The Environment Makes the Stem Cell

Intravital multiphoton imaging offers new insights on stem cell research

In many tissues of the body there is a huge turnover. The epithelial lining of the small intestine for example is totally renewed every 4 days. On top of this process are stem cells – cells that are infinitely dividing. In the small intestine, clusters of 14–16 stem cells are located in the base of the columnar crypts which comprises the intestinal stem cell niche. A recent publication in Nature [1] shows that all stem cells divide and compete for niche space by passively "kicking out" others so that eventually one stem cell takes over the whole niche. This means stem cells may lose stemness when they lose the environment of the niche. Jacco van Rheenen and Saskia Ellenbroek from the Hubrecht Institute in Utrecht, the Netherlands, talk about the new method of intravital imaging, which allows following the fate of individual stem cells over time in vivo and explains the new paradigm for stem cell development in the intestinal stem cell niche.



The stem cell niche – a "plate of marbles"

Until recently, most knowledge about stem cells came from research on the hematopoietic system. It bases on the assumption of asymmetric cell division which means the stem cell is dividing and produces 2 daughter cells: one new stem cell and a progenitor cell which can further divide and differentiate into tissue specific cell types.

Important seminal work by the lab of Prof. Clevers and others has shown that the stem cell system of the small intestine is different from the hematopoietic system. Stem cells are located in the bottom of the crypts between villi, small protrusions of the intestinal wall that magnify the intestinal surface. The basis of these crypts comprises the stem cell niche. 14–16 stem cells reside in one crypt. When they divide, there are too many cells to fit into the niche space. Cells are stochastically "kicked out" of the niche at the border where they start to differentiate. This competition ultimately leads to one winning stem cell that will give rise to all cells within the crypt and adjacent villus.

These findings have been shown previously in a fluorescent mouse model [2] that can express different colored fluorescent proteins in intestinal stem cells upon induction – the Lgr5eGFP-Ires-CreERT2/R26R-Confetti mice. In these mice, the 14–16 stem cells within each niche can be differently color-coded by administration of tamoxifen that activates the Confetti color-randomizer. Surprisingly, after two or three months, all cells in one crypt will have the same color which means that one cell has won the competition over the others. This finding means, in a niche with 14–16 different stem cells, there is only one stem cell with long-term stem cell potential.

"You can compare this to a plate of different colored marbles. The plate represents the niche and the marbles the stem cells. If you allow the marble to divide, at one point there are too many of them and they will fall off the plate due to limited space. If there is for example just one black marble on the plate and this falls off, you lose this color. In the end you will have a plate with only one color of marbles," explains Jacco van Rheenen.

How do you know which stem cell wins the competition?

The previous study [2] only showed snapshots of the different conditions. The differently colored stem cells shortly after activation of the color randomizer in the crypts were seen in one mouse, and in a different mouse all crypts were shown to be monoclonal and have only one color, three months after activation of the color randomizer. From these experiments it is clear that one stem cell wins, but the static images lack information about the position and history of this cell.

Intravital imaging allows following the stem cells over time. It showed that every stem cell in the niche has a certain probability to win the competition. But stem cells in the center of the niche have a higher probability to win than stem cells at the border. Nevertheless, these cells have the possibility to change places and gain a more favorable position. The reason for this is: upon every cell division, the position of each stem cell can be affected. Cells at the border can be pushed towards the center and regain full stem cell capacity. This means that cells with a less favorable position can still win the competition, although it is less likely.

A stem cell is a stem cell because of its environment

The current dogma of stem cell research is that a stem cell is a stem cell because it is intrinsically a stem cell. "It sounds very easy: I am Dutch because I am intrinsically Dutch," compares van Rheenen. But he and his group found out that a stem cell is a stem cell because of its environment. A stem cell can be more potent than others just because of its position within the niche. When it changes its position it can change its stem cell potential and it can either lose or gain stemness. In the words of the previous metaphor, this means: "I am Dutch because I am living in the Netherlands. If I moved to Germany, I would become German." "But this is also reversible: when a German would move to the Netherlands, he/she would become Dutch." And this is really a completely new way of thinking.

Previously, people could only look at snapshots of different conditions. One mouse was looked at shortly after switching on the different colors. Another mouse was examined much later when one colored cell gave rise to all the cells in one crypt. In the current study the dynamics of stem cells  were investigated over time in one animal. The history of individual stem cells was followed to see how that one winning stem cell actually is competing out all the others. This was achieved with intravital imaging. By following the progeny of stem cells over a long period, van Rheenen and colleagues could exclude the possibility that it is always one cell at the border or at the center of the stem cell niche that takes over the crypt.

How does intravital imaging work?

Usually, an animal is opened up in order to expose the tissue of interest to the microscope. However, you get an open wound and the animal can survive this only for a couple of hours. Van Rheenen and his team invented an abdominal imaging window (AIW) [3, 4]. With a relatively simple surgical procedure, a little glass window is inserted in the skin and abdominal wall. This tiny window does not affect the mice and allows imaging of the intestine over multiple days and multiple sessions using multiphoton microscopy, during which they are asleep. "The nice thing is, in between the imaging sessions, the mice are awake and can do what normal mice do. Just during imaging, the mouse is shortly put onto the microscope stage," says Saskia Ellenbroek, first author of the publication together with Laila Ritsma. With the help of the Leica microscope software, the position of the imaged field is remembered. As the intestine is very mobile, big blood vessels are used as a roadmap to find approximately the same imaging position. By the relative position of the different colors to each other the crypts can be retraced and imaged over multiple sessions. Since this technology allows studying the same stem cells over time in the same mouse, instead of different mice at various time points, it dramatically reduces the number of mice required for these types of studies.

Intravital imaging shows the big picture

By using intravital imaging and the abdominal imaging window, it is really possible to follow one individual cell over time and link the behavior of a cell to a certain position within the niche. "Instead of getting static images, we can now really see the dynamics over time", says Saskia Ellenbroek. "Imagine looking at an image of a very crowded shopping street. If you look at a snapshot, you can just see many people in one position. If you take a movie of the same street, you can suddenly see a guy running from a shop and someone running after him. Only then you realize that this person had stolen something from a shop. You would never see this at the moment you take a snapshot. That’s the big difference intravital imaging makes to our research," explains van Rheenen.

New opportunities for cancer research

With the finding that a stem cell can lose stemness as a consequence of a passive event – being kicked out of the niche by other dividing stem cells – the current Nature paper shifts the way of thinking about stem cell behavior. And intravital imaging offers plenty of possibilities for stem cell research.

The current study focuses on stem cells and showed that the microenvironment is important for the behavior of the stem cells. Consequentially, the next steps will be to manipulate the environment and the stem cell niche to investigate how this affects the stem cells. Will they lose stemness? What is going to happen with the tissue?

The second big question to be answered is: what happens in a developing tumor? Is there also a similar stem cell hierarchy where one stem cell is responsible for tumor growth and the others will die out because they divide only a couple of times and then are kicked out? We are curiously awaiting the answer.

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