Effectiveness of mesenchymal stem cells (MSCs) proliferation and differentiation was studied after addition of medicinal product containing human recombinant erythropoietin (rhEPO) into enriched culture medium with components for osteo- and angiogenesis. Cultivation of mesenchymal stem cells under static conditions using “cocktail” of growth factors and rhEPO allowed to populate allogenic discs-scaffolds with connective tissue formations with the participation of osteoblasts and elongated multicellular structures, which formed after endothelium-like cells fusio

Atamanyuk, E. A.

Guzyukina, S. A.

Nadezhdin, S. V.

Ovsyankin, A. V.

Zubareva, E. V.

English

DSpace at Belgorod State University

310Influence of recombinant erythropoietin on mesenchymal stem cells proliferation and differentiation during cultivation on allogenic bone matrixSergey V. Nadezhdin1, Ekaterina V. Zubareva1, Anatoliy V. Ovsyankin2, Svetlana A. Guzyukina2, Elena A. Atamanyuk3, Ekaterina S. Gnezdyukova4, Kseniya R. Zhukovskaya41Belgorod National Research University, 308015, Russia, Belgorod, Pobedy St. 85; https://orcid.org/0000-0001-7215-295X 1Belgorod National Research University, 308015, Russia, Belgorod, Pobedy St. 85; https://orcid.org/0000-0002-9557-145X 2Federal State Budgetary Institution “Federal Center for Traumatology, Orthopedics and Endoprosthetics” of the Ministry of Health of the Russian Federation. 214031, Russia, Smolensk, Stroiteley Ave. 29; https://orcid.org/0000-0002-0779-091X 2Federal State Budgetary Institution “Federal Center for Traumatology, Orthopedics and Endoprosthetics” of the Ministry of Health of the Russian Federation. 214031, Russia, Smolensk, Stroiteley Ave. 29; https://orcid.org/0000-0001-5652-9489 3Regional State Budgetary Institution of Health “Belgorod Regional Clinical Hospital of St. Ioasaph”. 308007, Russia, Belgorod, Nekrasova St. 8/9; https://orcid.org/0000-0002-4095-9473 4Belgorod National Research University, 308015, Russia, Belgorod, Pobedy St. 85; https://orcid.org/0000-0002-1672-3495 4Belgorod National Research University, 308015, Russia, Belgorod, Pobedy St. 85; https://orcid.org/0000-0002-2835-0630 *corresponding author: Sergey V. Nadezhdin, Belgorod National Research University, 308015, Russia, Belgorod, Pobedy St. 85. Email: nadezhdin@bsu.edu.ru Influencia de la erytropoietina recombinante en la proliferación y diferenciación de células madre mesequímicas durante el cultivo matriz alógena de huesoAbstractIntroductionffectiveness of mesenchymal stem cells (MSCs) proliferation and differentiation was studied after addition of medicinal product containing human recombinant erythropoietin (rhEPO) into enriched culture medium with components for osteo- and angiogenesis. Cultivation of mesenchymal stem cells under static conditions using “cocktail” of growth factors and rhEPO allowed to populate allogenic discs-scaffolds with connective tissue formations with the participation of osteoblasts and elongated multicellular structures, which formed after endothelium-like cells fusion. In the presence of rhEPO MSCs differentiates more effectively in osteogenic direction than in angiogenic.Keywords: mesenchymal stem cells (MSCs), recombinant erythropoietin, allogenic bone matrix, proliferation, differ-entiation.a efectividad de la proliferación y diferen-ciación de las células madre mesenquimales (MSC) se estudió después de la adición de un medicamento que contiene eritropoyetina recombinante humana (rhEPO) en un medio de cultivo enriquecido con componentes para osteo- y angiogénesis. El cultivo de células madre mesenquimáticas en condiciones estáticas utilizando un “cóctel” de factores de crecimiento y rhEPO permitió poblar los andamios-discos alogénicos con for-maciones de tejido conectivo con participación de osteo-blastos y estructuras multicelulares alargadas, que se for-maron después de la fusión de células de tipo endotelio. En presencia de rhEPO, las MSC se diferencian más eficaz-mente en la dirección osteogénica que en la angiogénica.Palabras clave: Células madre mesenquimales (MSC), eritropoyetina recombinante, matriz ósea alogénica, proli-feración, diferenciación.311Revista Latinoamericana de Hipertensión. Vol. 14 - Nº 4, 2019www.revhipertension.comecent advances in biotechnology allowed to synthesize and produce in an industrial large-scale human recombinant erythro-poietin (rhEPO, epoetin)1 and its analogs (erythropoiesis-stimulating agents (ESAs))2-4 to use as medicinal products for treatment of various diseases5,6, rhEPO was charac-terized as hematopoietic and tissue-protective factor3,7,8, and besides the properties similar to vascular endothelial growth factor (VEGF) were revealed6. It was discovered that erythropoietin could stimulate endothelial cells pro-liferation in vitro and mobilize endothelial progenitor cells from the bone marrow9-11, promote circulating endothe-lial progenitor cells adhesive and proliferative properties12. In most research papers it is stated that erythropoietin triggers mesenchymal stem cell differentiation into osteo-blasts in vitro and stimulation of bone tissue formation oc-curs because of increased VEGF expression13-16. This raises the question: does erythropoietin induce mesenchymal stem cell differentiation into osteoblasts or endothelio-cytes predominantly?Aim: The research aimed to study the effectiveness of mesenchymal stem cells proliferation and direction of their differentiation after the addition of erythropoietin into culture medium containing supplements for osteo- and angiogenesis. he experiment was divided into three parts: dur-ing the first part proliferative activity of cells was investigated (6 samples), at the second and the differentiation of the third parts of MSCs was studied in osteogenic (6 samples) and angiogenic directions (n=6) respectively. To perform the study at each part control and experimental groups were formed. Tests performed in triplicates for each group. The cells from the control group were cultured on bone discs-scaffolds in growth medium containing osteogenic and endotheliogenic supplements. The cells from the experimental group were cultured on bone discs-scaffolds in growth medium containing osteo-genic and endotheliogenic supplements and recombinant human erythropoietin.After the end of the cultivation period at the first part of the experiment proliferative activity of hAT-MSCs which were grown on bone, discs-scaffolds was studied using confocal laser scanning microscope DIGITAL ECLIPSE С1 plus (Nikon, Japan) with a fluorescent probe – Сalcein AM (Invitrogen, USA). The solution was prepared and the time of cultivation was determined according to the manufac-turer’s instructions. Fluorescent dye stock solution at a concentration of 5 mM was prepared in dimethyl sulfox-ide (DMSO). During the second and the third parts of the experiment bone discs-scaffolds with cells were washed with phos-phate-buffered saline (PBS) and fixed with 10% formalin solution for investigation of the direction of hAT-MSCs dif-ferentiation. For these purposes, Osterix (a marker of hAT-MSCs differentiation into osteoblasts) and CD31 (a marker of endothelial cells) were used. Software “Statistica 10” was used for statistical analysis of experimental data.Main part: Study of hAT-MSCs, osteoblasts and endothe-lial progenitor cells proliferative potential was performed via measuring of intensive proliferation centers areas be-cause the cells at the 20th day of cultivation on the bone discs-scaffolds united in connective tissue structures. In-crease of the area of new formed connective tissue struc-tures in the experimental group (16406.93±235.92 mkm2) in comparison with the control group (15250.97±182.34 mkm2) (р≤0.01) was revealed (Fig. 1).During the analysis of fluorescent images, it was revealed, that after 20 days of cultivation on bone trabeculae and intertrabecular space of the experimental group samples connective tissue formations appeared which had been formed due to mesenchymal stem cells, osteoblasts, and endothelial progenitor cells proliferation. increase of connective tissue structures area formed by osteoblasts (fluorescent probe Osterix) was noted in the experimen-tal group (17362.94±266.34 mkm2) in comparison with control (16274.89±247.98 mkm2) (р≤0.01) (Fig. 2.А). Be-sides the bone, connective tissue formations elongated multicellular structures containing endothelial-like cells (fluorescent probe CD31) were discovered in the experi-mental group samples. increase of the elongated multi-cellular formations area was revealed in the experimental group (164.19±4.63 mkm2) in comparison with the con-trol group (147.51±7.34 mkm2) (р≤0.01) (Fig. 2.B).Thus, the addition of rhEPO to the culture medium con-taining inducers of osteo- and angiogenesis leads to en-hancement of MSCs proliferation and differentiation in comparison with the control group. This effect could be described because rhEPO is a pleiotropic growth factor which stimulates the proliferation of both mesenchymal and hematopoietic stem cells17.(reference 26 not valid)Cultivation of mesenchymal stem cells in static conditions using “cocktail” of growth factors and medicinal prod-uct which includes rhEPO allowed to load allogenic discs-scaffolds with cells and accompanied by the formation of connective tissue formations with the participation of os-teoblasts and elongated multicellular structures appeared during endothelial-like cells fusion.IntroductionMaterials and methods312he addition of human recombinant erythro-poietin into growth medium increases mesen-chymal stem cells proliferation which accompa-nied by the formation of connective tissue and elongated multicellular structures.Differentiation of mesenchymal stem cells under the influ-ence of recombinant human erythropoietin is more effi-cient in osteogenic direction than in angiogenic direction.Figure 1. Optical slices series. Fluorescence of new formed connective tissue structures in bone disc-scaffold at the 20th day of cultivation (experimental group from the first part of the study, cells were stained with Сalcein AM). (DIGITAL ECLIPSE С1 plus, Nikon, Japan, Х 200 magnification).BFigure 2. Fluorescence of connective tissue structures – elements of the osteoid matrix (red fluorescence) (experimental group from the second part of the study) and elongated multicellular formations (green fluorescence) on the bone trabecula of disc-scaffold (experimental group from the third part of the study). (DIGITAL ECLIPSE С1 plus, Nikon, Japan, Х 200 magnification)Conclusions313Revista Latinoamericana de Hipertensión. Vol. 14 - Nº 4, 2019www.revhipertension.com1.  Flaharty, K. K., Caro, J., Erslev, A., Whalen, J. J., Morris, E. M., Bjorns-son, T. D., Vlasses, P. H. Pharmacokinetics and erythropoietic response to human recombinant erythropoietin in healthy men. Clinical Pharma-cology and Therapeutics. 1990; 47(5):557–564.2.  Rölfing, J.H.D., The effect of erythropoietin on the bone. Thesis. Acta Orthopaedica supplementum. 2014;353(85):1–29.3.  Elliott, S., Sinclair, A.M. The effect of erythropoietin on normal and neoplastic cells. Biologics. 2012; 6: 163–189.4.  Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fritsch, E. F., Kawakita, M., Shimizu, T., Miyake, T. Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature. 1985; 313(6005): 806–810.5.  Arcasoy, M. O. The non-hematopoietic biological effects of erythropoi-etin. British Journal of Haematology. 2008; 141(1):14–31.6.  Suk, K. K., Dunbar, J. A., Liu, A., Daher, N. S., Leng, C. K., Leng, J. K., Lim, P., Weller, S., Fayard, E. Human recombinant erythropoietin and the incidence of retinopathy of prematurity: a multiple regression model. Journal AAPOS: the Official Publication of the American Asso-ciation for Pediatric Ophthalmology and Strabismus. 2008; 12(3):233–238.7.  Ye, L., Chen, L., Yu, Q., Cheng, F. Effect of recombinant human eryth-ropoietin on the stemness of bone marrow-derived mesenchymal stem cells in vitro. International Journal of Stem Cells. 2010; 3(2):175–182.8.  Broudy, V. C., Lin, N., Brice, M., Nakamoto, B., Papayannopoulou, T. Erythropoietin receptor characteristics on primary human erythroid cells. Blood. 1991; 77(12):2583–2590.9.  Heeschen, C., Aicher, A., Lehmann, R., Fichtlscherer, S., Vasa, M., Ur-bich, C., Mildner-Rihm, C., Martin, H., Zeiher, A. M., Dimmeler, S., 2003. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood. 2003; 102(4):1340–1346.10.  Bahlmann, F. H., DeGroot, K., Duckert, T., Niemczyk, E., Bahlmann, E., Boehm, S. M., Haller, H., Fliser, D. Endothelial progenitor cell prolifera-tion and differentiation is regulated by erythropoietin. Kidney Interna-tional. 2003; 64(5):1648–1652.11.  Bahlmann, F. H., De Groot, K., Spandau, J. M., Landry, A. L., Hertel, B., Duckert, T., Boehm, S.M., Menne, J., Haller, H., Fliser, D. Erythropoietin regulates endothelial progenitor cells. Blood. 2004; 103(3): 921–926.12.  Ye, L., Chen, L., Yu, Q., Cheng, F. Effect of recombinant human eryth-ropoietin on the stemness of bone marrow-derived mesenchymal stem cells in vitro. International Journal of Stem Cells. 2010; 3(2):175–182.13.  Shiozawa, Y., Jung, Y., Ziegler, A. M., Pedersen, E. A., Wang, J., Wang, Z., Song, J., Wang, J., Lee, C. H., Sud, S., Pienta, K. J., Krebsbach, P. H., Taichman, R.S. Erythropoietin couples hematopoiesis with bone formation. PLoS One. 2010; 5(5): e10853:1–14.14.  Balaian, E., Wobus, M., Weidner, H., Baschant, U., Stiehler, M., Ehnin-ger, G., Bornhäuser, M., Hofbauer, L. C., Rauner, M., Platzbecker, U. 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Influence of recombinant erythropoietin on mesenchymal stem cells proliferation and differentiation during cultivation on allogenic bone matrix

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Influence of recombinant erythropoietin on mesenchymal stem cells proliferation and differentiation during cultivation on allogenic bone matrix

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