when a single cell divides to make two new cells: these cells are called daughter cells. However, the original cell is used up in this division process: it splits into two daughter cells, neither of which is necessarily identical to the parent. While these cells have the same genetic material, they are not necessarily the same, because different other bits of the cells will not necessarily divide evenly. (For instance, imagine arbitrarily dividing Canada in half. The resulting countries would still have the same constitution, but the Eastern half will have a lot more lakes, while the Western half will have a lot more grain.) Because the stem cell's job is to produce differentiated cells, it makes sense that at least one of the daughter cells produced by a stem cell would be a cell on its way to specialization (otherwise we'd have a body full of stem cells, but no cells with other functions.) However, if each stem cell divided to make two starting-to-specialize daughter cells, we would eventually run out of stem cells. This means that, for the stem cell population needs to be maintained over time, then at least some of the time a stem cell must divide so that at least one of its daughter cells is also a stem cell. This form of self replacement is referred to as self-renewal.

The gold standard test for self-renewal is serial transplantation. This means that we transplant a single cell into a recipient, and give it time to re-grow the system. Then, we harvest cells from this new system, and re-transplant into a new recipient. If this second recipient can re-grow their system from these cells, then the very first cell we transplanted into the first recipient must have been able to make new stem cells as it grew.

This is of course hard to test. For one thing, we can't perform transplantation assays on humans just to identify if there was a stem cell in a sample, and it can be difficult to transplant human cells into mice, since the mouse's immune system understandably recognizes the human cells as foreign. It's also much easier to harvest and transplant bone marrow stem cells (which are used to circulating through the blood and finding their way to where they are supposed to be) than stem cells which are found in specific solid-tissue organs (like breast tissue) and which don't move around. Scientists are getting better at developing mouse models that will accept some human cells, and finding substitute transplant locations where cells can develop into faux organs. That said, our detection systems are by no means perfect, and some stem cell types have better detection assays than others. 

We also have assays in the lab which attempt to look at self-renewal. Unfortunately, however, cell culture is not a perfect mimic of what happens in the body, and all of these assays will give some false positives - they pick up on very early progenitors, which are very closely related to stem cells but don't quite have all the stem cell capabilities. (Going back to our student metaphor, these early progenitors are analogous to first year university students partway through their first term - they are still not very specialized, but they have committed to certain first year classes and are therefore no longer quite stem cells.) Therefore, the goal for all stem cell fields is to move towards rigorous proof of self-renewal and differentiation potential in vivo ("in life", i.e. in a living model).