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.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Secondary release of the peripheral nerve with autologous fat derivates benefits for functional and sensory recovery.

The reconstruction of nerve continuity after traumatic nerve injury is the gold standard in hand surgery. Immediate, tension-free, end-to-end nerve suture ensures the best prognosis. The recovery is mostly promising; however, in a few cases, insufficient outcomes in motor or sensory function are observed. Intra- and extra-fascicular scarring accompanies the nerve regeneration process and limits final outcomes. Secondary nerve release in those cases is recommended. Unfortunately, scarring recurrence cannot be eliminated after secondary revision and neurolysis. The supportive influences of mesenchymal stem cells in the process of nerve regeneration were observed in many preclinical studies. However, a limited number of studies in humans have analyzed the clinical usage of mesenchymal stem cells in peripheral nerve reconstruction and revisions. The objective of this study was to evaluate the effects of undifferentiated adipose-derived stromal/stem cell injection during a last-chance surgery (neurolysis, nerve release) on a previously reconstructed nerve. Three patients (one female, two males; mean age 59 ± 4.5 years at the time of injury), who experienced failure of reconstructions of median and ulnar nerves, were included in this study. During the revision surgery, nerve fascicles were released, and adipose-derived stromal/stem cells were administered through microinjections along the fascicles and around the adjacent tissues after external neurolysis. During 36 months of follow-up, patients noticed gradual signs of sensory and in consequence functional recovery. No adverse effects were observed. Simultaneous nerve release with adipose-derived stromal/stem cells support is a promising method in patients who need secondary nerve release after nerve reconstruction. This method can constitute an alternative procedure in patients experiencing recovery failure and allow improvement in cases of limited nerve regeneration. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016.

Biomimetic microenvironmental preconditioning enhance neuroprotective properties of human mesenchymal stem cells derived from Wharton's Jelly (WJ-MSCs).

Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O2). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O2 conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

Bone Defect Repair Using a Bone Substitute Supported by Mesenchymal Stem Cells Derived from the Umbilical Cord.

OBJECTIVE: Bone defects or atrophy may arise as a consequence of injury, inflammation of various etiologies, and neoplastic or traumatic processes or as a result of surgical procedures. Sometimes the regeneration process of bone loss is impaired, significantly slowed down, or does not occur, e.g., in congenital defects. For the bone defect reconstruction, a piece of the removed bone from ala of ilium or bone transplantation from a decedent is used. Replacement of the autologous or allogenic source of the bone-by-bone substitute could reduce the number of surgeries and time in the pharmacological coma during the reconstruction of the bone defect. Application of mesenchymal stem cells in the reconstruction surgery may have positive influence on tissue regeneration by secretion of angiogenic factors, recruitment of other MSCs, or differentiation into osteoblasts. Materials and Methods. Mesenchymal stem cells derived from the umbilical cord (Wharton's jelly (WJ-MSC)) were cultured in GMP-grade DMEM low glucose supplemented with heparin, 10% platelet lysate, glucose, and antibiotics. In vitro WJ-MSCs were seeded on the bone substitute Bio-Oss Collagen® and cultured in the StemPro® Osteogenesis Differentiation Kit. During the culture on the 1st, 7th, 14th, and 21st day (day in vitro (DIV)), we analyzed viability (confocal microscopy) and adhesion capability (electron microscopy) of WJ-MSC on Bio-Oss scaffolds, gene expression (qPCR), and secretion of proteins (Luminex). In vivo Bio-Oss® scaffolds with WJ-MSC were transplanted to trepanation holes in the cranium to obtain their overgrowth. The computed tomography was performed 7, 14, and 21 days after surgery to assess the regeneration. RESULTS: The Bio-Oss® scaffold provides a favourable environment for WJ-MSC survival. WJ-MSCs in osteodifferentiation medium are able to attach and proliferate on Bio-Oss® scaffolds. Results obtained from qPCR and Luminex® indicate that WJ-MSCs possess the ability to differentiate into osteoblast-like cells and may induce osteoclastogenesis, angiogenesis, and mobilization of host MSCs. In animal studies, WJ-MSCs seeded on Bio-Oss® increased the scaffold integration with host bone and changed their morphology to osteoblast-like cells. CONCLUSIONS: The presented construct consisted of Bio-Oss®, the scaffold with high flexibility and plasticity, approved for clinical use with seeded immunologically privileged WJ-MSC which may be considered reconstructive therapy in bone defects.

Intrathecal Infusion of Autologous Adipose-Derived Regenerative Cells in Autoimmune Refractory Epilepsy: Evaluation of Safety and Efficacy.

Objective/Purpose. Evaluation of efficacy and safety of autologous adipose-derived regenerative cells (ADRCs) treatment in autoimmune refractory epilepsy. Patients. Six patients with proven or probable autoimmune refractory epilepsy (2 with Rasmussen encephalitis, 2 with antineuronal autoantibodies in serum, and 2 with possible FIRES) were included in the project with approval of the Bioethics Committee. METHOD: Intrathecal injection of autologous ADRC acquired through liposuction followed by enzymatic isolation was performed. The procedure was repeated 3 times every 3 months with each patient. Neurological status, brain MRI, cognitive function, and antiepileptic effect were monitored during 12 months. RESULTS: Immediately after the procedure, all patients were in good condition. In some cases, transient mildly elevated body temperature, pain in regions of liposuction, and slight increasing number of seizures during 24 hours were observed. During the next months, some improvements in school, social functioning, and manual performance were observed in all patients. One patient has been seizure free up to the end of trial. In other patients, frequency of seizures was different: from reduced number to the lack of improvement (3-year follow-up). CONCLUSION: Autologous ADRC therapy may emerge as a promising option for some patients with autoimmune refractory epilepsy. Based on our trial and other clinical data, the therapy appears to be safe and feasible. Antiepileptic efficacy proved to be various; however, some abilities improved in all children. No signs of psychomotor regression were observed during the first year following the treatment.

Human Somatic Stem Cell Neural Differentiation Potential.

Human somatic stem cells can be identified and isolated from different types of tissues and are grouped here based on their developmental maturation and ability to undergo neural differentiation. The first group will represent afterbirth somatic tissues, which are perinatal stem cells including placental blood and tissue, amniotic fluid and tissue, and umbilical cord blood- and umbilical cord tissue-derived cells. The second group of cells discussed in this chapter is the adult stem cells, generally those in a transient period of development, thus placing them in the special position of transitioning from the perinatal to young somatic tissue, and they include the menstrual blood-, the peripheral blood-, and the bone marrow-derived stem cells.

Intraspinal Transplantation of the Adipose Tissue-Derived Regenerative Cells in Amyotrophic Lateral Sclerosis in Accordance with the Current Experts' Recommendations: Choosing Optimal Monitoring Tools.

Stem cells (SCs) may constitute a perspective alternative to pharmacological treatment in neurodegenerative diseases. Although the safety of SC transplantation has been widely shown, their clinical efficiency in amyotrophic lateral sclerosis (ALS) is still to be proved. It is not only due to a limited number of studies, small treatment groups, and fast but nonlinear disease progression but also due to lack of objective methods able to show subtle clinical changes. Preliminary guidelines for cell therapy have recently been proposed by a group of ALS experts. They combine clinical, neurophysiological, and functional assessment together with monitoring of the cytokine level. Here, we describe a pilot study on transplantation of autologous adipose-derived regenerative cells (ADRC) into the spinal cord of the patients with ALS and monitoring of the results in accordance with the current recommendations. To show early and/or subtle changes within the muscles of interest, a wide range of clinical and functional tests were used and compared in order to choose the most sensitive and optimal set. Additionally, an analysis of transplanted ADRC was provided to develop standards ensuring the derivation and verification of adequate quality of transplanted cells and to correlate ADRC properties with clinical outcome.

Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017).

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version "Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)". The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility.

Neuroprotective Potential and Paracrine Activity of Stromal Vs. Culture-Expanded hMSC Derived from Wharton Jelly under Co-Cultured with Hippocampal Organotypic Slices.

Regardless of enormous translational progress in stem cell clinical application, our knowledge about biological determinants of transplantation-related protection is still limited. In addition to adequate selection of the cell source well dedicated to a specific disease and optimal standardization of all other pre-transplant procedures, we have decided to focus more attention to the impact of culture time and environment itself on molecular properties and regenerative capacity of cell cultured in vitro. The aim of this investigation was to determine neuroprotection-linked cell phenotypic and functional changes that could spontaneously take place when freshly isolated Wharton's jelly mesenchymal stem cell (WJ-MSC) undergo standard selection, growth, and spontaneous differentiation throughout passaging in vitro. For determining their neuroprotective potential, we used experimental model of human WJ-MSC co-culture with intact or oxygen-glucose-deprived (OGD) rat organotypic hippocampal culture (OHC). It has been shown that putative molecular mechanisms mediating regenerative interactions between WJ-MSC and OHC slices relies mainly on mesenchymal cell paracrine activity. Interestingly, it has been also found that the strongest protective effect is exerted by the co-culture with freshly isolated umbilical cord tissue fragments and by the first cohort of human mesenchymal stem cells (hMSCs) migrating out of these fragments (passage 0). Culturing of WJ-derived hMSC in well-controlled standard conditions under air atmosphere up to fourth passage caused unexpected decline of neuroprotective cell effectiveness toward OGD-OHC in the co-culture model. This further correlated with substantial changes in the WJ-MSC phenotype, profile of their paracrine activities as well as with the recipient tissue reaction evaluated by changes in the rat-specific neuroprotection-linked gene expression.

Phenotypic, Functional, and Safety Control at Preimplantation Phase of MSC-Based Therapy.

Mesenchymal stem cells (MSC) exhibit enormous heterogeneity which can modify their regenerative properties and therefore influence therapeutic effectiveness as well as safety of these cells transplantation. In addition the high phenotypic plasticity of MSC population makes it enormously sensitive to any changes in environmental properties including fluctuation in oxygen concentration. We have shown here that lowering oxygen level far below air atmosphere has a beneficial impact on various parameters characteristic for umbilical cord Wharton Jelly- (WJ-) MSC and adipose tissue- (AD-) derived MSC cultures. This includes their cellular composition, rate of proliferation, and maintenance of stemness properties together with commitment to cell differentiation toward mesodermal and neural lineages. In addition, the culture genomic stability increased significantly during long-term cell passaging and eventually protected cells against spontaneous transformation. Also by comparing of two routinely used methods of MSCs isolation (mechanical versus enzymatic) we have found substantial divergence arising between cell culture properties increasing along the time of cultivation in vitro. Thus, in this paper we highlight the urgent necessity to develop the more sensitive and selective methods for prediction and control cells fate and functioning during the time of growth in vitro.

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.

Induction of Endothelial Phenotype From Wharton's Jelly-Derived MSCs and Comparison of Their Vasoprotective and Neuroprotective Potential With Primary WJ-MSCs in CA1 Hippocampal Region Ex Vivo.

Ischemic stroke results in violent impairment of tissue homeostasis leading to severe perturbation within the neurovascular unit (NVU) during the recovery period. The aim of this study was to assess the potential of mesenchymal stem cells (MSCs) originating from Wharton's jelly (WJ) to differentiate into functionally competent cells of endothelial lineage (WJ-EPCs). The protective effect(s) of either primary WJ-MSCs or induced WJ-EPCs was investigated and compared after oxygen-glucose deprivation (OGD) of hippocampal organotypic slices (OHC) in the indirect coculture model. WJ-MSCs, primed in EGM-2 (Lonza commercial medium) under 5% O2, acquired cobblestone endothelial-like morphology, formed capillary-like structures and actively took up DiI-Ac-LDL. Both cell types (WJ-MSCs and WJ-EPCs) were positive for CD73, CD90, CD105, VEGFR-2, and VEGF, but only endothelial-like culture expressed vWF and PECAM-1 markers at significant levels. In the presence of either WJ-MSCs or WJ-EPCs in the compartment below OGD-injured slices, cell death and vascular atrophy in the hypoxia-sensitive CA1 region were substantially decreased. This suggests that a paracrine mechanism may mediate WJ-MSC- and WJ-EPC-dependent protection. Thus, finally, we estimated secretion of the neuro/angio/immunomodulatory molecules IL-6, TGF-ß1, and VEGF by these cell cultures. We have found that release of TGF-ß1 and IL-6 was TLR ligand [LPS and Poly(I:C)] concentration dependent and stronger in WJ-EPC than WJ-MSC cultures. Simultaneously, the uneven pattern of TLR receptors and modulatory cytokine gene expression was confirmed also on qRT-PCR level, but no significant differences were noticed between WJ-EPC and primary WJ-MSC cultures.

Complex assessment of distinct cognitive impairments following ouabain injection into the rat dorsoloateral striatum.

A stroke in humans may induce focal injury to the brain tissue resulting in various disabilities. Although motor deficits are the most discernible, cognitive impairments seem to be crucial for patients mental well-being. The current lack of effective treatments encourages scientists and clinicians to develop novel approaches. Before applying them in clinic, testing for safety and effectiveness in non-human models is necessary. Such animal model should include significant cognitive impairments resulting from brain lesion. We used ouabain stereotactic injection into the right dorsolateral striatum of male Wistar rats, and enriched environment housing. To confirm the brain injury before cognitive testing, rats were given a beam-walking task to evaluate the level of sensorimotor deficits. To determine the cognitive impairment after focal brain damage, rats underwent a set of selected tasks over an observation period of 30 days. Brain injury induced by ouabain significantly impaired the acquisition of the T-maze habit learning task, where 'win-stay' strategy rules were applied. The injured rats also showed significant deficits in the performance of the T-maze switching task, which involved shifting from multiple clues previously relevant to the only one important clue. Focal brain injury also significantly changed 'what--where' memory, tested in the object exploration task, in which a novel object consecutively appeared in the same place while the location of a familiar item was continuously changed. In conclusion, we developed an animal model of distinct cognitive impairments after focal brain injury that provides a convenient method to test the effectiveness of restorative therapies.

Long-term MRI cell tracking after intraventricular delivery in a patient with global cerebral ischemia and prospects for magnetic navigation of stem cells within the CSF.

BACKGROUND: The purpose of the study was to evaluate the long-term clinical tracking of magnetically labeled stem cells after intracerebroventricular transplantation as well as to investigate in vitro feasibility for magnetic guidance of cell therapy within large fluid compartments. METHOD: After approval by our Institutional Review Board, an 18-month-old patient, diagnosed as being in a vegetative state due to global cerebral ischemia, underwent cell transplantation to the frontal horn of the lateral ventricle, with umbilical cord blood-derived stem cells labeled with superparamagnetic iron oxide (SPIO) contrast agent. The patient was followed over 33 months with clinical examinations and MRI. To evaluate the forces governing the distribution of cells within the fluid compartment of the ventricular system in vivo, a gravity-driven sedimentation assay and a magnetic field-driven cell attraction assay were developed in vitro. RESULTS: Twenty-four hours post-transplantation, MR imaging (MRI) was able to detect hypointense cells in the occipital horn of the lateral ventricle. The signal gradually decreased over 4 months and became undetectable at 33 months. In vitro, no significant difference in cell sedimentation between SPIO-labeled and unlabeled cells was observed (p = NS). An external magnet was effective in attracting cells over distances comparable to the size of human lateral ventricles. CONCLUSIONS: MR imaging of SPIO-labeled cells allows monitoring of cells within lateral ventricles. While the initial biodistribution is governed by gravity-driven sedimentation, an external magnetic field may possibly be applied to further direct the distribution of labeled cells within large fluid compartments such as the ventricular system.

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.

[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.

Encapsulation of mesenchymal stem cells by bioscaffolds protects cell survival and attenuates neuroinflammatory reaction in injured brain tissue after transplantation.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin-laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Human mesenchymal stem cells in the treatment of neurological diseases.

Here we provide a comprehensive data on the unique features of mesenchymal stem cells (MSCs) which makes them feasible and preferred candidate for cell-based therapy in neurological clinic. From this point of view the most important features of these cells are: (1) availability from autologous sources independently from age of patient; (2) extensive expansion in vitro; (3) immunomodulatory "bystander" function after transplantation in vivo; (4) potentiality to protect, repair or eventually replace impaired or dysfunctional host cells. For complete this last task of functional regeneration of central nervous system, we have to take advantages of MSCs capability for transient, time-locked proliferation, migration to site of injury and their commitment to neuronal differentiation. However, if we are to make progress in the use of MSCs for therapy in the clinic it will be necessary to establish more unified, advanced standards for cells processing in vitro as well as safer and improved procedures for their delivery in vivo.

Differentiation of glia-committed NG2 cells: the role of factors released from hippocampus and spinal cord.

The NG2-positive cells are the oligodendrocyte precursors, which, when terminally differentiated, are capable of myelinating the central nervous system. There is however an ever-growing list of evidences that NG2 cells actually possess an intrinsic neurogenic potential and they are capable of neuronal differentiation in response to environmental stimuli. To address the question, we have established a model of an indirect co-culture system of the freshly isolated rat neonatal NG2 cells and organotypic slices derived from two distinct CNS regions (hippocampus and spinal cord) to mimic the nervous tissue microenviroment. The cell differentiation in microenvironment of OGD-injured hippocampal slices has been studied as well. The molecular analysis of selected trophic factors has been performed to determine the patterns of their expression. Indeed, the comparison of the cell commitment and development in various microenvironments has pointed to significant dissimilarities. First of all, the medium being continuously conditioned by the hippocampal slices efficiently promoted neurogenesis. The effect has been significantly abolished in co-cultures with the injured tissue. The less pronounced susceptibility to adopting neuronal phenotype and the considerable slowdown of oligodendroglial development was observed in the co-cultures with the spinal cord slices. The role of BDNF in oligodendroglial progenitor commitment and development has been investigated proving that it is one of the key players in the examined processes. The specificity of the instructive clues cocktail might module the fate choice of mobilized endogenous or transplanted cells, which should be taken into consideration while planning neurorepair strategies.