Oxygen-glucose deprivation in organotypic hippocampal cultures leads to cytoskeleton rearrangement and immune activation : link to the potential pathomechanism of ischaemic stroke

Ischaemic stroke is characterized by a sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. As a result of this process, neurons in the ischaemic core are deprived of oxygen and trophic substances and are consequently destroyed. Tissue damage in brain ischaemia results from a complex pathophysiological cascade comprising various distinct pathological events. Ischaemia leads to brain damage by stimulating many processes, such as excitotoxicity, oxidative stress, inflammation, acidotoxicity, and apoptosis. Nevertheless, less attention has been given to biophysical factors, including the organization of the cytoskeleton and the mechanical properties of cells. Therefore, in the present study, we sought to evaluate whether the oxygen-glucose deprivation (OGD) procedure, which is a commonly accepted experimental model of ischaemia, could affect cytoskeleton organization and the paracrine immune response. The abovementioned aspects were examined ex vivo in organotypic hippocampal cultures (OHCs) subjected to the OGD procedure. We measured cell death/viability, nitric oxide (NO) release, and hypoxia-inducible factor $1\alpha$ (HIF-$1\alpha$) levels. Next, the impact of the OGD procedure on cytoskeletal organization was evaluated using combined confocal fluorescence microscopy (CFM) and atomic force microscopy (AFM). Concurrently, to find whether there is a correlation between biophysical properties and the immune response, we examined the impact of OGD on the levels of crucial ischaemia cytokines (IL-$1\beta$, IL-6, IL-18, TNF-$\alpha$, IL-10, IL-4) and chemokines (CCL3, CCL5, CXCL10) in OHCs and calculated Pearsons’ and Spearman’s rank correlation coefficients. The results of the current study demonstrated that the OGD procedure intensified cell death and nitric oxide release and led to the potentiation of HIF-$1\beta$ release in OHCs. Moreover, we presented significant disturbances in the organization of the cytoskeleton (actin fibers, microtubular network) and cytoskeleton-associated protein 2 (MAP-2), which is a neuronal marker. Simultaneously, our study provided new evidence that the OGD procedure leads to the stiffening of OHCs and a malfunction in immune homeostasis. A negative linear correlation between tissue stiffness and branched IBA1 positive cells after the OGD procedure suggests the pro-inflammatory polarization of microglia. Moreover, the negative correlation of pro- and positive anti-inflammatory factors with actin fibers density indicates an opposing effect of the immune mediators on the rearrangement of cytoskeleton induced by OGD procedure in OHCs. Our study constitutes a basis for further research and provides a rationale for integrating biomechanical and biochemical methods in studying the pathomechanism of stroke-related brain damage. Furthermore, presented data pointed out the interesting direction of proof-of-concept studies, in which follow-up may establish new targets for brain ischemia therapy

Multilineage differentiation potential of human dental pulp stem cells : impact of 3D and hypoxic environment on osteogenesis in vitro

Human dental pulp harbours unique stem cell population exhibiting mesenchymal stem/stromal cell (MSC) characteristics. This study aimed to analyse the differentiation potential and other essential functional and morphological features of dental pulp stem cells (DPSCs) in comparison with Wharton’s jelly-derived MSCs from the umbilical cord (UC-MSCs), and to evaluate the osteogenic differentiation of DPSCs in 3D culture with a hypoxic microenvironment resembling the stem cell niche. Human DPSCs as well as UC-MSCs were isolated from primary human tissues and were subjected to a series of experiments. We established a multiantigenic profile of DPSCs with CD45−/CD14−/CD34−/CD29+/CD44+/CD73+/CD90+/CD105+/Stro-1+/HLA-DR− (using flow cytometry) and confirmed their tri-lineage osteogenic, chondrogenic, and adipogenic differentiation potential (using qRT-PCR and histochemical staining) in comparison with the UC-MSCs. The results also demonstrated the potency of DPSCs to differentiate into osteoblasts in vitro. Moreover, we showed that the DPSCs exhibit limited cardiomyogenic and endothelial differentiation potential. Decreased proliferation and metabolic activity as well as increased osteogenic differentiation of DPSCs in vitro, attributed to 3D cell encapsulation and low oxygen concentration, were also observed. DPSCs exhibiting elevated osteogenic potential may serve as potential candidates for a cell-based product for advanced therapy, particularly for bone repair. Novel tissue engineering approaches combining DPSCs, 3D biomaterial scaffolds, and other stimulating chemical factors may represent innovative strategies for pro-regenerative therapies

Comparison of phenotype and biological properties of human dental pulp stem cells and umbilical cord mesenchymal stem cells in vitro

Ludzkie komórki macierzyste miazgi zęba (ang. human dental pulp stem cells- hDPSCs) stanowią populację komórek rezydujących w komorach zębowych ssaków. Posiadają one pochodzenie ektodermalne, ponieważ wywodzą się z grzebienia nerwowego. Dzięki swojej zdolności do proliferacji, adhezji do plastiku, różnicowania w wiele linii komórkowych oraz charakterystycznego fenotypu, DPSCs zalicza się do mezenchymalnych komórek macierzystych (ang. mesenchymal stem cells - MSCs). Innym przykładem tego typu komórek są mezenchymalne komórki macierzyste sznura pepowinowego (ang. umbilical cord mesenchymal stem cells- UC-MSCs), które charakteryzują się wielokierunkową zdolnością do różnicownia, a także wyższą proliferacją w porównaniu do komórek ze szpiku kostnego. Zarówno hDPSCs jak i UC-MSCs stanowią alterantywne źródło komórek, które ze względu na łatwą dostępność oraz nieinwazyjną izolację mogą zostać potencjalnie wykorzystane w regeneracji tkanek. Dużą zaletą jest również brak zdolności do tworzenia potworniaków, czyli łagodnych guzów nowotworowych, co jest charakterystyczną cechą pluripotencjalnych komórek macierzystych oraz brak kontrowersji etycznych związanych z pochodzeniem komórek hDPSCs oraz UC-MSCs.Celem przeprowadzonych badań było porównanie fenotypu oraz właściwości biologicznych ludzkich komórek hDPSCs i UC-MSCs in vitro. Źródłem hDPSCs była .miazga izolowana z zębów ludzkich, a komórki pozyskane zostały na dwa sposoby: metodą trawienia enzymatycznego i metodą eksplantów. Natomiast źródłem UC-MSCs była pępowina, z której komórki wyizolowano metodą eksplantów. Dwa typy komórek poddano różnicowaniu w osteocyty, chondrocyty, adipocyty, kardiomiocyty oraz komórki endotelialne w celu porównania właściwości różnicowania. Przeprowadzono również analizę fenotypu wieloantygenowego z zastosowaniem cytometrii przepływowej oraz analizę ekspresji charakterystycznych dla danego różnicowania genów.Wyniki badań wykazały, że zarówno hDPSCs oraz UC-MSCs zróżnicowały w osteocyty, chondrocyty oraz adipocyty. Dodatkowo komórki UC-MSCs wykazywały potencjał do różnicowania w kardiomiocyty oraz komórki endotelialne. Jednak w przypadku hDPSCs zdolność do różnicowania zmieniała się w zależności od poszczególnych linii, co może świadczyć o dużej zmienności pomiędzy dawcami, co wymaga dalszych badań. Komórki hDPSCs stanowią alternatywne źródło mezenchymalnych komórek macierzystych w regeneracji tkanek, szczególnie tkanki chrzęstnej i kostnej.Human dental pulp stem cells (hDPSCs) represent the population of cells that reside in mammalian chambers of teeth. They have ectodermal origin, because they are orginating from neural crest. Thanks to their ability to proliferate, plastics adhesion, multi-cell differentiation, and characteristic phenotype, hDPSCs could be classified as mesenchymal stem cells (MSCs). Another example of this type of cells are umbilical-cord mesenchymal stem cells (UC-MSCs), which are characterized by high ability to differentiate into various lineages and also higher proliferation compared to bone marrow cells. Both hDPSCs and UC-MSCs are alternative sources of cells for tissue regeneration because of their accessibility and non-invasive isolation. The great advantage is also lack of capacity for teratoma formation, which is characteristic for pluripotent stem cells and the lack of ethical controversy associated with the origin of hDPSCs and UC-MSCs cells.The aim of the study was to compare the phenotype and biological properties of hDPSCs and UC-MSCs cells in vitro. The source of hDPSCs was a human dental pulp and cells were obtained in two ways: via enzymatic digestion and explants methods. On the other hand, the source of UC-MSCs was umbilical cord and the cells were isolated via explants method. These two types of cells were differentiated into osteocytes, chondrocytes, adipocytes, cardiomyocytes and endothelial cells to compare their differention capacity. Phenotypic analysis by flow cytometry and gene expression analysis was also performed for both types of cells.The results indicated that both hDPSCs and UC-MSCs may differentiate into osteocytes, chondrocytes and adipocytes. In addition, UC-MSCs cells exhibited the potential for differentiation into cardiomyocytes and endothelial cells. However, in case of hDPSCs, the ability to differentiate varies between cell lines, which may indicate high variability between donors that needs to be further investigated. hDPSCs are an alternative source of mesenchymal stem cells for tissue regeneration, especially cartilage and bone tissue

Application of stem cells in the treatment of Parkinson's Disease

Komórki macierzyste stanowią populację komórek, które mogą różnicować się w inne typy komórek oraz mają zdolność do proliferacji. Obecnie wiadomo, że można znaleźć je w każdej tkance. Ich potencjał do regeneracji jest zróżnicowany w zależności od tkanki, z której są izolowane. Istnieją cztery typy komórek macierzystych: totipotencjalne, które mają największy potencjał do różnicowania, pluripotencjalne, multipotencjalne i unipotencjalne posiadające najmniejszy potencjał do różnicowania. Również w mózgu znajdują się komórki macierzyste, które mogą różnicować się we wszystkie typy komórek ośrodkowego układu nerwowego. U ssaków występują one w dwóch niszach: strefie okołokomorowej komór bocznych (SVZ) i strefie podziarnistej zakrętu zębatego hipokampa (SGZ). Nisze te tworzą specjalne środowisko, które utrzymuje je w stanie multipotencji. Izolacja neuralnych komórek macierzystych z mózgu jest inwazyjną procedurą związaną z dużym ryzykiem dla pacjenta. Innym źródłem komórek o charakterze neuronalnym są embrionalne komórki macierzyste, które w odpowiednich warunkach mogą się różnicować w kierunku komórek układu nerwowego. Jednakże wykorzystanie ludzkich embrionalnych komórek macierzystych stwarza kontrowersje etyczne. Innym źródłem komórek pluripotencjalnych są indukowane pluripotencjalne komórki macierzyste (komórki iPS). Można je pozyskać z dorosłych komórek człowieka bez problemów natury etycznej. Istnieją dwa główne sposoby wykorzystania komórek iPS w leczeniu chorób neurodegeneracyjnych. Wykorzystanie ich jako modelu w badaniach in vitro, różnicując je w neurony oraz przeszczepianie zróżnicowanych komórek iPS do uszkodzonych obszarów układu nerwowego. Neuralne komórki macierzyste mogą być użyte w leczeniu chorób neurodegeneracyjnych np. choroby Parkinsona (PD). Jej przyczyną jest degeneracja neuronów dopaminergicznych istoty czarnej. Obecnie nie da się całkowicie wyleczyć PD, stąd poszukuje się skutecznej metody regeneracji uszkodzonych neuronów. Badania in vivo na szczurzym modelu choroby Parkinsona wykazały, że u szczurów, którym podano komórki macierzyste pojawiła się regresja choroby. Ludzkie komórki iPS mogą również być różnicowane w neurony dopaminergiczne jako model in vitro do badań nad chorobą Parkinsona. Pokazuje to, że komórki macierzyste stanowią obiecujące narzędzie w leczeniu choroby Parkinsona.Stem cells are population of cells which can differentiate into other cell types and have capability to proliferation. Stem cells can be found in every tissue in the organism. In various organs, potential of stem cells to regenerate them is different. There are four types of stem cells: totipotent, which have the most potential to differentiate, pluripotent, multipotent and unipotent, which have the least potential to differentiate. Also in a brain there are stem cells which can differentiate into all types of central nervous system cells. They are located in two main niches: subventricular zone (SVZ) and subgranular zone (SGZ). These niches create special micro enviroment which maintains them at the multipotent stage.Obtaining neural stem cells from a brain is impossible without damaging it. Therefore, scientists are reprograming stem cells with higher potential for differentiation into neurons. Pluripotent embryonic stem cells can be used in this procedure. However, using human embryonic stem cells is ethically controversion. Another source of pluripotential cells are induced pluripotent stem cells (iPS-cells) which are obtained from adult tissues without ethical issues. There are two main ways of using iPS-cells in neurodegenerative disease research: using them as a model in vitro, differentiating them into neurons and grafting into degenerated areas of nervous system.Neural stem cell can be used in neurodegenerative disease treatment for instance in Parkinson disease (PD), in which degeneration of dopaminergic neurons occurs in midbrain. Nowadays PD is incurable, so scientists are looking for a method in which neurons can be renewed. The research into rodents indicated that rats which suffered from Parkinson disease showed the regression of this disease after an injection of neural stem cells. Independent researcher groups examined dopaminergic neurons from iPS-cells in vitro to model molecular mechanisms of PD. These results indicated that using stem cells in Parkinson disease treatment is very promising

Mechanobiology of Dental Pulp Cells

The dental pulp is the inner part of the tooth responsible for properly functioning during its lifespan. Apart from the very big biological heterogeneity of dental cells, tooth microenvironments differ a lot in the context of mechanical properties—ranging from 5.5 kPa for dental pulp to around 100 GPa for dentin and enamel. This physical heterogeneity and complexity plays a key role in tooth physiology and in turn, is a great target for a variety of therapeutic approaches. First of all, physical mechanisms are crucial for the pain propagation process from the tooth surface to the nerves inside the dental pulp. On the other hand, the modulation of the physical environment affects the functioning of dental pulp cells and thus is important for regenerative medicine. In the present review, we describe the physiological significance of biomechanical processes in the physiology and pathology of dental pulp. Moreover, we couple those phenomena with recent advances in the fields of bioengineering and pharmacology aiming to control the functioning of dental pulp cells, reduce pain, and enhance the differentiation of dental cells into desired lineages. The reviewed literature shows great progress in the topic of bioengineering of dental pulp—although mainly in vitro. Apart from a few positions, it leaves a gap for necessary filling with studies providing the mechanisms of the mechanical control of dental pulp functioning in vivo

Role of Polyinosinic:Polycytidylic Acid-Induced Maternal Immune Activation and Subsequent Immune Challenge in the Behaviour and Microglial Cell Trajectory in Adult Offspring: A Study of the Neurodevelopmental Model of Schizophrenia

Multiple lines of evidence support the pathogenic role of maternal immune activation (MIA) in the occurrence of the schizophrenia-like disturbances in offspring. While in the brain the homeostatic role of neuron-microglia protein systems is well documented, the participation of the CX3CL1-CX3CR1 and CD200-CD200R dyads in the adverse impact of MIA often goes under-recognized. Therefore, in the present study, we examined the effect of MIA induced by polyinosinic:polycytidylic acid (Poly I:C) on the CX3CL1-CX3CR1 and CD200-CD200R axes, microglial trajectory (MhcII, Cd40, iNos, Il-1β, Tnf-α, Il-6, Arg1, Igf-1, Tgf-β and Il-4), and schizophrenia-like behaviour in adult male offspring of Sprague-Dawley rats. Additionally, according to the “two-hit” hypothesis of schizophrenia, we evaluated the influence of acute challenge with Poly I:C in adult prenatally MIA-exposed animals on the above parameters. In the present study, MIA evoked by Poly I:C injection in the late period of gestation led to the appearance of schizophrenia-like disturbances in adult offspring. Our results revealed the deficits manifested as a diminished number of aggressive interactions, presence of depressive-like episodes, and increase of exploratory activity, as well as a dichotomy in the sensorimotor gating in the prepulse inhibition (PPI) test expressed as two behavioural phenotypes (MIAPPI-low and MIAPPI-high). Furthermore, in the offspring rats subjected to a prenatal challenge (i.e., MIA) we noticed the lack of modulation of behavioural changes after the additional acute immune stimulus (Poly I:C) in adulthood. The important finding reported in this article is that MIA affects the expression and levels of the neuron-microglia proteins in the frontal cortex and hippocampus of adult offspring. We found that the changes in the CX3CL1-CX3CR1 axis could affect microglial trajectory, including decreased hippocampal mRNA level of MhcII and elevated cortical expression of Igf-1 in the MIAPPI-high animals and/or could cause the up-regulation of an inflammatory response (Il-6, Tnf-α, iNos) after the “second hit” in both examined brain regions and, at least in part, might differentiate behavioural disturbances in adult offspring. Consequently, the future effort to identify the biological background of these interactions in the Poly I:C-induced MIA model in Sprague-Dawley rats is desirable to unequivocally clarify this issue

Role of Polyinosinic:Polycytidylic Acid-Induced Maternal Immune Activation and Subsequent Immune Challenge in the Behaviour and Microglial Cell Trajectory in Adult Offspring: A Study of the Neurodevelopmental Model of Schizophrenia

Multiple lines of evidence support the pathogenic role of maternal immune activation (MIA) in the occurrence of the schizophrenia-like disturbances in offspring. While in the brain the homeostatic role of neuron-microglia protein systems is well documented, the participation of the CX3CL1-CX3CR1 and CD200-CD200R dyads in the adverse impact of MIA often goes under-recognized. Therefore, in the present study, we examined the effect of MIA induced by polyinosinic:polycytidylic acid (Poly I:C) on the CX3CL1-CX3CR1 and CD200-CD200R axes, microglial trajectory (MhcII, Cd40, iNos, Il-1β, Tnf-α, Il-6, Arg1, Igf-1, Tgf-β and Il-4), and schizophrenia-like behaviour in adult male offspring of Sprague-Dawley rats. Additionally, according to the “two-hit” hypothesis of schizophrenia, we evaluated the influence of acute challenge with Poly I:C in adult prenatally MIA-exposed animals on the above parameters. In the present study, MIA evoked by Poly I:C injection in the late period of gestation led to the appearance of schizophrenia-like disturbances in adult offspring. Our results revealed the deficits manifested as a diminished number of aggressive interactions, presence of depressive-like episodes, and increase of exploratory activity, as well as a dichotomy in the sensorimotor gating in the prepulse inhibition (PPI) test expressed as two behavioural phenotypes (MIAPPI-low and MIAPPI-high). Furthermore, in the offspring rats subjected to a prenatal challenge (i.e., MIA) we noticed the lack of modulation of behavioural changes after the additional acute immune stimulus (Poly I:C) in adulthood. The important finding reported in this article is that MIA affects the expression and levels of the neuron-microglia proteins in the frontal cortex and hippocampus of adult offspring. We found that the changes in the CX3CL1-CX3CR1 axis could affect microglial trajectory, including decreased hippocampal mRNA level of MhcII and elevated cortical expression of Igf-1 in the MIAPPI-high animals and/or could cause the up-regulation of an inflammatory response (Il-6, Tnf-α, iNos) after the “second hit” in both examined brain regions and, at least in part, might differentiate behavioural disturbances in adult offspring. Consequently, the future effort to identify the biological background of these interactions in the Poly I:C-induced MIA model in Sprague-Dawley rats is desirable to unequivocally clarify this issue

Time-Dependent Protective and Pro-Resolving Effects of FPR2 Agonists on Lipopolysaccharide-Exposed Microglia Cells Involve Inhibition of NF-κB and MAPKs Pathways

Prolonged or excessive microglial activation may lead to disturbances in the resolution of inflammation (RoI). The importance of specialized pro-resolving lipid mediators (SPMs) in RoI has been highlighted. Among them, lipoxins (LXA4) and aspirin-triggered lipoxin A4 (AT-LXA4) mediate beneficial responses through the activation of N-formyl peptide receptor-2 (FPR2). We aimed to shed more light on the time-dependent protective and anti-inflammatory impact of the endogenous SPMs, LXA4, and AT-LXA4, and of a new synthetic FPR2 agonist MR-39, in lipopolysaccharide (LPS)-exposed rat microglial cells. Our results showed that LXA4, AT-LXA4, and MR-39 exhibit a protective and pro-resolving potential in LPS-stimulated microglia, even if marked differences were apparent regarding the time dependency and efficacy of inhibiting particular biomarkers. The LXA4 action was found mainly after 3 h of LPS stimulation, and the AT-LXA4 effect was varied in time, while MR-39′s effect was mainly observed after 24 h of stimulation by endotoxin. MR-39 was the only FPR2 ligand that attenuated LPS-evoked changes in the mitochondrial membrane potential and diminished the ROS and NO release. Moreover, the LPS-induced alterations in the microglial phenotype were modulated by LXA4, AT-LXA4, and MR-39. The anti-inflammatory effect of MR-39 on the IL-1β release was mediated through FPR2. All tested ligands inhibited TNF-α production, while AT-LXA4 and MR-39 also diminished IL-6 levels in LPS-stimulated microglia. The favorable action of LXA4 and MR-39 was mediated through the inhibition of ERK1/2 phosphorylation. AT-LXA4 and MR39 diminished the phosphorylation of the transcription factor NF-κB, while AT-LXA4 also affected p38 kinase phosphorylation. Our results suggest that new pro-resolving synthetic mediators can represent an attractive treatment option for the enhancement of RoI, and that FPR2 can provide a perspective as a target in immune-related brain disorders