Triggering effect of “therapeutic MSC”

V. A. Kordium, D. M. Irodov, Yu. B. Chaikovsky © 2017 V. A. Kordium et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio­ polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577

The use of MSCs as a therapeutic agent is proven to be highly successful in many cases. However, the effects obtained are often temporary, and not leading to complete recovery. The reasons of such phenomenon are discussed in the article and the concept of "triggering effects" of MSCs is substantiated. The effect consists of the fact, that signal molecules, secreted by MSCs after administration, on one hand reduce the cell damage severity, supports and heals the cells. And on the other hand, MSCs induce mobilization and activation of the own (resident) stem cells, which replace the damaged cells. The realization of the therapeutic effect depends on the presence or absence of genetically determined disorders. The last fifteen years have witnessed the in tense study of mesenchymal stem cells (MSC) as a promising remedy of treating a wide range of human diseases. This trend is based on the extensive fundamental research and techno logical elaborations [1], evolving into the in creasing number of clinical trials [2][3][4]. It takes a therapeutic preparation a long time to enter the market. However, the first samples of MSC have already become commercial products, although this evident success is accompanied with rather numerous reports about low effi ciency of MSC, short duration or absence of the therapeutic effect [5]. The interpretation of even the most successful clinical results of MSC application is complicated due to sig nificant changes in the original ideas about MSC nature [6][7][8] and the mechanisms of their action [9]. Modern views do not deny the ca pability of MSC to get differentiated into var ious specialized cells, but relate their signifi cance in the organism mainly to such signa lingtrophic functions as the induction of re programming both immune system cells and tissue cells in the damaged zone via signaling molecules released by MSC [7,[10][11][12][13]. When the organism is damaged, MSC rebuild their metabolism on their own [14].
Different protocols of applying MSC are reported in the scientific literature [15; 16].
There are differences in doses, terms, fre quency, sources, conditions of obtaining, pa thologies, recipients, efficiency of obtained results, etc. [8]. Currently the available works do not provide specific unambiguous conclu sions. Regardless of numerous detailed studies on the mechanisms of MSC action [17], it is yet to be defined why "therapeutic MSC", introduced from outside, have medicinal effect, whereas the inner ones do not. Moreover, many cases prove the efficiency of the inner MSC after they have been extracted and then introduced back.
To find out possible reasons of this phenom enon, we have conducted several series of experiments with laboratory animals. The task was to check our hypothesis that the single step introduction of a large amount of MSC from outside (regardless of their nature) leads to changing metabolism of the whole organism towards activation, reprogramming and mo bilization of its own systems of damage resto ration, which have been inactive or blocked in a chronically sick organism. These mobilized and reprogrammed cells of the mentioned sick organism start restoring it on their own.
The series of experiments on laboratory models of chronic pathologies were conducted to analyze the MSC action. One model simu lated well-known in scientific literature liver damage, cirrhosis, in rats caused by carbon tetrachloride. According to histological analy ses, these injections during one month resulted in the development of fibrosis, in 12-14 weeks of the experiment -in formation of cirrhosis, thereafter the introduction of CCl 4 was stopped. MSC suspension was introduced once into the caudal vein of the experimental group of ani mals, at the rate of 7 10 6 /kg of animal's weight.
The injection of saline solution was given to the animals of control group. The changes, occurring in the liver of animals, were studied in dynamics for 13 weeks. Gradual restoration of liver was registered in experimental group; with the approximation to the norm for all the defined indices by the end of the experiment. There were no restoration changes in the con trol group [18].
Another model simulated chronic systemic damage of the organism. The object was the mice, injected twice a week for 12 weeks with carbon tetrachloride in the dose of 1.5 µl of 30 % solution of CCl 4 per one gram of the bodyweight. This procedure induced a severe damage of practically the whole organism [19]. 12 weeks later the mice of experimental group were injected with MSC, in dose of 10 5 per animal. In different schemes of independent experiments, these were either fetal allogenic MSC, obtained from the embryo of the second line animal, or xenogenic MSC from Wharton's jelly of the human umbilical cord. Some ani mals were taken out of the experiment after 3, 9 and 12 weeks and the state of their organs was evaluated. The restoration of damaged organs was observed in MSCinjected animals. In the mice of control group there were either no positive changes or these were partial. The effect obtained on the models using other ani mals and other experimental pathologies was practically the same. For instance, the intro duction of MSC at rhinitis mucosa damage completely restored damaged tissues of mice and rats whereas there was no selfcure in the control group [20,21]. The systemic or local introduction of MSC at the simulated allergic encephalomyelitis in rats resulted in clinical recovery of animals, whereas in the control group the acute phase turned into the recurrent motor impairment [22; 23].
Noteworthy, in all these experiments, both on rats and mice, only single MSC injection was done after the pathology development. As the introduced cells were nonautologous, their replacement action via differentiation and res toration of damaged organs was almost impos sible. The results of PCR analysis, obtained by us on different models, demonstrated the dis appearance of the detected material 1-5 days after the MSC introduction [24]. The restora tion process without MSC injection lasted for many weeks.
The confirmation of the involvement of exogenous MSC in the mobilization of resident MSC was experimentally obtained. A matrix was implanted under the skin of the animal, and a damaging signal was imitated via the introduction of SDF1 source. Such carrier with chemokine was implanted under the skin of the ICR line mouse, imitating a damage zone. Then bone marrow MSC of GFPtransgenic mice were introduced into the caudal vein of the mice of experimental group. Several days later the explant was removed and analyzed for the presence of MSC. GFP marker was used to differentiate between resident and non resident MSC. It was established that in the variants with introduced exogenous cells the amount of matrixattracted MSC was higher compared to the control, which proved the mobilization of resident MSC, mediated by the introduction of the exogenous ones [25].
The analysis of the obtained results, the literature data and current views on MSC sta tus in the organism allows us to draw several key assumptions. One of them is that MSC, introduced from outside (regardless of their nature, including autologous ones), and resi dent MSC, present in the organism all the time, are different cells by many biological proper ties. As has already been stated in the litera ture, MSC cultivated in laboratories on plastic, in nutrient media, in "clean" homogeneous culture outside the organism, are "a phenom enon of in vitro cultures" [26]. However, it is not the only problem. Prior to the introduction into the organism, MSC, extracted from it (or from a donor) are reproduced up to the amount of the "therapeutic dose". The number of cells in this "dose" exceeds considerably the number of the resident cells, present at each given mo ment in the organism. "The dose" is usually calculated taking at least 10 6 MSC per 1 kg of bodyweight, which is 7 · 10 7 MSC for an "ave rage" human organism. As for systemic intro duction, the minimal singlestep introduced doses are usually 5-10 times higher.
It may be stated that MSC in the organism are transitory. Their amount in the normal con ditions is not very large, reflecting some kind of "onduty" state. In normal life of healthy individuals, MSC are massively formed out of their predecessors (progenitors) in the required amounts only in case of emergency needswounds, fractures, burns, etc. [27]. In such acute states, there is a release of a specific complex of signaling and informational molecules, gov erning the mobilization and rearrangement of metabolism of different cells of the systems of protection, pre ser vation and restoration, includ ing the stem cells [28]. The same is observed in the experimentally damaged organism. However, on the other hand, it was demon strated that in case of chronic impairments, stem cells may not be determined in blood at all [29], which may testify to the fact that the signaling molecules of chronically damaged tissues either switch off or block the response of stem cells to the activation. One of these mechanisms, invol ving dipeptidyl peptidase4, which blocks SDF1, was described by Jixin Zhong and Sanjay Rajagopalan [30]. In this respect, MSC of the chronically sick organism are also "sick" -they are in a "chronically blocked or inactive" state, i.e. unable to function. As for MSC, cultivated in vitro, they are not affected by the organism and thus selftune to the culti vation conditions rather fast. As a result, they differ in terms of status and condition from those still present in the organism. The inner rearrangements in MSC are almost not studied. However, as the conditions in different labora tories are similar, the standardisation of MSC preparation during reprogramming seems rath er possible.
Many researchers often describe the results of a single introduction of MSC at chronic pathological processes, which in many cases (although not always) is sufficient for inducing a reliable therapeutic effect. In our experiments we also used a single introduction of allo genic and xenogenic MSC to avoid the effect of engraftment and differentiation. In our ex periments a single introduction of MSC was proven to be sufficient for therapeutic effect to start and develop, intensifying over the whole term of observation -up to 13 weeks. However, the allogenic and moreover xeno genic cells introduced from outside were shown in many experiments to be present in the recipient organism only for a few first days. Their main bulk does not enter the damage zone, being stuck in the lungs [30]. Our ex periments demonstrated that they were absent in the damaged organs as early as after five days whereas the therapeutic effect developed for months, increasing week by week. This takes place at the introduction of any MSC from outside, including the cases when "exo ge nous", foreign, ones are almost completely eliminated from the recipient organism just a few days after the introduction.
As for the "own" MSC of the organism, they may be formed from progenitors in un limited amount during any period, required for the restoration of any damage. Noteworthy, three basically different types of MSC are acknowledged in current literature (Fig. 1).
MSC of the first type, localized in the bone marrow, are the closest to the traditional no tions about stem cells and the most homoge neous, though they have some subpopulations of different origin [32; 33]. One of subpopula tions originates from neural crest in early em bryogenesis, the second from some yet un known place but not neural crest, and the third one is composed of Schwann cells, marked as a specific subpopulation. There are no clear answers why there are three different MSC types, what their functions are and how they differ by their action in the organism.
MSC of the second type are welldescribed regional MSC, localized in different tissues, e.g. oval hepatocytes, stem cells of eyes, brain, etc. They fulfill a replacement function, i.e. if necessary differentiate into highly specialized cells and replace the dying or damaged ones. At first, they were considered as the derivatives of the "real" stem cells and the bone marrowtheir central storage. Therefore, the stem cells in the organs were called "regional". However, it is not a common belief, and the origin of stem cells in different tissues is yet to be established.
Most of MSC relate to the third type and actually they exist in the form of their prede cessors (progenitors) in the endothelium of small and medium vessels [6]. Vessels perme ate all tissues of all organs. Therefore, if re quired this source of MSC may be almost unlimited. However, this potential is realized mainly in response to the emergency signal, otherwise only an insignificant number of MSC "on duty" circulates. The place of "emer gency" is determined in the organism by the gradient of signaling molecules from the da ma ged zone, where the relevant chemokines are released. MSC are formed, mobilized and enter the affected zone to restore a specific damage in a specific organ [34]. The signaling molecules from the damaged zone cells "li cense" (commit, prime, polarize, etc.) MSC.
In addition to these described and localized sources of MSC there are some MSC, involved in the restoration of tissues and organs, which are formed by the potentially unlimited system of epithelialmesenchymal/mesenchymalepi thelial transitions [35][36][37][38][39][40].
Talking about restoration, one should men tion another, quite unique, group of selfrepro ducing differentiated cells (i.e. selfpreserving and self-restoring their deficiency in case of pathologies), which is composed of the cells of non-bone-marrow origin, defined as macro phages, originating from the yoke sac (prior to the growth of hematopoietic stem cells) [41][42][43]. These are Kupffer cells, brain glia, alveolar macrophages, etc., which do not own any SC, reproducing themselves. Additionally, some highly differentiated cells (hepatocytes, for ex ample) are also capable of reproducing without de/redifferentiation, i.e. the differentiated cell divides into the differentiated duplicate.
Summarizing the abovementioned, the hypothesis of the mechanism of action of "therapeutic MSC" may be formulated.
The restoration of damage, occurring after the introduction of "therapeutic MSC" from outside, is realized via the triggering mecha nism. The introduced MSC mobilize and acti vate the resident stem cells "onduty" and launch the intense formation of own MSC out of progenitors of the vascular endothelium. When there is acute damage, this accelerates the curing and restoring process in injured tis sues (organs). In case of any chronic damage, lasting for a long time due to various reasons, a powerful impulse of signaling, trophic, and other biological molecules from the introduced MSC reprograms, mobilizes resident MSC, which had become insensitive due to chronic impairment, and they ensure their selfforma tion and restoration of damaged tissues (or gans) further on in the selfsupporting mode. It has already been described in the literature that endothelial progenitor cells have their receptors of emergency (including SDF1 re ceptor) and are capable of getting activated and mobilized [44]. It may be assumed that other (hematopoietic, regional) stem cells are activated as well, ensuring the selfcure.
The abovementioned is true for the orga nism without any additional fundamental im pairments. If the latter are present, the mecha nism of selfrestoration either fails to repair the damage, or is partially blocked. There may be two main types of such impairments.
The first one is conditioned by genetically determined impairments of the functions of a tissue or an organ. In extreme cases, these are clearly manifested hereditary diseases. In a milder (and more widespread) variant, these are "hereditary predispositions". When imple mented (usually with the individual's aging), there is a constant source of damage which is constitutive by its impact. Here the own cells of the organism are not capable of eliminating this source, no matter how much they are ac tivated or mobilized. However, MSC, intro duced from outside, can transfer the organism into a healthy phenotype at least for some time, activating the restoring, compensatory func tions of the cells of a chronically sick organ, enhancing their abilities of eliminating the pathological phenotype. Here a single intro duction of MSC will have only a temporary effect. Durable return to the functional norm would require the periodic introduction of MSC with the interval depending on the degree of impairment, determined by the "predisposi tion" gene.
The efficiency of multiple introduction of MSC was demonstrated by us in the experi ments using mice with a severe degree of systemic chronic damage. Single introduction of allogenic or xenogenic MSC is insufficient for restoration and some animals still perish, a larger number in the control, and a smaller in the experiment. When allogenic MSC were introduced three times, all the animals survived and restored the damaged organs. This is ex pected in case of various "predispositions", affecting the restoration systems. A single in troduction of MSC induced the mobilization and activation of the resident SC. With no predisposition, such induction was sufficient for reprogramming for further selfsupport of the restoring status of SC and the organism was cured. With predisposition, the induction diminished and the initially weaker animals, which had more severe pathology, died.
However, the threetime introduction of MSC resulted in 3time periodical induction of the restoration systems and the restoration process was going on. There are some communications in the literature, stating that standard systems of treatment cannot be of the same efficacy for different individuals. Thus, an individualized analytical base should be developed] [45].
The second extreme type of impairments is related to the source of stable or increasing impairment, which cannot be eliminated by the organism itself. In this variant, the intro duced MSC cannot adjust the metabolism of a constitutively selfsupported impairment. In case of allogenic origin, they are transitory in the organism, while the source of impairment works constantly. Even if syngen or autologous MSC are introduced into such an organism, the signaling and informational products of the constitutive source of damage will reconstruct the metabolism of the introduced MSC, ma king them either inactive, or "acting on the contrary", supporting the pathology. Tumors are the most vivid [46; 47], although not the only example of the second type of impair ments. The chronic infections, leading to the death of all or most key differentiated cells of different organs (kidneys, glands of internal secretion, mucosa, etc.), such as hepatitis B virus, hepatitis C virus, HIV and others, will act in the same way. The scheme of MSC ap plication should be modified depending on the reasons of chronic disease. With no fundamen tal impairments, a stable effect may be ex pected from a single introduction of MSC in the restoring status. In case of impairments, caused by a constitutively acting damaging factor, MSC may be in another state (in ano ther polarization, as they now say) and serve merely as an additional mean. Another pro mi sing method is the transfection using a target gene, the product of which promotes the eli mi na tion of a constitutively acting damaging factor of nonautogenetic nature. At geneti cally determined predispositions, the introduc tion of MSC will be highly effective only in case of periodic application or in the variant of replacement action. However, regular mul tiple introduction of allogenic MSC may cause the avalanche of autoimmune diseases. So, it is necessary to use either only autologous, genetically modified MSC, or the allogenic ones, along with the procedures, eliminating the development of immune impairments. At present this is only a perspective at the level of rare laboratory investigations.