Лабораторія клітинних та тканинних культур відділу клітинних та тканинних технологій, Інститут генетичної та регенеративної медицини НАМН України, Київ, Україна Фенотипові зміни і проліферативний потенціал мезенхімальних стовбурових клітин з вартонових драглів пуповини людини в умовах культивування Анотація Мета. Розробити протокол отримання мезенхімальних стовбурових клітин (МСК) з вартонових драглів пуповини людини, дослідити їх фенотипові зміни і проліферативний потенціал в умовах культивування. Матеріали і методи. МСК пуповини людини культивували протягом 6 пасажів. Для дослідження морфологічних характеристик клітин у культурі застосовували методики забарвлення гематоксиліном та еозином, адаптовані для використання у культурах клітин, за Романовським-Гімзе, толуїдиновим синім. Для забарвлення ядерної ДНК використовували реагенти-інтеркалятори: Hoechst 33342, DАРІ та Еthidium bromid. Експресію поверхневих маркерів МСК (CD105, CD90, CD73 , CD34) досліджували за допомогою FACS-аналізу. Результати. В культурі МСК зберігають морфологічну ідентичність протягом 2 пасажів. В подальшому спостерігають тенденцію до зменшення інтенсивності експресії маркерів МСК, ознаки деградації культури, появу на пізніх пасажах спонтанного адипогенного та хондрогенного диференціювання. Висновки. При культивуванні МСК пуповини людини протягом 2 пасажів зберігають морфологічні характеристики і проліферативний потенціал, в подальшому вони втрачають мезенхімальний мультипотентний фенотип.
Стаття містить рисунки, які відображаються в друкованій версії у відтінках сірого, в електронній -у кольорі.with intravenous introduction of HSC (20 million cells) and suboccipital transplantation of FNC was associated with temporary significant deterioration after HSC administration and rapid recovery after FNC application (on the 29 th day). In all treatment options we revealed the tendency to reduction of pathologically changed neurocyts and gliocyts number. Conclusion.Suboccipital administration of allogeneic FNC improves the clinical condition of rats with EAE. The best results of experimental EAE treatment were observed at intravenous administration of HSC (20 million cells) followed by FNC neurotransplantation, particularly on the 63 rd day.
On the model of experimental analogue of human multiple sclerosis we studied the effects of the mesenchymal stem cells transfected with plasmid vector containing gene IL-10 (MSCs-T) on the functional state of the CNS in rats.Materials and methods. The experimental allergic encephalomyelitis (EАЕ) was induced with spinal cord homogenate of rats with complete Freund’s adjuvant. The mesenchymal stem cells (MSCs) were isolated by the explants technique from Wharton’s jelly of the human umbilical cord and culture up to two passages. Then the MSCs of second passage were transfected of plasmid vector with gene IL-10 and marker gene of green fluorescent protein. Cell transplantation was performed suboccipitally on the 17th day at a dose of 1 million cells in 100 µl of saline per animal.Results. In the open field test we have established that the use of MSCs-T transfected with gene IL-10 suppressed the vertical locomotor activity and elevated the emotional activity as well as partially corrects horizontal locomotor activity indexes which approach the indexes of intact animals.Conclusions. The use of MSCs transfected with plasmid vector with gene IL-10 in the rats with induced EAE is more effective method than treatment using non-transfected MSCs. Combined treatment with IL-10 + MSCs in ЕАЕ rats is more effective than treatment with transfected МSCs.
The urgent problem of long-term storage of multipotent mesenchymal stromal cells (MMSCs) is to improve the protocol of their cryopreservation for further application maintaining the therapeutic properties and minimizing the risks of adverse effects on the health of the recipient. As a standard cryoprotectant, a mixture of 90 % fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO) is used, which, however, can cause a variety of adverse reactions. Therefore, it is important to study the possibility of reducing the concentration of potentially dangerous DMSO by adding other components to the mixture for cell cryopreservation. Purpose. To determine the efficiency of cryopreservation of human Wharton's jelly MMSCs using cryoprotectants of different composition by studying the proliferative activity, phenotype and features of cell morphology in culture in vitro. Materials and methods. The cryoprotective effect of various combinations of DMSO, ethylene glycol, sucrose and trehalose was studied. The efficacy was assessed by cell viability, their adhesive properties, expansion rate and monolayer formation, as well as the expression of main MMSCs markers. Results. It is shown that the most effective combination is 4 % DMSO with 6 % trehalose which provides the highest level of preservation of cell viability, as well as their adhesive and proliferative properties during thawing. Other combinations of the cryoprotectant components showed a much slower cell division, in some cases, the monolayer was not formed at all. For all investigated variants, the main surface markers of MMSCs were preserved. Conclusions. The obtained results indicate the possibility of reducing the concentration of DMSO to 4 % in the freezing medium for MMSCs cryopreservation while maintaining their viability, proliferative activity and common surface markers.
Motor disability is a common outcome of spinal cord injury (SCI). The recovery of motor function after injury depends on the severity of neurotrauma; motor deficit can be reversible, at least partially, due to the innate tissue capability to recover, which, however, deteriorates with age. Pain is often a comorbidity of injury, although its prediction remains poor. It is largely unknown whether pain can attend motor dysfunction. Here, we implemented SCI for modelling severe and moderate neurotrauma and monitored SCI rats for up to 5 months post-injury to determine the profiles of both motor deficit and nociceptive sensitivity. Our data showed that motor dysfunction remained persistent after a moderate SCI in older animals (5-month-old); however, there were two populations among young SCI rats (1 month-old) whose motor deficit either declined or exacerbated even more over 4–5 weeks after identical injury. All young SCI rats displayed changed nociceptive sensitivity in thermal and mechanical modalities. The regression analysis of the changes revealed a population trend with respect to hyper- or hyposensitivity/motor deficit. Together, our data describe the phenotypes of motor deficit and pain, the two severe complications of neurotrauma. Our findings also suggest the predictability of motor dysfunction and pain syndromes following SCI that can be a hallmark for long-term rehabilitation and recovery after injury.
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