Stroke is a global medical and socio-economic problem and a great demand for alternative therapies, the leading one being stem cell (SC) therapy. Pathogenetic processes in ischemic stroke (II) trigger the mechanisms of necrotic and apoptotic death of neurons with the formation of the central infarct zone («core of ischemia») and the ischemic «penumbra» zone; the severity and reversibility of the injury directly depends on the duration of ischemia. In parallel with pathogenetic processes, endogenous neurogenesis occurs – the proliferation of neurogenic stem and progenitor cells (NSC/NPC) and their migration into the ischemic focus; however, most NSCs and newly formed neurons undergo apoptosis and recovery of lost functions does not occur. Significant efforts are being made to find ways to control neurogenesis, in particular through the transplantation of exogenous SCs. The main factors preventing the use of SCs in humans are moral, ethical, religious and legal aspects related to the source and method of obtaining cells, as well as possible immunocompromised complications due to incompatibility of donor cells with the recipient of the main histocompatibility complex antigens. The safest is the use of autologous SCs (the patient’s own cells), as it does not require the use of immunosuppressive protocols. Due to the relative safety and ease of production, the most common are multipotent mesenchymal stem cells (MSCs), namely MSCs of the bone marrow (BM). Numerous preclinical studies in experimental animals with modeled II, as well as clinical trials conducted over the past 15 years, have shown the safety and feasibility of transplantation of autologous MSCs in patients with severe neurological deficits after II. Two different approaches to the use of MSCs are discussed: neuroprotection in the acute phase and neurorestoration in the chronic phase II. Proposals are currently being developed for phase II/III clinical trials in acute and chronic stroke using BM MSCs, the results of which will form the basis for certified standardized II treatment protocols.

The aim of the work was to study the impact of fetal rat brain cell supernatant (FRBCS) on the expression of transforming growth factor β1 (TGF-β1) and p53 in C6 cells of rat glioma in vitro. Materials and Methods: FRBCS was obtained from suspensions of fetal rat brain cells on the 14th (E14) day of gestation. C6 glioma cells were cultured for 48 h in the presence of FRBCS or FRBCS + anti-TGF-β1 monoclonal antibody. Immunocytochemical staining for TGF-β1 and p53 was performed. Results: The proportion of TGF-β1-immunopositive tumor cells in C6 glioma cultures was statistically significantly higher than in the control cell cultures of normal fetal rat brain. FRBCS reduced the proportion of TGF-β1-immunopositive tumor cells and increased the proportion of p53-immunopositive cells in C6 glioma cultures. In cells cultured with FRBCS + anti-TGF-β1 monoclonal antibody, the above effects of FRBCS were abrogated. Conclusion: The obtained results suggest that TGF-β1 seems to be responsible for decrease in TGF-β1 expression and increase in p53 expression in C6 glioma cells treated with FRBCS.

To evaluate the infl uence of the rat progenitor neurogenic cells supernatant (RPNS) on the transplantable rat malignant brain glioma cells (strain 101.8) under conditions of cultivation. Methods. primary cultures were obtained from glioma 101.8 fragments (n = 12) and intact brain of newborn rats (n = 9). RPNS was received from neurogenic cell suspensions of fetal rat brain on 8-11 th (E8-11) and 12-16 th (E12-16) days of gestation. Results. RPNS (E8-11) as well as RPNS (E12-16) showed a cytotoxic effect on the glioma 101.8 cells in short-term cultures, the level of which was dose-dependent and intensifi ed with increasing duration of incubation. RPNS (E12-16) had a more pronounced cytotoxic action on the cells of glioma 101.8 compared with RPNS (E8-11). The cytotoxic index (CI) of RPNS (E12-16) on the glioma 101.8 cells was signifi cantly higher than CI determined in cell suspensions of normal rat brain (CI was (91.99 ± 2.37) % and (22.9 ± 4.97) % respectively over 48 h incubation with RPNS). After RPNS (E8-11) infl uence on the glioma 101.8 primary cultures the signs of dose-dependent cytotoxic effects were observed: the thinning of growth areas, appearance of dystrophic and necrobiotic changes in tumour cells and decreasing of a mitotic index. These features were strengthened under the RPNS (E12-16) infl uence. Conclusions. fetal RPNS showed dose-dependent cytotoxic and antiproliferative effects on the cultivated glioma 101.8 cells, which were intensifi ed with the increasing of rat brain gestational age and lengthening of the incubation duration. A prerequisite for such effects is likely the NPC ability to produce the substances with antitumour activity. K e y w o r d s: progenitor neurogenic cells, rat fetal brain, supernatant, glioma 101.8, cytotoxic index, mitotic index.

No abstract

Вивчали дію супернатанта прогеніторних нейроклітин (СНК) на цитотоксичну функцію лімфоцитів у щурів за умов фізіологічної норми та експериментально змодельованого пухлинного процесу (гліома головного мозку, штам 101.8 ВСТУПВажливою проблемою імунології злоякісних пухлин і, зокрема, гліом головного мозку є визначення механізмів їх «вислизання» з-під нагляду імунної системи та її пригні-чення. Відомо, що при гліомах -найбільш загальних первинних пухлинах мозку, які характеризуються інфільтративним ростом, резистентністю до лікування і мають не-сприятливий прогноз для життя пацієнтів, виявляються зміни у всіх ланках імунітету [1]. Гліома формує імуносупресивне оточен ня секрецією різних молекул (простагландину Е2, інтерлейкіну-10 -IL-10, трансформую-чого ростового фактора b -TGF-b, гангліо-зидів) для послаблення імунної відповіді [2]. Поряд зі зниженням кількості та функції імунокомпетентних клітин (відносна та аб-солютна лімфопенія, знижений вміст лімфо-цитів CD4 + , CD8 + , CD16 + , HLA-DR + -клітин, гальмування проліферативної здатності лімфоцитів, зниження секреції ефекторних цитокінів), підвищується кількість Т-регуля-торних клітин CD4 + CD25 + FoxP3 + , що проду-кують імуноінгібувальні медіатори [3]. При гліомах, особливо злоякісних, відбувається часткова або повна втрата клітинами пухлин експресії антигенів І та/або ІІ класу головно-

Cellular and molecular similarities between brain tumor stem cells (BTSCs) and normal neurogenic stem cells (NSCs) motivate the search for new methods of treatment of malignant glioma using NSCs. CD133 molecule could be one of the most typical markers of BTSCs and considered as a target for therapy of brain tumors.The aim of this study was to evaluate the effect of rat neurogenic cells supernatant (NCsS) on the content of CD133+ cells in glioma C6 cell cultures.Materials and methods. The cells of rat brain glioma C6 were used as the source for the cultivation; for comparative assessment of tested compound impact on the intact nervous system the fetal rat brain cells on 14th (E14) day of gestation were used. The study was performed in control cultures under standard culture conditions without NCsS adding and tested cultures with adding NCsS (0.10 mg/ml of protein) for 48 hours. NCsS was received from suspensions of rat brain neurogenic cells (E14).Results. CD133-positive cells were 12.05 ± 4.77 % of the total number of cells in C6 glioma culture and 37.36 ± 12.33 % of the total number of cells in fetal rat brain culture. CD133-positive cells had a smaller size than negative cells (average values of cross-sectional area of cells and nucleus) and greater nuclear-cytoplasmic ratio. The cell and nucleus sizes of CD133-positive cells in cell cultures of fetal rat brain were twice larger than sizes of such cells in cultures of glioma C6.Under the conditions of NCsS for 48 hours the reducing in the number of CD133-positive cells in rat glioma C6 cell cultures (2.88 ± 0.41 %) and lack of such effects in cell cultures of fetal rat brain (E14) were found.Conclusion. The morphological differences of CD133-positive cells in glioma C6 cultures and in cell cultures of fetal rat brain (E14) were detected. The decrease of CD133-positive cells in glioma C6 cells culture under the influence of neurogenic cells supernatant was shown.

В огляді представлено аналіз сучасного стану розробки проблеми імунобіологічних властивостей нейрогенних стовбурових та прогеніторних клітин (НСК/НПК) головного мозку. У частині ІІ наведено дані щодо імунних реакцій in vitro при сумісному культивуванні НСК/НПК та клітин імунної системи, а також особливостей їх розвитку in vivo після трансплантації НСК/НПК. Розглянуто трансформацію уявлень щодо імунної відповіді в ЦНС при введенні НСК/НПК: від концепції «абсолютної імунопривілейованості» мозку до концепції «функціональної» або «терапевтичної пластичності», що враховує різні аспекти і механізми взаємодії клітин імунної системи і НСК/ НПК. Узагальнено дані стосовно імунної відповіді при трансплантації ало-та ксеногенних НСК/ НПК та її залежність від імунного статусу реципієнтів. Підкреслено актуальність досліджень імунобіологічних властивостей нейрогенних клітин фетального мозку, які сприяють розкриттю механізмів дії НСК/НПК та надають змогу оцінити доцільність їх практичного використання для клітинної терапії уражень ЦНС. Ключові слова: нейрогенні стовбурові клітини; нейрогенні прогеніторні клітини; алотрансплантація; ксенотрансплантація; імунна відповідь; імуномодуляція.

A potential strategy for recovery and regeneration of brain damage due to traumatic brain injury is considered to be the transplantation of neurogenic stem and/or progenitor cells (NSCs/NPCs). The key factors of the regenerative non-targeted effects of NSCs/NPCs (so-called bystander effects) include the signal molecules produced by them into the extracellular environment (secretome). The purpose is to study the regenerative bystander effects of rat fetal brain neurogenic cells (FBNCs) in the in vitro model of neurotrauma. Materials and methods. In cell culture of FBNCs from rat fetuses (E14-16), neurotrauma was modeled in vitro by mechanical scratching of monolayer and conditioned medium obtained from 24-h cultures of rat FBNCs was added. Cell phenotype was evaluated by morphological features and by immunocytochemical staining for Nestin and GFAP. The density and length of processes, migration capacity, the cell growth rate and monolayer density in the scratched area were compared. Morphometric study included analysis of the width of the scratched area, the number of migrating cells, the distance of migration and mitotic activity in the intact monolayer. Results. Under the conditions of the nutrient medium of standard composition in the scratched area the signs of endogenous regeneration are shown during 24-48 h of cultivation. The overgrowth of cell processes from monolayer and short distance migration of single undifferentiated or poorly differentiated cells were shown. In the next 72-96 h of observation, the degeneration of migrated cells and processes in the scratched area was detected. Under the influence of conditioned media from 24-h cultures of FBNCs by single addition immediately after scratching at dose of 0.1 mg/ml for protein content the stimulation of regeneration were detected up to 96 hours of cultivation. The migration of cell processes from the monolayer simultaneously with undifferentiated or poorly differentiated cells at 24 hours was shown. The formation of cell clusters and their differentiation (at 48 h), as well as migration of differentiated cells with partial or complete overgrowth of scratched area (72-96 h) were observed. The morphological signs of degeneration of migrated cells in the scratched area appeared only on the 8th day of cultivation. Conditioned media does not affect qualitative and quantitative properties of the culture of rat FBNCs in the intact area where mitotic activity was average. Conclusions. Conditioned medium from 24-h cultures of rat FBNC can stimulate reparation in the in vitro model of neurotrauma in neural cell culture for at least 7 days at a single addition, without affecting the cellular composition and mitotic activity of the intact monolayer.