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HayKOBMM BiCHMK ^tBiBCtKoro Ha^OHa^tHoro yHiBepcMTeTy
BeTepMHapHoi Megw^HM Ta öioTexHO^oriw iMem C.3. I^M^Koro
Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies
ISSN 2518-7554 print ISSN 2518-1327 online
doi: 10.15421/nvlvet8369 http://nvlvet.com.ua/
UDC 576.6-57.085.23
The system influence of allogeneic adipose tissue derived mesenchymal stem cells on the functional state of immune organs
L.V. Kladnytska, A.Y. Mazurkevych, M.O. Maluk, V.B. Danilov, Iu.O. Kharkevych, S.V. Velychko, D.A. Shelest, V.S.Velychko
National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine
Article info
Received 01.02.2018 Received in revised form
06.03.2018 Accepted 12.03.2018
National University of Life and Environmental Sciences of Ukraine, Heroiv Oborony Str., 15, Kyiv, 03041, Ukraine. Tel.: +38-063-186-62-33 E-mail: [email protected]
Kladnytska, L.V., Mazurkevych, A.Y., Maluk, M.O., Danilov, V.B., Kharkevych, Iu.O., Velychko, S.V., Shelest, D.A., & Velychko, V.S. (2018). The system influence of allogeneic adipose tissue derived mesenchymal stem cells on the functional state of immune organs. Scientific Messenger of Lviv National University of Veterinary Medicine and Biotechnologies. 20(83), 347-351. doi: 10.15421/nvlvet8369
The studies were conducted on 2-3-months-old males of mice weighing 20-24 g. Our work was to study the functional state of the organs of the immune system of C57BI/6 mice after introduction of allogeneic MSCs of adipose tissue origin. Obtaining and cultivating of MSCs were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. C57Bl/6 mice adipose tissue cultured in a CO2 incubator at 37 °C and5% CO2 inDMEM with 10-15% of fetal bovine serum, 1% of antibiotic-antimycotic solution (Sigma-Aldrich, USA). The following groups of animals were formed: 1 group - intact (control group); 2 group - animals, to whom 0.5 ml of 0.9% NaCl solution (placebo) were injected into the caudal vein; 3 group - animals, to whom were injected 104 of allogeneic MSCs from adipose tissue in 0.5 ml of phosphate buffer solution into the caudal vein. The weight index, content of lymphoid cells of thymus and spleen in C57Bl/6 mice investigated after the introduction of MSCs on 7, 18 and 25 days. To assess the content of lymphocytes in lymphoid organs, the latter were weighed. Whole thymus and 50 mg of spleen were triturated and filtered through the kapron tissue. After that, the tissue homogenate was applied to the gradient of ficoll-urografin (density 1.077) in a ratio of 3:2. The test tubes were centrifuged at a rate of 1500 rpm for 30-40 minutes. After centrifugation the layer of lymphocytes which was above the gradient was collected by a Pasteur pipette and washed twice with an arbitrary amount of Hanks' solution by centrif-ugation at a rate of 1500for 10 minutes. 1 ml of Hanks's solution was added to lymphocytes after washing. Lymphocytes were counted in the Goryaev chamber. Calculation of the cells of lymphoid organs was performed on 1 mg of tissue. The administration of allogenic adipose derived mesenchymal stem cells affects on the central and peripheral organs of the immune system. Administration of allogenic adipose derived mes-enchymal stem cells cause a significant increase in the content of lymphoid cells in the thymus at 7, 18 and 25 days by 71, 57 and 53% respectively (P < 0.05) compared to the control. Weight index of the thymus directly correlates with the content of lymphoid cells and its value was r = 0.57, P < 0.01 on 7 day and 18 day r = 0.50, P < 0.05. Quantity lymphoid cells in the spleen significant increase at the 7 and 18 days of the immune response by 33 and 19%, respectively (P < 0.01, P < 0.05) compared to the control under administration of allogenic adipose derived mesenchymal stem cells. On the 25th day of experience, the content of lymphoid cells in spleen and spleen index values return to normal. Weight index of the spleen directly correlates with the content of lymphoid cells - r = 0.91-0.94 (P < 0.001).
Key words: mice, weight index, lymphoid cells, thymus, spleen, allogenic mesenchymal stem cells, adipose tissue.
Ефект системного впливу алогенних мезенхiмальних стовбурових з жирово'1 тканини на показники функщонального стану iMyHH^ оргашв
Л.В. Кладницька, А.Й. Мазуркевич, М.О. Малюк, В.Б. Даншов, Ю.О. Харкевич, С.В. Величко, Д.В. Шелест, В С. Величко
Нацюнальний yuieepcumem 6iopecypcie i природокористування Украти, м. Кшв, Украна
До^дження проводили на самцях мишей C57BL /6 вагою 20-24г eiKOM 2-3 мгсящ Мантуляци з отримання первичного мате-piany та культивування МСК здшснювали в стерильному бокЫ з дотриманням ycix правил асептики й антисептики. Абдомтальну жирову тканину мишей C57BL /6 культивували за температури 37 °C, 5% CO2, 95% вологостi у CO2 iнкyбaтоpi у сеpедовищi DMEM, з додаванням 10-15% фетальног сироватки бичтв, 1% антибютика-антимтотика (Sigma-Aldrich, USA). Для проведення до^джень було сформовано наступт групи тварин: 1-ша група - ттактт (контрольна група); 2-га група - тварини, яким у хвостову вену вводили 0,5 мл 0,9% розчину NaCl (плацебо); 3-тя група - тварини, яким у хвостову вену вводили 104 алогенних МСК з жировог тканини в 0,5 мл фосфатно-буферного розчину. До^джували ваговий тдекс, вмкт лмфогдних клтин тимусу та селезтки мишей C57BI/6 за введення МСКз жировог тканини. Для оцтки вм^ту лiмфоцитiв в лмфогдних органах, остант зважу-вали (тимус повтстю), а селезтку - по 50 мг, потм розтирали та фтьтрували через капронову тканину. Пкля цього гомогенат тканини наносили на гpaдieнт фтол-верографн (щтьтсть 1,077) у сniввiдношеннi 3:2. Пpобipки з вмктом центрифугували зi швидтстю 1500 об/хв., протягом 30-40 хвилин. Шсля центрифугування над шаром градшнта залишаеться плазма i лмфоцити (не менш 90%), яю збирали naстеpiвською птеткою i двiчi вiдмивaли довтьною ктьюстю розчину Хенкса шляхом центрифугування при швидкостi обертання 1500-1800 об/хв. протягом 10 хвилин. Пкля вiдмивaння до лiмфоцитiв додавали 1 мл розчину Хенкса i тдраховували гх кмьтсть в кaмеpi Горяева. Розрахунок клiтинностi лмфогдних оргатв проводили на 1 мг тканини. Транспланта-щя алогенних мезенхмальних стовбурових клтин з жировог тканини чинить вплив на централью i периферичж органи мунног системи. За впливу алогенних МСК з жировог тканини вiдбyвaеться достовipне тдвищення вмкту лмфогдних клтин тимусу на paннiх i тзшх етапах iмyнног вiдnовiдi на на 7, 18 та 25 добу на 71, 57 i 53% вiдnовiдно (P < 0,05) nоpiвняно з контролем. Юль-тсть лмфогдних клтин у селезтщ достовipно зростала на 7 та 18 добу iмyнног вiдnовiдi на 33 та 19%, вiдnовiдно (P < 0,01, P < 0,05) nоpiвняно з контролем при введены алогенних мезенхмальних стовбурових клтин, одержуваних з жировог тканини. На 25-ту добу показники вмкту лмфогдних клтин та тдексу селезтки повертаються до норми. 1ндекси ваги тимуса i селезтки прямо корелюють з вмктом лмфогдних клтин в цих органах.
Ключовi слова: мишi, ваговий тдекс, лмфогдж клтини, тимус, селезтка, алогенн мезенхiмaльнi стовбypовi клтини, жирова тканина
Introduction
Important biological features of MSCs, in particular, the ability to migrate to the inflammation site, low immunogenic^, immunomodulatory activity, and the ability to stimulate hemopoies make them potentially active regulators of reparative processes (Kladnytska et al., 2014).
At the present stage of the development of biological sciences, different approaches develops for the use of MSCs in the treatment of various diseases, and a number of preclinical and clinical trials have already been conducted, the results of which have shown the effectiveness of the use of these cells (Haghighat et al., 2011; Reich et al., 2012; Arnhold and Wenisch, 2015; Kathrine et al., 2017).
As an alternative source of MSC is adipose tissue, which contain stem cells in a higher percentage than bone marrow. Obtaining of adipose tissue is less traumatic procedure for the donor than the obtaining of bone marrow both during the process of obtaining the primary material and during the postoperative period (Marx et al., 2014; Kladnytska et al., 2017).
Regardless of the origin of MSCs, they have pronounced immunosuppressive activity: they block in vitro differentiation of naive CD4 + T cells in Th17 and suppress the synthesis a lot of cytokines like IL-17, IL-22, interferon-gamma and TNFa (Bartholomew et al., 2002; Hryshchenko and Tomchuk, 2013).
Despite the large number of publications confirming the immunosuppressive properties of MSC, there are works that deny such effect on immune responses (Di Nicola et al., 2002; Aggarwal and Pittenger, 2005; Djouad et al., 2005).
Thus, it was found that transplantation of MSC stimulates antibody production and increases the cellularity of the bone marrow of recipients. With increasing the number of introduced cells, a significant increase in thymus cellularity and decrease cellularity of the spleen was recorded. The authors suggest that a significant dose of the MSC creates suppressive microenvironment for lymphoid
cells, which is accompanied by inhibition of the immune response (Le Blanc et al., 2003; Batten et al., 2006; Lu et al., 2009).
Opposite, a small number of transplanted cells, by virtue of homing, is collected in the bone marrow niches, contributing to hematopoiesis, where the myeloid sprout can act as a spectacular factor in natural immunity (Nikolskaya et al., 2012).
So, it is not well known about the effect of MSC on the response of the immune organs, in particular on the functional state of thymus and spleen. Taking into account such controversial data on the influence of MSCs on the organs of the immune system, these issues require further research.
The purpose of our work was to study the functional state of the organs of the immune system of C57Bl/6 mice after introduction of allogeneic adipose MSCs.
Materials and methods
The studies were conducted on 2-3-months-old males of C57BL / 6 mice weighing 20-24 g. All studies were conducted in accordance with the Rules of Good Laboratory Practice and Use of Experimental Animals and in accordance to Compliance with the Law of Ukraine "On the Protection of Animals from Cruel Treatment" and the «International European Convention on the Protection of Animals Used for Experimental and Other Scientific Purposes».
MSCs obtaining from adipose tissue
Obtaining and cultivating of adipose MSCs (aMSCs) were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. The mice were euthanized, samples of abdominal adipose tissue were washed three times with sterile phosphate buffer solution with the addition of 1% antibiotic-antimycotic solution (Sigma-Aldrich, USA). Then samples of adipose tissue were chopped into pieces of 1-3 mm3 and placed to culture dishes filled with DMEM, 10-15% of fetal bovine serum, 1% of antibiotic-antimycotic solution (Sigma-
Aldrich, USA) and cultured in a CO2 incubator at 37 °C and 5% CO2. The culture medium was partially or completely changed by fresh medium every 3 days during cultivation. After formation of cells mo nolayer at 8090%, cells were removed with trypsin-ethylenediaminetetraacetic acid solution (EDTA), washed with phosphate buffer and placed in Petri dishes for futher cultivation. Passaging the cells provided a reduction of heterogeneity of cell culture and the development of biological material for transplantation (Kladnitska et al., 2016). For transplantation were used MSCs of the 4 passage.
MSCs administration to mice
The following groups of animals were formed: 1 group - intact (control group); 2 group - animals, to whom 0.5 ml of 0.9% NaCl solution (placebo) were injected into the caudal vein; 3 group - animals, to whom 104 of allogenic aMSCs in 0.5 ml of phosphate buffer solution were injected into the caudal vein.
Estimation of weight index of thymus and spleen of mice after introduction of aMSCs
Indicators of the weight of peripheral lymphoid organs relative to the body weight (weight index) of animals were evaluated at 7, 18 and 25 days after the introduction of aMSCs. The mice were pre-weighed for weight control. At each study period in each group 3 animals were euthanized and the weight index of lymphoid organs and their cellularity were studied.Euthanasia of animals was carried out with using of carbon dioxide, lymphoid organs - thymus and spleen - were removed and determined its mass. Indices of lymphoid organs in
relation to the weight of the animal were calculated according to the formula:
Weight index (%) = weight of the lymphoid organ / weight of the animal * 100.
Evaluation of cellularity of the thymus and spleen after the introduction of aMSCs
To assess the content of lymphocytes in lymphoid organs, the latter were weighed. Whole thymus and 50 mg of spleen were triturated and filtered through the kapron tissue. After that, the cell homogenate was applied to the gradient of ficoll-urografin (density 1.077) in a ratio of 3:2. The test tubes were centrifuged at a rate of 1500 rpm for 30-40 minutes. After centrifugation the layer of lymphocytes which was above the gradient was collected by a Pasteur pipette and washed twice with an arbitrary amount of Hanks' solution by centrifugation at a rate of 1500 for 10 minutes. 1 ml of Hanks's solution was added to lymphocytes after washing. Lymphocytes were counted in the Goryaev chamber. Calculation of the cells of lymphoid organs was performed on 1 mg of tissue.
Results and discussion
The functional state of the organs of immunogenesis largely depends on the ratio of the processes of proliferation and apoptosis of the immune cells, that are practically not studied after administration MSC.
After the introduction of allogeneic aMSCs at the 7, 18 and 25 day, the content of lymphoid cells in the thymus was significantly increased compared with control animals at 71, 57 and 53% respectively (table 1).
Table 1
The content of lymphoid cells and the weight index of thymus of C57BI / 6 mice after administration of allogeneic aMSCs, M + m, n = 9, x106 / mg,%
Groups of an animals / Terms of study Intact (n = 6) (x106 / mg) Administration of 0.89% NaCl, placebo (n = 9) (x106 / mg) Administration of aMSCs (n = 9) (X106/MT) Weight index of thymus after administration of aMSC,%
7 day 1.4 ± 0.1 1.9 ± 0.2 2.7 ± 0.1*v 0.19 ± 0.03*v
18 day 1.4 ± 0.1 1.3 ± 0.1 2.2 ± 0.1*v 0.16 ± 0.01*v
25 day 1.7 ± 0.1 1.4 ± 0.1 2.6 ± 0.3*w 0.17 ± 0.01*v
*- P < 0.05, compared to a group of intact animals; v - P < 0.5, compared to placebo group
Compared with placebo group the content of lym-phoid cells in the thymus was significantly increased at 42, 69 and 86% respectively. The increase of cellularity of thymus is due to the activation of proliferation of resi-dental thymocytes due to antigenic stimulation by MSC,
consistent with studies by Huang Y., Johnston P., Za-kari A. et al. (Huang et al., 2009). The thymus contains T-lymphoblasts, ripening and mature lymphocytes, supporting and secretory cells of the thymus stromal component (Figure 1).
Fig. 1. The cellularity of thymus on the 7 day of the study: A - intact group, B - after the introduction of aMSC,
(smear-imprint, x 400)
A positive correlation between content of lymphoid cells and weight index of thymus was established at 7 days after the introduction of aMSC. Weight index of the thymus directly correlates with the content of lymphoid cells and its value was r = 0.57, P < 0.01 on 7 day and r = 0.50, P < 0.05 on 18 day of experience.
Under the influence of MSCs from adipose tissue, the
indicator of the weight index of the spleen was significantly increased until the 18 day of the experiment (table 2). At the 25 day the weight index of spleen was not significantly differ from that in experimental group and placebo animals, but only observed a tendency to increase it.
Table 2
The content of lymphoid cells and the weight index of spleen of C57BI / 6 mice after administration of allogeneic aMSCs, M + m, n = 9, x106 / mg,%
Groups of an animals / Terms of study
Intact (n = 6)
(x106 / mg)
Administration of 0.89% NaCl, placebo (n = 9) (x106 / mg)
Administration of aMSCs (n = 9) (X!06/MT)
Weight index of spleen after administration of aMSC,%
7 day 18 day 25 day
2.7 ± 0.1 2.7 ± 0.1 2.7 ± 0.1
2.9 ± 0.1 2.8 ± 0.4 2.5 ± 0.1
3.6 ± 1.1**vv 3.2 ± 0.1*v 2.9 ± 0.1
0.79 ± 0.04* v 0.79 ± 0.04* v 0.46 ± 0.03
* - P < 0.05, ** - P <0.01, compared to a group of intact animals; v - P < 0.5, compared to placebo group
The spleen, as the peripheral organ of the immune system, is also involved in the process of forming an immune response to the antigen. After the administration of MSC, the content of lymphoid cells in the spleen significantly exceeded the parameters of cellularity of spleen of intact animals (table 2). Spot-imprint contains erythroid cells, neutrophil granulocytes, monocytes and lymphoid cells.
The number of lymphoid cells significantly increased by 33% and 24% compared to intact animals and the placebo group on the 7 day of the study. On the 18 day of experiment the cellularity of spleen under the influence of MSCs from adipose tissue was significantly higher at 19 and 14% respectively. At the 25 day of the experiment, the lymphoid cell count was higher by 7 and 15% within the tendency.
Fig. 2. The cellularity of spleen on the 7 day of the study: A - intact group, B - after the introduction of aMSC,
(smear-imprint, x 400)
The weight index of the spleen directly correlates with the content of lymphoid cells in it r = 0.91 (P < 0.001), r = 0,94 (P < 0.001), r = 0.92 (P < 0.001) on the 7, 18 and 25 day of experience respectively. Such changes indicate a direct reaction of the spleen to the introduction of al-logeneic stem cells from adipose tissue.
Discussion
Thus, the administration of allogeneic MSCs isolated from adipose tissue of the C57Bl/6 mice causes systemic effects on the thymus and the spleen. As a result of anti-genic stimulation by allogeneic stem cells, there is an increase in mitotic activity of thymocytes and spleno-cytes. Despite numerous publications that reveal the im-munological properties of cells and confirm the presence of immunosuppressive effects, the results of individual scientific studies show that MSCs under certain conditions can be eliminated by cells of the immune system of
the animal recipient, since they have signs of foreignness (Huang et al., 2009).
The increase of the content of lymphoid cells of the thymus and spleen after the introduction of aMSCs of our mind may be due to the heterogeneity of introduced culture cultures, insufficient number of introduced cells for the implementation of immunosuppressive effect, as well as low concentration of immunosuppressive factors synthesized by MSCs.
Conclusion
1. The administration of allogenic adipose derived mesenchymal stem cells affects on the central and peripheral organs of the immune system.
2. Administration of allogenic adipose derived mesen-chymal stem cells cause a significant increase in the content of lymphoid cells in the thymus at 7, 18 and 25 days by 71, 57 and 53% respectively (P < 0.05) compared to the control.
3. Weight index of the thymus directly correlates with the content of lymphoid cells and its value was r = 0.57, P < 0.01 on 7 day and 18 day r = 0,50, P < 0.05.
4. Quantity lymphoid cells in the spleen significant increase at the 7 and 18 days of the immune response by 33 and 19%, respectively (P < 0.01, P < 0.05) compared to the control under administration of allogenic adipose derived mesenchymal stem cells.
5. On the 25th day, the content of lymphoid cells in spleen and spleen index values return to normal.
6. Weight index of the spleen directly correlates with the content of lymphoid cells in its r = 0.91-0.94, P < 0.001.
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