Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

Home/2021, Vol. 9, No. 1/Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

Cell and Organ Transplantology. 2021; 9(1):12-19.
DOI: 10.22494/cot.v9i1.117

Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols

Tsymbaliuk V.1, Vaslovych V.1, Pichkur L.1, Liubych L.1, Malysheva T.1, Verbovska S.1, Egorova D.1, Lontkovskkiy Yu.2

  • 1A. P. Romodanov State Institute of Neurosurgery, National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine
  • 2Medical Center “MEDLON”, Kamianets-Podilsky, Ukraine

Abstract

The transplantation of multipotent mesenchymal stromal cells (MMSCs) is considered to be a possible therapy of multiple sclerosis. For the clinical application of human umbilical cord-derived MMSCs (UC-MMSCs) it is necessary to develop a method of their cryopreservation taking into account the type of cryoprotective media and to investigate the possibility of using these cells for therapeutic purposes in vivo.
The purpose of the study was to investigate the effect of UC-MMSCs, cryopreserved in solutions of different composition, on the processes of demyelination and remyelination of the spinal cord of rats with experimental allergic encephalomyelitis (EAE) as a model of multiple sclerosis.
Materials and methods. The EAE was modeled by subcutaneous administration of homogenized spinal cord of adult rats with complete Freund’s adjuvant. On the 18th day rats with moderate relapsing-remitting form of EAE were suboccipitally injected 1·106 UC-MMSCs, cryopreserved in cryoprotective media containing dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), ethylene glycol, trehalose and sucrose at different composition. On the 35th and 60th days, the studies of ultrastructural changes of the lumbar spinal cord (L3-L5) were performed, assessing the degree of demyelination of nerve fibers by the ratio of myelin sheath (MS) thickness to the diameter of the axis cylinder (AC) of axons.
Results. In rats with moderate EAE from the 35th to the 60th day after the modelling of the disorder, destructive changes and signs of demyelination in the spinal cord increased; the MS/AC index corresponded to the average degree of axon demyelination. Suboccipitally administered cryopreserved UC-MMSCs to EAE rats, depending on the used cryopreservation solution, slowed or stopped the demyelination, decreased the MS/AC index to a low degree of axonal demyelination. Reducing the concentration of DMSO in the cryopreservation medium from 10 % to 4 % and adding 6 % trehalose provided a better effectiveness of UC-MMSCs in decreasing the degree of demyelination in EAE. At the same time, the standard solution (10 % DMSO, 90 % FBS) provided these effect, but to a lesser extent. The use of a multicomponent cryopreservation medium containing 15 % ethylene glycol, 3 % DMSO, 10 % sucrose, 12 % trehalose and 60 % FBS did not achieve the goal of maintaining the effects of UC-MMSCs to reduce the degree of demyelination in EAE.
Conclusions. To maintain the therapeutic properties of UC-MMSCs, it is advisable to add a reduced concentration of DMSO (4 %) and 6 % trehalose to the cryopreservation medium, supplemented with 90 % fetal bovine serum.

Key words: demyelination; remyelination, multipotent mesenchymal stromal cells; cell cryopreservation

Full Text PDF

1. Pichkur LD, Verbovs’ka SA, Akinola ST, Chitaeva GE. The main pathogenetic mechanisms of the demyelination process in the central nervous system and the possibility of its correction. Ukr neurological journal. 2017;(2):12-19. Ukrainian.
2. Scuteri A, Donzelli E, Rigolio R, Ballarini E, Monfrini M, Crippa L, Chiorazzi A, Carozzi V, Meregalli C, Canta A, Oggioni N, Tredici G, Cavaletti G. Therapeutic Administration of Mesenchymal Stem Cells Abrogates the Relapse Phase in Chronic Relapsing-Remitting EAE. J Stem Cell Res Ther. 2015; 5:262 http://dx.doi.org/10.4172/2157-7633.1000262
https://doi.org/10.4172/2157-7633.1000262
3. Fong CY, Chak LL, Biswas A, Tan JH, Gauthaman K, Chan WK, Bongso A. Human Wharton’s jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells. Stem Cell Rev. 2011;7:1-16. doi: 10.1007/s12015-010-9166-x
https://doi.org/10.1007/s12015-010-9166-x
PMid:20602182
4. Kovalchuk MV, Deryabina OG, Pichkur LD, Verbovskaya SA, Shuvalova NS, Pichkur OL, Kordium VA. Distribution of transplanted human mesenchymal stem cells from Wharton’s Jelly in the central nervous systems of the EAE rats. Biopolym. Cell. 2015;31(5):371-378. doi: 10.7124/bc.0008F9.
https://doi.org/10.7124/bc.0008F9
5. Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, Giunti D, Ceravolo A, Cazzanti F, Frassoni F, Mancardi G, Uccelli A. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood. 2005;106(5):1755-1761. doi: https://doi.org/10.1182/blood-2005-04-1496
https://doi.org/10.1182/blood-2005-04-1496
PMid:15905186
6. Anderson P, Gonzalez-Rey E, O’Valle F, Martin F, J.Oliver F, Delgado M. Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function. Stem Cells International. 2017; Article ID 2389753, 15 p. https://doi.org/10.1155/2017/2389753
https://doi.org/10.1155/2017/2389753
PMid:28250776 PMCid:PMC5303870
7. Vaslovych VV, Pichkur LD, Malysheva TA, Akinola ST, Verbovs’ka SA, Toporova OK, Shuvalova NS. Ul’trastrukturni zminy spynnoho mozku shchuriv z eksperymental’nym alerhichnym entsefalomiyelitom pid vplyvom mezenkhimal’nykh stovburovykh klityn ta interleykina-10. Zhurnal klynycheskykh i eksperymental’nykh medytsynskykh issledovanyy. 2018;1:17-30. Ukrainian.
8. Tsymbalyuk VI, Pichkur LD, Verbovs’ka SA, Akinola ST, Vaslovych VV, Deryabina OH, Pokholenko YaO, Toporova OK, Shuvalova NS, Kordyum VA. Vplyv ksenohennoyi transplantatsiyi natyvnykh i transfikovanykh henom interleykina 10 mezenkhimal’nykh stovburovykh klityn na perebih eksperymental’noho alerhichnoho entsefalomiyelitu. Visnyk problem biolohiyi i medytsyny. 2018;2(1): 227-234. Ukrainian.
9. Tsymbaliuk VI, Velychko OM, Pichkur OL, Verbovska SA, Pichkur LD, Shuvalova NS. Effects of human Wharton’s jelly-derived mesenchymal stem cells and interleukin-10 on behavioural responses of rats with experimental allergic encephalomyelitis. Cell and Organ Transplantology. 2015; 3(1):46-51. doi: 10.22494/COT.V3I1.19
https://doi.org/10.22494/COT.V3I1.19
10. Kassis I, Petrou P, Halimi M, Karussis D. Mesenchymal Stem Cells (MSC) derived from mice with Experimental Autoimmune Encephalomyelitis (EAE) suppress EAE and have similar biological properties with MSC from healthy donors. Immunol Lett. 2013;154(1-2):70-6. doi: 10.1016/j.imlet.2013.06.002. PubMed PMID: 23994102.
https://doi.org/10.1016/j.imlet.2013.06.002
PMid:23994102
11. Rudenko VA, Gnedkova IO, Pichkur DL, Verbovska SA, Pokholenko YaO. Influence of xenogenic transplantation of mesenchymal stem cells and Il- 10 on cellular immunity in rats with experimental allergic encephalomyelitis. Collection of scientific works of staff members of NMAPE. 2014;23(2):434-441. Ukrainian. https:// https://nuozu.edu.ua/zagruzka/zbornikNMAPO23_2.pdf Ukrainian
12. Rudenko VA, Belska LM, Verbovska SA, Pichkur OL. Neuroautoimmune reactions in rats with experimental allergic encephalomyelitis after treatment by xenogeneic MSCs and ІL 10. Collection of scientific works of staff members of NMAPE. 2014;23(3):447-452. Ukrainian. https://nuozu.edu.ua/zagruzka/zbornikNMAPO23_3.pdf Ukrainian
13. Akinola ST, Verbovs’ka SA, Vaslovych VV, Pichkur LD. Doslidzhennya vplyvu stovburovykh klityn na perebih eksperymental’noho alerhichnoho entsefalomiyelitu i morfofunktsional’nyy stan nervovykh volokon spynnoho mozku. Zb. nauk. prats’ spivrobitnykiv NMAPO im. P. L. Shupika. 2017;(28):5-17. Ukrainian.
14. Skiles ML, Marzan AJ, Brown KS, Shamonki JM. Comparison of umbilical cord tissue-derived mesenchymal stromal cells isolated from cryopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model. Cytotherapy. 2020;22(10):581-591, ISSN 1465-3249, https://doi.org/10.1016/j.jcyt.2020.06.002
https://doi.org/10.1016/j.jcyt.2020.06.002
PMid:32718875
15. Maslova O, Novak M, Kruzliak P. Umbilical cord tissue-derived cells as therapeutic agents. Stem Cells International. 2015; 2015:1-10. doi:10.1155/2015/150609
https://doi.org/10.1155/2015/150609
PMid:26246808 PMCid:PMC4515303
16. Best BP. Cryoprotectant toxicity: facts, issues, and questions. Rejuvenation Res. 2015; 18(5):422-36. doi:10.1089/rej.2014.1656. PMID: 25826677. PMCID: PMC4620521.
https://doi.org/10.1089/rej.2014.1656
PMid:25826677 PMCid:PMC4620521
17. Bersenev AV. Convulsions and coma as complications of DMSO-associated toxicity during infusion of hematopoietic cells in bone marrow transplantation. Cell Transplantology and Tissue Engineering. 2006; 1: 31-32.
18. Mantri S, Kanungo S, Mohapatra P. Cryoprotective Effect of Disaccharides on Cord Blood Stem Cells with Minimal Use of DMSO. Indian Journal of Hematology and Blood Transfusion. 2014;31(2):206-212. doi:10.1007/s12288-014-0352-x.
https://doi.org/10.1007/s12288-014-0352-x
PMid:25825559 PMCid:PMC4375150
19. Tsymbaliuk V, Deryabina O, Shuvalova N, Verbovska S, Pichkur L, Oleksenko N, Kordium V. Cryopreservation of human Wharton’s jelly multipotent mesenchymal stromal cells with reduced concentration of dimethyl sulfoxide. Cell and Organ Transplantology. 2020; 8(1):51-57. doi:10.22494/cot.v8i1.109.
https://doi.org/10.22494/cot.v8i1.109
20. Pichkur LD, Semenova VM, Velychko OM, Verbovs’ka SA, Yehorova DM, Akinola ST, Vaslovych VV. Optymizatsiya modelyuvannya eksperymental’noho alerhichnoho entsefalomiyelita z khronichnym retsydyvuyuchym perebihom. Eksperymental’na i klinichna medytsyna. 2017;4(77):4-14. Ukrainian
21. Palade GE. A study of fixation for electron microscopy. J. Exp.Med. 1952;95(3):285-298.
https://doi.org/10.1084/jem.95.3.285
PMid:14927794 PMCid:PMC2212069
22. Gayer G. Electronic histochemistry. Moscow: Mir, 1974: 488 p. Russian
23. Reynolds ES. The use of lead citrate at high pH as an electronopague stain in electron microscopy. J Cell Biol. 1963;17:208-212.
https://doi.org/10.1083/jcb.17.1.208
PMid:13986422 PMCid:PMC2106263
24. Tsimbalyuk VI, Markova OV, Pichkur LD, Vaslovich VV et al. A method of assessing the degree of demilinization of axons in experimental allergic encephalomies. Declarative patent for useful model No. 17499. Ukraine, IPC G 01N 23/02.- З.№ 2006 05697; declared 05.24.06; publ. 09.15.06, Bul. No. 9. Ukrainian
25. Rebrova OY. Statistical analysis of medical data. Application of the STATISTICA Application Package), Moscow: MediaSfera, 2002: 312 p. Russian
26. Shabanov YES. Online synopsis of the course “Biometric data processing in zoology and ecology” 2011. E-book, access modeЖ: https://batrachos.com/biometria Russian
27. Mastitsky SE, Shitikov VK. Statistical Analysis and Data Visualization with R. 2014. E-book, Access Mode: http: //r-analytics.blogspot.com. Russian
28. Afanasyev YuI, Yurina NA, Kotovsky EF et al. Histology, embryology, cytology: textbook, 6th ed.: ed. Yu. I. Afanasyeva, N, A. Yurina. Moscow: GEOTAR-Media, 2012: 800 p. Russian
29. Bogolepov NN. Brain ultrastructure during hypoxia. M .: Medicine, 1979: 168 p. Russian
30. Li Q, Weiland A, Chen X, Lan X, Han X, Durham F, Liu X, Wan J, Ziai WC, Hanley DF, Wang J. Ultrastructural Characteristics of Neuronal Death and White Matter Injury in Mouse Brain Tissues After Intracerebral Hemorrhage: Coexistence of Ferroptosis, Autophagy, and Necrosis. Front Neurol. 2018;9:581. doi: 10.3389/fneur.2018.00581. PMID: 30065697; PMCID: PMC6056664.
https://doi.org/10.3389/fneur.2018.00581
PMid:30065697 PMCid:PMC6056664
31. Frumkina LE, Yakovleva NI, Bogolepov NN. Mekhanizmy razvitiya sinapsa kak osnova ego involyutsii. Byulleten’ eksperimental’noy biologii. 1993;116:214-217. Russian
https://doi.org/10.1007/BF00786090
32. Multiple sclerosis. Selected questions of theory and practice. Ed. IA Zavalishin, VI Golovkin. Moscow, 2000: 637 p. Russian
33. Chekhonin VP, Davydovskaya MV, Lebedev SV et al. Neurobiological bases of remyelination in the central nervous system. // Bulletin of the Russian Academy of Medical Sciences. 2003;8:43-52.
34. Zhivolupov SA, Samartsev IN, Syroezhkin FA. Sovremennaya kontseptsiya neyroplastichnosti (teoreticheskie aspekty i prakticheskaya znachimost’). Zhurnal nevrologii i psikhiatrii im. S.S. Korsakova. 2013;10:102-108.
35. Meerlo P, Mistlberger RE, Jacobs BL, Heller HC, McGinty D. New neurons in the adult brain: the role of sleep and consequences of sleep loss. Sleep Med Rev. 2009;13(3):187-194. doi: 10.1016/j.smrv.2008.07.004.
https://doi.org/10.1016/j.smrv.2008.07.004
PMid:18848476 PMCid:PMC2771197
36. Gomzikova MO, James V, Rizvanov AA. Therapeutic Application of Mesenchymal Stem Cells Derived Extracellular Vesicles for Immunomodulation. Front. Immunol. 2019;10:1-9.
https://doi.org/10.3389/fimmu.2019.02663
PMid:31849929 PMCid:PMC6889906
37. Zanier ER, Montinaro M, Vigano M, Villa P, Fumagalli S, Pischiutta F, Longhi L, Leoni ML, Rebulla P, Stocchetti N, et al. Human umbilical cord blood mesenchymal stem cells protect mice brain after trauma. Crit. Care Med. 2011;39:2501-2510.
https://doi.org/10.1097/CCM.0b013e31822629ba
PMid:21725237
38. Hsieh JY, Fu YS, Chang SJ, Tsuang YH, Wang HW. Functional module analysis reveals differential 0steogenic and stemness potentials in human mesenchymal stem cells from-bone marrow and Wharton’s jelly of the umbilical cord. Stem Cells Dev. 2010;19:1895-1910. https://doi.org/10.1089/scd.2009.0485. PMID:20367285
https://doi.org/10.1089/scd.2009.0485
PMid:20367285
39. Konala VB, Mamidi MK, Bhonde R, Das AK, Pochampally R, Pal R. The current landscape of the mesenchymal stromal cell secretome. Cytotherapy. 2016;18:13-24. DOI:10.1016/j.jcyt.2015.10.008
https://doi.org/10.1016/j.jcyt.2015.10.008
PMid:26631828 PMCid:PMC4924535
40. Putra A, Ridwan ВR, Putridewi AI, Kustiyah AR, Wirastuti K, Sadyah NA, et al. The role of TNF-alpha induced MSCs on suppressive inflammation by increasing TGF-beta and IL-10. Open Access Maced J Med Sci. 2018;6(10):1779-1783.
https://doi.org/10.3889/oamjms.2018.404
PMid:30455748 PMCid:PMC6236029
41. Laroni A, Kerlego de Rosbo N, Uccelli A. Mesenchymal stem cells for the treatment of neurological diseases: immunoregulation beyond neuroprotection. Immunology letter. 2015;168:183-190. http://dx.doi.org/10.1016/j.imlet.2015.08.007.
https://doi.org/10.1016/j.imlet.2015.08.007
PMid:26296458
42. Bahsoun S, Coopman K, Elizabeth C, Akam EC. The impact of cryopreservation on bone marrow derived mesenchymal stem cells: a systematic review. J Transl Med. 2019;17:397. https://doi.org/10.1186/s12967-019-02136-7.
https://doi.org/10.1186/s12967-019-02136-7
PMid:31783866 PMCid:PMC6883667

Tsymbaliuk V, Vaslovych V, Pichkur L, Liubych L, Malysheva T, Verbovska S, Egorova D, Lontkovskkiy Yu. Ultrastructural changes in the spinal cord of rats with experimental allergic encephalomyelitis under the influence of human umbilical cord-derived multipotent mesenchymal stromal cells cryopreserved according to different protocols. Cell Organ Transpl. 2021; 9(1):12-19. doi:10.22494/cot.v9i1.117

Creative Commons License
Is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.