Three times marked

Scientists have a "third eye" to observe stem cells

MIPT blog, Naked Science

Scientists from MIPT, Stony Brook University (USA), Cold Spring Harbor Laboratory (USA) and the Institute of Developmental Biology. Koltsova RAS (part of the work was carried out on the basis of the Kurchatov Institute) learned to mark dividing stem cells with three different labels – before that, it was possible to use a maximum of two labels at the same time. The new method will increase the accuracy and speed of stem cell division analysis. In the article Podgorny et al.Triple S-Phase Labeling of Dividing Stem Cells, published in Stem Cell Reports, researchers demonstrated that using a triple label, it is possible to study the reproduction of stem cells in different tissues.

"Reincarnation" of cells

Stem cells have been found in many tissues and organs that provide tissue repair. Even in the adult brain, contrary to the popular statement that nerve cells do not regenerate, there are areas where the so–called neurogenesis occurs - the development of new neurons. Neurogenesis is believed to play an important role in learning and memory, stress response, and regeneration of damaged nerve tissue. Usually, tissue regeneration occurs as follows: stem cells divide, some of them continue to divide, renewing the stock of stem cells, and some give rise to new adult cells. In different tissues, this process proceeds in different ways, the task of scientists is to find out exactly how it proceeds and how different factors can affect it. To find out how many divisions stem cells undergo in a particular tissue, what is the length of the cell cycle, where newborn cells move and what they turn into, we need a way to observe them.

The cell cycle. During the G1 phase, preparation for division takes place, the contents of the cell, except DNA, are doubled. During phase S, DNA is "copied" – molecular machinery builds daughter DNA in the image and likeness of the DNA of a cell. G2 is the final stage of preparation for division, that is, for mitosis (M). During the G1 phase, the cell can exit the cycle and go into the G0 resting phase, that is, stop dividing.

Odd label

When DNA doubling occurs in a dividing cell, you can trick the system and slip it a labeled "brick" for daughter DNA. To do this, an analog of thymidine, one of the four components that make up DNA, is injected into the body, and this analog is embedded in the daughter DNA instead of thymidine. The remaining "bricks" are involved in many other reactions in the cell, therefore, in order to mark only newly synthesized DNA, thymidine analogues are used. At the time of administration of the thymidine analogue, only those cells that are in the S-phase of the cell cycle are marked, because it is during this phase that DNA doubling occurs. Moreover, when the marked cell divides and gives offspring, it will also be marked. Next, scientists can take the tissue for analysis and detect the tags using fluorescent antibodies.

Antibodies are proteins that are produced by cells of the immune system, recognize molecules alien to the body and sit on them. Antibodies selectively bind to their targets, and this property is widely used in biology and medicine. So, with the help of fluorescent derivatives of antibodies, it is possible to recognize certain components inside organisms.

Thus, it is possible to trace the fate of cells that were in the S-phase during the introduction of the label. In principle, this can be done using a single label on several groups of mice. For example, take the first group for analysis 2 hours after entering the label, the second – after 24, the third – after 48 hours, and then see where the labeled cells were and understand how they moved. However, in this case, too many mice are needed, and accuracy suffers: we monitor cells in more than one organism. If there are several different tags, then you can enter them at different time intervals into one animal. Until now, scientists have been able to use a maximum of double tagging. But to determine the most important parameters using a double label, it still takes a lot of time and several groups of animals.

Photo of the intestine: blue is the first label; green is the label introduced 24 hours after the first; red is 24 hours after the second; white cells are cells in any phase of the cycle except G0 (i.e. cells involved in division)

The Power of Three

Tags, which are used as thymidine analogues, are recognized with the help of antibodies. But the antibodies are imperfect and can bind not only to "their" target, but also to a similar one. If the same antibodies land on both one and the other label, then it will be impossible to distinguish them. Due to the fact that thymidine analogues are similar and in most cases the antibodies confuse them, until recently only two labels could be used. Not so long ago, a new analogue of thymidine appeared, which can be detected not with antibodies, but with the help of so-called click chemistry, when the addition of a fluorescent dye occurs by chemical reaction. The problem with this dye was that with triple labeling, it left "unpainted" areas on which antibodies against the other two labels landed, as a result of which the labels could not be distinguished. However, scientists have found a way to clog these areas – with the help of a dye-related, but colorless substance.

Scientists have tried the triple tagging method on stem cells of the brain, intestines and testes. The most obvious result was obtained on the intestine: the labels introduced at different times were distributed in accordance with the known data on the movement of cells in the intestine. In addition, with the help of a triple label, it turned out to determine new important parameters of neurogenesis with high accuracy. Thus, the authors of the article have developed a tool for the study of stem cells in any tissue, which surpasses the previous one in accuracy, accelerates and facilitates obtaining results.

Oleg Podgorny, Senior Researcher at the MIPT Brain Stem Cell Laboratory and one of the authors of the article:

 "We are now widely using the triple label in the analysis of the neurogenesis of mice under various influences: radiation exposure, diet, the use of various drugs, including antitumor therapy. In addition, we use this technology in order to test how neurogenesis is affected by those substances that are used to treat Alzheimer's disease and other diseases of the nervous system. In Alzheimer's disease, a person's cognitive abilities and memory decrease, and today there is a lot of data indicating that neurogenesis is very important for the implementation of these brain functions."

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