This thesis is devoted to the characterization of the biological effects of non-activated mesenchymal stem cells (MSCs) exometabolites on the manifestations of the fibrotic process on the experimental model of tetrachloromethane fibrosis and to elucidate the contribution of individual immunological indicators to these effects.
MSCs are a population of highly plastic cells that take part in regenerative processes and regulation of body homeostasis. The mechanisms of their action can be divided into contact (direct intercellular contacts) and distant (excretion of a wide range of exometabolites). MSCs can also differentiate into cells of the damaged tissue in small quantities. Numerous biological and even clinical effects of stem cells have been shown. The regulatory effect of exometabolites (secretome) of stem cells has been studied much less than contact interactions. Elucidating the role of the secretome is important both for understanding the mechanism of stem cells action and for their practical application in biotechnology. The use of the secretome is not related to the potential risk of living cell therapy and solves most bioethical problems.
Exometabolites secreted by MSCs contain soluble components – cytokines, chemokines, growth factors – and vesicles (miRNAs), and are collectively called the secretome. The influence of LPS, inflammatory cytokines, activates MSCs and changes the profile of their secretome, which cannot be reliably predicted. The study of the biological effect of the secretome of non-activated MSCs, obtained under relatively standardized conditions, was almost never carried out. To understand the mechanism of action and potential use of secretome components, it is advisable to develop effective and relatively inexpensive methods of fractionating, e.g., membrane filtration.
Liver fibrosis was chosen as an experimental model. A liver disease is a common cause of death in the world. The main pathological process in these diseases is exactly fibrosis with a potential transition to a cirrhosis. Although the liver has a high capacity for regeneration, in conditions of intense fibrosis accompanied by persistent inflammation, the recovery of damaged tissue is ineffective. It is important to search for substances and compositions that can effectively shift the balance of "fibrosis–regeneration" towards the latter process and reduce inflammatory manifestations.
Persistent inflammation is a factor that supports the fibrotic process, and cells of the immune system can produce an excess of pro-inflammatory cytokines, causing a "cytokine storm" that leads to tissue damage. There are probably other immunological mechanisms supporting liver fibrosis. One of the mechanisms of MSCs action and their secretome is exactly the immunomodulatory and anti-inflammatory effect. The study of the MSCs secretome effect on the dynamics of inflammation changes, the intensity of antibody production, the phagocytic activity of neutrophils and cellular reactions in the model of delayed type hypersensitivity allows to obtain the data necessary for a comprehensive understanding of their antifibrotic action. So, the topic of the thesis is relevant and will help to solve the problems of practical application of MSCs.
A method for obtaining MSCs exometabolites of bone marrow origin has been developed and included cryogenic storage of cells. It is better to collect MSCs conditioned medium with exometabolites from cultures of passages 3–5, for 48h growth provided there are 5–6 million cells/ml. Exometabolites can be separated by mass by ultrafiltration.
A comparison of the secretome fractions absorption spectra shows a greater number of proteins in the whole fraction of the secretome and their difference from the control medium in amino acid composition.
The xenogeneic MSCs exometabolites stimulate in vitro formation of a growth zone around liver fragments obtained from intact animals. The fraction with a mass <10kDa showed the highest activity, the fraction with a mass >30kDa showed the lowest one.
For the first time, the effect of the secretome with a mass <10kD on the fibrotic liver in vivo was studied. A decrease in endogenous intoxication by liver autolysis products, fibrotic processes and anemic manifestations were recorded. Compensatory enhancement of liver regenerative activity ex vivo was also observed.
Non-activated xenogeneic MSCs secretome enhances antibody production. The effect of i/m administration is more pronounced. MSCs exometabolites are able to compensate for the immunodeficiency state.
The impact of the MSCs secretome on cellular responses, on the contrary, is more pronounced in case of s/c administration. The secretome of xenogeneic non-activated MSCs has neither a suppressive nor an activating effect on the phagocytic activity of neutrophils.
MSCs exometabolites have a pronounced anti-inflammatory action, which begins later than the action of cyclooxygenase inhibitors and is more gradual.
