Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult-to-heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non-healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long-term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre-defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non-invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Evaluation of the Optimal Manufacturing Protocols and Therapeutic Properties of Mesenchymal Stem/Stromal Cells Derived from Wharton's Jelly.

Wharton's jelly (WJ) from the umbilical cord (UC) is a good source of mesenchymal stem/stromal cells (MSCs), which can be isolated and used in therapy. Current knowledge shows that even small changes in the cell environment may result in obtaining a subpopulation of cells with different therapeutic properties. For this reason, the conditions of UC transportation, cell isolation, and cultivation and the banking of cells destined for clinical use should be unified and optimized. In this project, we tried various protocols for cell vs. bioptat isolation, banking, and transport in order to determine the most optimal. The most efficient isolation method of WJ-MSCs was chopping the whole umbilical matrix with a scalpel after vessel and lining membrane removal. The optimal solution for short term cell transportation was a multi-electrolyte fluid without glucose. Considering the use of WJ-MSCs in cell therapies, it was important to investigate the soluble secretome of both WJ bioptats and WJ-MSCs. WJ-MSCs secreted higher levels of cytokines and chemokines than WJ bioptats. WJ-MSCs secreted HGF, CCL2, ICAM-1, BDNF, and VEGF. Since these cells might be used in treating neurodegenerative disorders, we investigated the impact of cerebrospinal fluid (CSF) on WJ-MSCs' features. In the presence of CSF, the cells expressed consecutive neural markers both at the protein and gene level: nestin, ß-III-tubulin, S-100-ß, GFAP, and doublecortin. Based on the obtained results, a protocol for manufacturing an advanced-therapy medicinal product was composed.

Ischemia/Reperfusion-Induced Translocation of PKCßII to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus.

Emerging reports indicate that activated PKC isoforms that translocate to the mitochondria are pro- or anti-apoptotic to mitochondrial function. Here, we concentrate on the role of PKCß translocated to mitochondria in relation to the fate of neurons following cerebral ischemia. As we have demonstrated previously ischemia/reperfusion injury (I/R) results in translocation of PKCß from cytoplasm to mitochondria, but only in ischemia-resistant regions of the hippocampus (CA2-4, DG), we hypothesize that this translocation may be a mediator of a protective signaling mechanism in this region. We have therefore sought to demonstrate a possible relationship between PKCßII translocation and ischemic resistance of CA2-4, DG. Here, we reveal that I/R injury induces a marked elevation of PKCßII protein levels, and consequent enzymatic activity, in CA2-4, DG in the mitochondrial fraction. Moreover, the administration of an isozyme-selective PKCßII inhibitor showed inhibition of I/R-induced translocation of PKCßII to the mitochondria and an increase in neuronal death following I/R injury in CA1 and CA2-4, DG in both an in vivo and an in vitro model of ischemia. The present results suggest that PKCßII translocated to mitochondria is involved in providing ischemic resistance of CA2-4, DG. However, the exact mechanisms by which PKCßII-mediated neuroprotection is achieved are in need of further elucidation.

Enhanced neuro-therapeutic potential of Wharton's Jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture.

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency.

Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner.

BACKGROUND AIMS: As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly-derived MSCs (WJ-MSCs). METHODS: Primary cultures growing in 21% oxygen were either transferred into 5% O2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α-3α were evaluated. RESULTS: Lowering O2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. CONCLUSIONS: A physiologically relevant microenvironment of 5% O2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α-mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

Stemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.

Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

[Bioengineering of neural stem cell niche]. / Bioinzynieria niszy neuralnych komórek macierzystych.

Maintenance of developmental and regenerative capability of the tissue highly depends upon mutual interaction of the stem cells with the components of their microenvironment (niche). The nature of this interaction is determined by the biochemical and biophysical properties of the niche constituencies. Although knowledge about the components of the stem cell microenvironment and their architecture is growing quickly, we still need to unravel the mechanisms underlying the control of the niche functioning, enabling stem cells differentiation and homeostasis of the tissue. Advancement in biotechnology provides tools to build up in vitro "biomimetic" microenvironments resembling a natural stem cell niche, where the cell is provided with diverse extracellular signals exerted by soluble and structural cues, mimicking those found in vivo. To obtain such microenvironment in vitro emerging nano/biotechnology methods were applied, using biomaterials of new generation, which enable controlling of the stem cell differentiation by time and special related release of the active factors. This article is providing an overview of the new research strategies for the bioengineering of the stem cell niche and gives the examples of the cell/biomaterial 2D and 3D complex systems used for basic and preclinical research as well as entering clinical applications for the therapy of the nervous system.

Understanding Intra- and Inter-Species Variability in Neural Stem Cells' Biology Is Key to Their Successful Cryopreservation, Culture, and Propagation.

Although clinical trials on human neural stem cells (hNSCs) have already been implemented in the treatment of neurological diseases and they have demonstrated their therapeutic effects, many questions remain in the field of preclinical research regarding the biology of these cells, their therapeutic properties, and their neurorestorative potential. Unfortunately, scientific reports are inconsistent and much of the NSCs research has been conducted on rodents rather than human cells for ethical reasons or due to insufficient cell material. Therefore, a question arises as to whether or which conclusions drawn on the isolation, cell survival, proliferation, or cell fate observed in vitro in rodent NSCs can be introduced into clinical applications. This paper presents the effects of different spatial, nutritional, and dissociation conditions on NSCs' functional properties, which are highly species-dependent. Our study confirmed that the discrepancies in the available literature on NSCs survival, proliferation, and fate did not only depend on intra-species factors and applied environmental conditions, but they were also affected by significant inter-species variability. Human and rodent NSCs share one feature, i.e., the necessity to be cultured immediately after isolation, which significantly maintains their survival. Additionally, in the absence of experiments on human cells, rat NSCs biology (neurosphere formation potential and neural differentiation stage) seems closer to that of humans rather than mice in response to environmental factors.

Critical points for optimizing long-term culture and neural differentiation capacity of rodent and human neural stem cells to facilitate translation into clinical settings.

Despite several decades of research on the nature and functional properties of neural stem cells, which brought great advances in regenerative medicine, there is still a plethora of ambiguous protocols and interpretations linked to their applications. Here, we present a whole spectrum of protocol elements that should be standardized in order to obtain viable cell cultures and facilitate their translation into clinical settings. Additionally, this review also presents outstanding limitations and possible problems to be encountered when dealing with protocol optimization. Most importantly, we also outline the critical points that should be considered before starting any experiments utilizing neural stem cells or interpreting their results.

Promising Markers in the Context of Mesenchymal Stem/Stromal Cells Subpopulations with Unique Properties.

The heterogeneity of the mesenchymal stem/stromal cells (MSCs) population poses a challenge to researchers and clinicians, especially those observed at the population level. What is more, the lack of precise evidences regarding MSCs developmental origin even further complicate this issue. As the available evidences indicate several possible pathways of MSCs formation, this diverse origin may be reflected in the unique subsets of cells found within the MSCs population. Such populations differ in specialization degree, proliferation, and immunomodulatory properties or exhibit other additional properties such as increased angiogenesis capacity. In this review article, we attempted to identify such outstanding populations according to the specific surface antigens or intracellular markers. Described groups were characterized depending on their specialization and potential therapeutic application. The reports presented here cover a wide variety of properties found in the recent literature, which is quite scarce for many candidates mentioned in this article. Even though the collected information would allow for better targeting of specific subpopulations in regenerative medicine to increase the effectiveness of MSC-based therapies.

The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue.

INTRODUCTION: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. RESULTS: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. CONCLUSION: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

Deciphering the impact of cerebrospinal fluid on stem cell fate as a new mechanism to enhance clinical therapy development.

Neural stem cells (NSCs) hold a very significant promise as candidates for cell therapy due to their robust neuroprotective and regenerative properties. Preclinical studies using NSCs have shown enough encouraging results to perform deeper investigations into more potential clinical applications. Nevertheless, our knowledge regarding neurogenesis and its underlying mechanisms remains incomplete. To understand them better, it seems necessary to characterize all components of neural stem cell niche and discover their role in physiology and pathology. Using NSCs in vivo brings challenges including limited cell survival and still inadequate integration within host tissue. Identifying overlooked factors that might influence these outcomes becomes pivotal. In this review, we take a deeper examination of the influence of a fundamental element that is present in the brain, the cerebrospinal fluid (CSF), which still remains relatively unexplored. Its role in neurogenesis could be instrumental to help find novel therapeutic solutions for neurological disorders, eventually advancing our knowledge on central nervous system (CNS) regeneration and repair.

Advances in Neurorestoratology-Current status and future developments.

Neurorestoratology constitutes a novel discipline aimed at the restoration of damaged neural structures and impaired neurological functions. This area of knowledge integrates and compiles all concepts and strategies dealing with the neurorestoration. Although currently, this discipline has already been well recognized by physicians and scientists throughout the world, this article aimed at broadening its knowledge to the academic circle and the public society. Here we shortly introduced why and how Neurorestoratology was born since the fact that the central nervous system (CNS) can be repaired and the subsequent scientific evidence of the neurorestorative mechanisms behind, such as neurostimulation or neuromodulation, neuroprotection, neuroplasticity, neurogenesis, neuroregeneration or axonal regeneration or sprouting, neuroreplacement, loop reconstruction, remyelination, immunoregulation, angiogenesis or revascularization, and others. The scope of this discipline is the improvement of therapeutic approaches for neurological diseases and the development of neurorestorative strategies through the comprehensive efforts of experts in the different areas and all articulated by the associations of Neurorestoratology and its journals. Strikingly, this article additionally explores the "state of art" of the Neurorestoratology field. This includes the development process of the discipline, the achievements and advances of novel neurorestorative treatments, the most efficient procedures exploring and evaluating outcome after the application of pioneer therapies, all the joining of a multidisciplinary expert associations and the specialized journals being more and more impact. We believe that in a near future, this discipline will evolve fast, leading to a general application of cell-based comprehensive neurorestorative treatments to fulfill functional recovery demands for patients with neurological deficits or dysfunctions.

Critical factors responsible for the therapeutic effect of mesenchymal stem/stromal cells in central nervous system disorders.

Nowadays it is observed that the number of stem-cell based experimental therapies in neurodegenerative disorders is massively increasing. Most of the clinical trials registered to date have been based on autologous mesenchymal stem/stromal cells (MSC) obtained from somatic tissues. In the conducted clinical trials neither serious side effects, nor statistically significant improvement were observed. The lack of statistical significance could result from a relatively small number of patients involved in clinical trials or highly incoherent study protocols. However, most clinical groups describe a trend towards improvement in MSC-treated patients. Hence, the question arises which factors associated with MSC-based therapy may be the key and result in better therapeutic response. In the presented paper, we summarize, in our opinion, the most important factors that could increase the effectiveness of this therapy.

Interaction of Neural Stem Cells (NSCs) and Mesenchymal Stem Cells (MSCs) as a Promising Approach in Brain Study and Nerve Regeneration.

Rapid developments in stem cell research in recent years have provided a solid foundation for their use in medicine. Over the last few years, hundreds of clinical trials have been initiated in a wide panel of indications. Disorders and injuries of the nervous system still remain a challenge for the regenerative medicine. Neural stem cells (NSCs) are the optimal cells for the central nervous system restoration as they can differentiate into mature cells and, most importantly, functional neurons and glial cells. However, their application is limited by multiple factors such as difficult access to source material, limited cells number, problematic, long and expensive cultivation in vitro, and ethical considerations. On the other hand, according to the available clinical databases, most of the registered clinical trials involving cell therapies were carried out with the use of mesenchymal stem/stromal/signalling cells (MSCs) obtained from afterbirth or adult human somatic tissues. MSCs are the multipotent cells which can also differentiate into neuron-like and glia-like cells under proper conditions in vitro; however, their main therapeutic effect is more associated with secretory and supportive properties. MSCs, as a natural component of cell niche, affect the environment through immunomodulation as well as through the secretion of the trophic factors. In this review, we discuss various therapeutic strategies and activated mechanisms related to bilateral MSC-NSC interactions, differentiation of MSCs towards the neural cells (subpopulation of crest-derived cells) under the environmental conditions, bioscaffolds, or co-culture with NSCs by recreating the conditions of the neural cell niche.

Effect of Long-Term 3D Spheroid Culture on WJ-MSC.

The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.

Biochemical aspirin resistance in acute stroke patients and its association with clinical factors: a prospective pilot study.

INTRODUCTION: Aspirin is still widely used in treatment and prevention of cardiovascular diseases. To predict which patients cannot benefit from aspirin due to aspirin resistance remains a great clinical challenge. MATERIAL AND METHODS: Fifty one acute stroke/transient ischemic attack (TIA) patients (ASG) with a history of regular aspirin intake for the previous 7 days or more were included to the study within 24 hours of symptoms onset. Twenty nine patients admitted to our department for other reasons were the controls (CG). Each patient underwent routine blood tests (white blood cells, platelets, total cholesterol, C-reactive protein) and additional blood test: glycated haemoglobin (HbA1c), insulin, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP). Biochemical aspirin resistance was measured using the VerifyNow Aspirin platelet function analyzer. RESULTS: There were 9 aspirin resistance patients in ASG (17.5%) and 3 in CG (10.3%) (p = 0.38). There were no differences in either age or gender between those groups. Twelve aspirin-resistant patients differed from aspirin nonresistant patients in age, NT-proBNP and total cholesterol levels (univariate model, p = 0.004, 0.04, 0.02, respectively). In a multivariate model patients aged 76 years and more would likely to be aspirin resistant with odds ratio = 9 (95% confidence interval: 1-78). CONCLUSIONS: Patients aged 76 and more can be more likely aspirin resistant than younger patients. We believe that especially in the elderly with congestive heart failure there is a strong need for further investigations in this field, including searching for alternative antiplatelet therapies.

The Effect of Proinflammatory Cytokines on the Proliferation, Migration and Secretory Activity of Mesenchymal Stem/Stromal Cells (WJ-MSCs) under 5% O2 and 21% O2 Culture Conditions.

Treatment with Mesenchymal Stem/Stromal Cells (MSCs) in clinical trials is becoming one of the most-popular and fast-developing branches of modern regenerative medicine, as it is still in an experimental phase. The cross-section of diseases to which these cells are applied is very wide, ranging from degenerative diseases, through autoimmune processes and to acute inflammatory diseases, e.g., viral infections. Indeed, now that first clinical trials applying MSCs against COVID-19 have started, important questions concern not only the therapeutic properties of MSCs, but also the changes that might occur in the cell features as a response to the "cytokine storm" present in the acute phase of an infection and capable of posing a risk to a patient. The aim of our study was thus to assess changes potentially occurring in the biology of MSCs in the active inflammatory environment, e.g., in regards to the cell cycle, cell migration and secretory capacity. The study using MSCs derived from Wharton's jelly (WJ-MSCs) was conducted under two aerobic conditions: 21% O2 vs. 5% O2, since oxygen concentration is one of the key factors in inflammation. Under both oxygen conditions cells were exposed to proinflammatory cytokines involved significantly in acute inflammation, i.e., IFNγ, TNFα and IL-1ß at different concentrations. Regardless of the aerobic conditions, WJ-MSCs in the inflammatory environment do not lose features typical for mesenchymal cells, and their proliferation dynamic remains unchanged. Sudden fluctuations in proliferation, the early indicator of potential genetic disturbance, were not observed, while the cells' migration activity increased. The presence of pro-inflammatory factors was also found to increase the secretion of such anti-inflammatory cytokines as IL-4 and IL-10. It is concluded that the inflammatory milieu in vitro does not cause phenotype changes or give rise to proliferation disruption of WJ-MSCs, and nor does it inhibit the secretory properties providing for their use against acute inflammation.

Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue.

Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells' neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O2). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen-glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders.