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.

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.

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.

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.

A human stem cell-based model for identifying adverse effects of organic and inorganic chemicals on the developing nervous system.

The aim of our study was to investigate whether a human neural stem cell line derived from umbilical cord blood (HUCB-NSC) can serve as a reliable test model for developmental neurotoxicity (DNT). We assessed the sensitivity of HUCB-NSCs at different developmental stages to a panel of neurotoxic (sodium tellurite, methylmercury chloride, cadmium chloride, chlorpyrifos, and L-glutamate) and non-neurotoxic (acetaminophen, theophylline, and D-glutamate) compounds. In addition, we investigated the effect of some compounds on key neurodevelopmental processes like cell proliferation, apoptotic cell death, and neuronal and glial differentiation. Less differentiated HUCB-NSCs were generally more sensitive to neurotoxicants, with the notable exception of L-glutamate, which showed a higher toxicity to later stages. The relative potencies of the compounds were: cadmium chloride > methylmercury chloride >> chlorpyrifos >> L-glutamate. Fifty nanomolar methylmercury chloride (MeHgCl) inhibited proliferation and induced apoptosis in early-stage cells. At the differentiated stage, 1 muM MeHgCl induced selective loss of S100 beta-expressing astrocytic cells. One millimolar L-glutamate did not influence the early stages of HUCB-NSC development, but it affected late stages of neuronal differentiation. A valuable system for in vitro DNT assessment should be able to discriminate between neurotoxic and non-neurotoxic compounds and show different susceptibilities to chemicals according to developmental stage and cell lineage. Although not exhaustive, this work shows that the HUCB-NSC model fulfils these criteria and may serve as a human in vitro model for DNT priority setting.

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.

Laminin promotes oligogliogenesis and increases MMPs activity in human neural stem cells of HUCB-NSC line.

Oligodendrocytes, the cells responsible for myelin formation and maintenance in CNS, are depleted in many acute and chronic conditions. The stem/progenitor cells stimulation or transplantation might be seriously considered as a long hoped for therapeutic perspective. Better understanding of the mechanism(s) regulating the activation of the cell lineage from the endogenous progenitor reservoir might be helpful. Therefore an efficient source of donor cells for transplantation in humans is being craved for. In this study we show that the application of extracellular matrix component-laminin promotes oligogliogenesis from neural stem-like cells of human cord blood cells (HUCB-NSC). Although oligodendrocytes constitute a minor subpopulation of spontaneously differentiated HUCB-NSC, the manipulation of active compounds regulating the process of cell commitment results in a several fold increase in their number. Thus cells of the HUCB-NSC line could be considered as a potential source of glial cells, fulfilling the suitable candidate criteria for oligodendrocyte replacement therapy.

Housing conditions influence motor functions and exploratory behavior following focal damage of the rat brain.

The present study investigated influence of housing conditions on motor functions recovery and exploratory behavior following ouabain focal brain lesion in the rat. During 30 days post-surgery period rats were housed individually in standard cages (IS) or in groups in enriched environment (EE) and behaviorally tested. The EE lesioned rats showed enhanced recovery from motor impairments in walking beam task, comparing with IS animals. Contrarily, in the open field IS rats (both lesioned and control) traveled a longer distance, showed less habituation and spent less time resting at the home base than the EE animals. Unlike the EE lesioned animals, the lesioned IS rats, presented a tendency to hyperactivity in postinjury period. Turning tendency was significantly affected by unilateral brain lesion only in the EE rats. We can conclude that housing conditions distinctly affected the rat's behavior in classical laboratory tests.

Patterned growth and differentiation of human cord blood-derived neural stem cells on bio-functionalized surfaces.

Bio-functionalized surfaces were prepared to study the adherence and differentiation capacity of neural stem cells derived from human umbilical cord blood (HUCB-NSC). Cell growth platforms containing arranged arrays of adhesive molecules were created by microcontact printing on a biologically inert surface. Biomolecules used to prepare microarray platforms included the extracellular matrix protein fibronectin and the polyaminoacid poly-L-lysine. HUCB-NSC plated on microplatforms at various serum conditions showed serum and molecule type dependent capacity for adhesion and differentiation. Poly-L-lysine allowed the maintenance of stem-like non differentiated cells attached to the surface, whereas fibronectin promoted spreading and neural commitment. Serum deprivation did not influence the attachment of HUCB-NSC to fibronectin, but significantly enhanced the attachment to poly-L-lysine and promoted dBcAMP induced neuronal differentiation. A bio-pattern of squares with interconnecting lines was used to guide neuronal differentiation by directing cell protrusion outgrowth. Tailoring the geometry of the bio-pattern enabled directing and monitoring of the neural stem cells. development in the large scale multiparameter biotests.

A novel, neural potential of non-hematopoietic human umbilical cord blood stem cells.

From the time of discovery that among the cord blood mononuclear cell population there are cells capable of changing their fate towards the neural lineage and producing functional neurons and macroglial cells, our attempts have been focused on the understanding of the underlying mechanism of this transition. We have deciphered the first steps of neural stem/progenitor gene induction in aggregating culture of cord blood mononuclear cells, their rapid phenotypic conversion under the influence of neuromorphogenic signals due to mitogen activation and their ability to expand and develop a prototypic, long-living line with neural stem cell properties. Evidence has accumulated that human umbilical cord-derived and neurally committed cells, due to their capacity for self-renewal, multilineage differentiation, plasticity and ability for long-lasting growth in vitro, provide unique material for the cell therapy of a wide spectrum of neurological diseases. The putative regenerating potential of these cord blood-derived neural stem/progenitor cells was evaluated after transplantation in experimental models of brain injury. In spite of initial promising data, the results indicate an urgent need to improve available animal model protocols in order to increase immuno-tolerance toward transplanted human cells.

Neurotransplantation in mice: the concorde-like position ensures minimal cell leakage and widespread distribution of cells transplanted into the cisterna magna.

The access of transplanted cells to large areas of the CNS is of critical value for cell therapy of chronic diseases associated with widespread neurodegeneration. Intrathecal cell application can match this requirement. Here we describe an efficient method for cell injection into the cisterna magna and the assessment of the cell distribution within subarachnoidal space in mice. In order to maximize cell distribution we applied a "concord-like" position, where the cisterna magna is nearly the highest point of the animal's body. A drop of saline on the needle insertion site avoided the outflow of transplanted cells from subarachnoidal space with CSF during surgery. Twenty-four hours later the preparation of the CNS with an intact dura mater by a suitable dissection technique (described in detail) revealed approx. 80% of the injected cells (100,000 cells per animal) within the subarachnoidal space ranging from the skull base (olfactory nerve to premedullary cisterns) to the IV ventricle, and to both the ventral and dorsal surfaces of the spinal cord. Thus the "concorde-like" position proved to be very useful for intrathecal cell application leading to a widespread cell distribution within the subarachnoidal space.

Structural and functional characteristic of a model for deep-seated lacunar infarct in rats.

Deep-brain lacunar infarct represents a significant clinical problem as it produces severe symptoms highly resistant to rehabilitation. The limited area of necrosis may facilitate neurorepair via the action of various novel neuroprotective strategies including cell-based therapies. The lesion was induced by stereotactic injection of ouabain into adult rat brains. Subsequent behavioral testing involved beam walking task, rotarod, visual discrimination task and apomorphine rotation. For morphological and topographical analysis brain slices were stained with H-E and evaluated under light microscopy. Lesion size was measured in absolute terms and in relation to the whole brain volume. Immunohistochemical analysis for the co-localization of BrdU with specific cell-type markers (PSA-NCAM, NG2, beta-tubulin III, GFAP, ED1) have has been performed, to determine the fate of newly generated cells with emphasis on evidence of neurogenesis. The lesion involved the basal ganglia, basal forebrain nuclei, internal capsule and striatum (just 1-2% of total brain volume). Significant and relatively stable behavioral deficits were observed up to 30 days. Furthermore, large numbers of cells are seen to be newly generated in response to injury with a significant proportion of these being present on account of neurogenesis.

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.

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.

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.