Osteogenic potential of multipotent mesenchymal stromal cells from human exfoliated deciduous teeth before and after cryopreservation

The use of multipotent mesenchymal stromal cellsfrom human exfoliated deciduous teeth (SHED) to stimulate bone regeneration requires data on the influence of cryopreservation on the osteogenic differentiation capacity of these cells. SHED were subjected to cryopreservation. Before freezing and after thawing, cell cultures were exposed osteogenic differentiation with vitamin D3 or dexametasone and assessed for expression of the osteogenic markers osteocalcin, alkaline phosphatase, BMP-2 and RunX2 using real-Time qPCR. Extracellular matrix (ECM) mineralization was evaluated by Alizarin red staining. Supplementation of osteogenic medium with vitamin D3 increased the expression of the osteogenic markers osteocalcin, alkaline phosphatase, BMP-2 and RunX2 as well as promoted an increase in the synthesis and mineralization of ECM in the cells both before and after cryopreservation. In the presence of vitamin D3 gene expression of alkaline phosphatase, BMP-2 and RunX2 after cryopreservation was higher than before freezing. Gene expression of osteocalcin, BMP2 RunX2 in osteogenic medium with vitamin D3was higher compared with dexamethasone for 14 days differentiation both before or after cryopreservation. The maintenance of SHED osteogenic differentiation potential after long-Term cryopreservation provides a basis for banking of these cellsfor further auto- or allotransplantation

Osteogenic potential of multipotent mesenchymal stromal cells from human exfoliated deciduous teeth before and after cryopreservation

The use of multipotent mesenchymal stromal cellsfrom human exfoliated deciduous teeth (SHED) to stimulate bone regeneration requires data on the influence of cryopreservation on the osteogenic differentiation capacity of these cells. SHED were subjected to cryopreservation. Before freezing and after thawing, cell cultures were exposed osteogenic differentiation with vitamin D3 or dexametasone and assessed for expression of the osteogenic markers osteocalcin, alkaline phosphatase, BMP-2 and RunX2 using real-Time qPCR. Extracellular matrix (ECM) mineralization was evaluated by Alizarin red staining. Supplementation of osteogenic medium with vitamin D3 increased the expression of the osteogenic markers osteocalcin, alkaline phosphatase, BMP-2 and RunX2 as well as promoted an increase in the synthesis and mineralization of ECM in the cells both before and after cryopreservation. In the presence of vitamin D3 gene expression of alkaline phosphatase, BMP-2 and RunX2 after cryopreservation was higher than before freezing. Gene expression of osteocalcin, BMP2 RunX2 in osteogenic medium with vitamin D3was higher compared with dexamethasone for 14 days differentiation both before or after cryopreservation. The maintenance of SHED osteogenic differentiation potential after long-Term cryopreservation provides a basis for banking of these cellsfor further auto- or allotransplantation

Comparative Analysis of the Paracrine Action of Neuronal and Glial Progenitor Cells Derived from Induced Human Pluripotent Stem Cells

We performed comparative analysis of paracrine activity of neuronal and glial progenitors derived from induced pluripotent stem cells under conditions of hypoxia modeled by addition of cobalt dichloride. Neuronal and glial progenitors produced neuroprotective and neurotrophic effects on SHSY-5Y neuroblastoma cells in co-culture during the post-hypoxic recovery and reduced the number of apoptotic and necrotic cells. Moreover, they produced a neurotrophic effect and promote the formation and growth of neurites in neuroblastoma cells. The paracrine effect of glial progenitors was more pronounced, which can be explained by more intensive expression and secretion of neurotrophic factors in these cells. © 2020, Springer Science+Business Media, LLC, part of Springer Nature

Сравнительный анализ паракринного действия нейрональных и глиальных предшественников, полученных из индуцированных плюрипотентных стволовых клеток человека

The article is focused on comparative analysis of the in vitro paracrine activity shown by the induced pluripotent stem cell-derived neuronal and glial progenitors and directed towards SHSY-5Y neuroblastoma cells during the recovery from hypoxic stress modeled by the exposure to cobalt dichloride. Co-cultivation with neuronal and glial progenitors exerted neuroprotective and neurotrophic effects on SHSY-5Y cells during the post-hypoxic recovery, reducing the number of apoptotic and necrotic cells and supporting neurite outgrowth. Moreover, the paracrine effect of glial progenitors was more pronounced compared to neuronal progenitors, which may be explained by a higher level of expression and secretion of neurotrophic factors by glial progenitors.Проведён сравнительный анализ паракринной активности нейрональных и глиальных предшественников, полученных из индуцированных плюрипотентных стволовых клеток, при моделировании гипоксии добавлением дихлорида кобальта. Сокультивирование с нейрональными и глиальными предшественниками оказывало нейропротективное действие на клетки нейробластомы SHSY-5Y, снижая количество апоптотических и некротических клеток в культуре. Кроме того, сокультивирование обеспечивало нейротрофический эффект, способствуя нейритогенезу в клетках линии SHSY-5Y. При этом паракринное влияние глиальных предшественников было более выраженным, что может объясняться более высоким уровнем экспрессии нейротрофических факторов в данных клетках

Comparative impact analysis of neuronal and glial progenitors conditioned medium on cerebellar neurons under glutamate exitotoxicity

One of the main causes of cell death in neurodegenerative diseases is excitotoxicity. Today the potential directions of treatment neurodegenerative diseases are including cell therapy, the purpose of which is to replace lost nerve tissue with donor cells. Transplanted cells along with replaced lost tissues have a paracrine effect, which requires careful study. The aim of this work was to study the effect of conditioned media, obtaining from neuronal and glial progenitor cells, on a primary culture of cerebellar neurons in a model of glutamate excitotoxicity. The cell viability, expression of marker genes for apoptosis and neuritogenesis, and the number of necrotic and apoptotic cells were determined in the culture of cerebellar neurons. The composition of the studied conditioned media was analyzed for the content of neurotrophins. A comparative analysis was revealed differences in the secretion of neurotrophins between the obtained cultures: the amount of brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor and glial neurotrophic factor was higher in the secretion of glial progenitors. It was shown that the addition of conditioned media from neuronal cells does not significantly affect the viability of cerebellar neurons, whereas preincubation with media from glial progenitors has a neuroprotective effect by increasing the viability of cerebellar neurons, and during long-term cultivation promotes the growth of neurites by increasing the expression level of MAP2 and GAP43 genes. © 2019, Human Stem Cell Institute. All rights reserved

ТЕРАПЕВТИЧЕСКАЯ ЭФФЕКТИВНОСТЬ ВНУТРИАРТЕРИАЛЬНОГО ВВЕДЕНИЯ КОНДИЦИОНИРОВАННОЙ СРЕДЫ, ПОЛУЧЕННОЙ ПРИ КУЛЬТИВИРОВАНИИ ГЛИАЛЬНЫХ КЛЕТОК-ПРЕДШЕСТВЕННИЦ, ПРИ ОСТРОМ ЭКСПЕРИМЕНТАЛЬНОМ ИШЕМИЧЕСКОМ ИНСУЛЬТЕ У КРЫС

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years and the results are promising. Recently, most researchers are inclined to assume that the therapeutic effect of stem cell therapy is based on the mechanism of paracrine action associated with the secretion wide set of regulatory proteins. The aim of this study was to evaluate therapeutic effects of iPSC-derived glial progenitor cells conditioned medium in the rat middle cerebral artery occlusion model of the ischemic stroke. We showed that intra-arterial administration of glial progenitor cells conditioned medium promoted faster decrease of neurological deficit compared to the control group. Moreover, expression of gap43, bax, and tnfa genes involved in neuritogenesis, apoptosis and neuroinflammation was altered. However, no significant enhanced reduction of the infarct volume was registered. Our results demonstrated that administration of glial progenitor cells conditioned medium induced functional recovery after experimental stroke and may affect brain plasticity. © 2020, Human Stem Cell Institute. All rights reserved.На сегодняшний день перспективным подходом к терапии ишемического инсульта является трансплантация различных типов стволовых клеток, результаты которой продемонстрировали свою эффективность. В последнее время большинство исследователей склоняются к предположению, что терапевтический эффект клеточной терапии в большей степени основан на механизме паракринного действия, связанного с секрецией клетками широкого набора регуляторных белков. Недавние исследования показали, что введение кондиционированных сред, полученных при культивировании стволовых клеток, может быть эффективным при терапии заболеваний центральной нервной системы. Целью настоящей работы является оценка терапевтических эффектов кондиционированной среды, содержащей продукты секреции глиальных клеток-предшественниц, полученных из индуцированных плюрипотентных стволовых клеток, на модели экспериментального ишемического инсульта у крыс. Было обнаружено, что внутриартериальное введение кондиционированной среды, полученной при культивировании глиальных клеток-предшественниц, способствовало более быстрому снижению степени неврологического дефицита по сравнению с контрольной группой. Кроме того, были выявлены изменения в экспрессии генов gap43, bax и tnfa. однако значимого уменьшения объема очага инфаркта зарегистрировано не было. Наши результаты показали, что введение кондиционированной среды, содержащей продукты секреции глиальных клеток-предшественниц, индуцирует функциональное восстановление и может повлиять на пластичность мозга после экспериментального инсульта