Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood.

Stem cell therapy is a potential treatment for spinal cord injury and different stem cell types have been grafted into animal models and humans suffering from spinal trauma. Due to inconsistent results, it is still an important and clinically relevant question which stem cell type will prove to be therapeutically effective. Thus far, stem cells of human sources grafted into spinal cord mostly included barely defined heterogeneous mesenchymal stem cell populations derived from bone marrow or umbilical cord blood. Here, we have transplanted a well-defined unrestricted somatic stem cell isolated from human umbilical cord blood into an acute traumatic spinal cord injury of adult immune suppressed rat. Grafting of unrestricted somatic stem cells into the vicinity of a dorsal hemisection injury at thoracic level eight resulted in hepatocyte growth factor-directed migration and accumulation within the lesion area, reduction in lesion size and augmented tissue sparing, enhanced axon regrowth and significant functional locomotor improvement as revealed by three behavioural tasks (open field Basso-Beattie-Bresnahan locomotor score, horizontal ladder walking test and CatWalk gait analysis). To accomplish the beneficial effects, neither neural differentiation nor long-lasting persistence of the grafted human stem cells appears to be required. The secretion of neurite outgrowth-promoting factors in vitro further suggests a paracrine function of unrestricted somatic stem cells in spinal cord injury. Given the highly supportive functional characteristics in spinal cord injury, production in virtually unlimited quantities at GMP grade and lack of ethical concerns, unrestricted somatic stem cells appear to be a highly suitable human stem cell source for clinical application in central nervous system injuries.

Hepatocyte growth factor-mediated attraction of mesenchymal stem cells for apoptotic neuronal and cardiomyocytic cells.

Human bone marrow-derived mesenchymal stem cells (MSC) home to injured tissues and have regenerative capacity. In this study, we have investigated in vitro the influence of apoptotic and necrotic cell death, thus distinct types of tissue damage, on MSC migration. Concordant with an increased overall motility, MSC migrated towards apoptotic, but not vital or necrotic neuronal and cardiac cells. Hepatocyte growth factor (HGF) was expressed by the apoptotic cells only. MSC, in contrast, revealed expression of the HGF-receptor, c-Met. Blocking HGF bioactivity resulted in significant reduction of MSC migration. Moreover, recombinant HGF attracted MSC in a dose-dependent manner. Thus, apoptosis initiates chemoattraction of MSC via the HGF/c-Met axis, thereby linking tissue damage to the recruitment of cells with regenerative potential.

A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential.

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Trauma-associated inflammatory response impairs embryonic stem cell survival and integration after implantation into injured rat brain.

Pluripotent embryonic stem cells were shown to survive and differentiate into mature neuronal cells after implantation in experimental models of Parkinson disease and cerebral ischemia. Embryonic stem cell transplantation has also been proposed as a potential therapy for cerebral trauma, characteristic of massive loss of multiple cell types due to primary insult and secondary sequelae. Green fluorescent protein (GFP)-transfected murine embryonic stem cells were implanted into the ipsi or contralateral cortex of male Sprague-Dawley rats 72 h after fluid-percussion injury. Animals were sacrificed at day 5 or week 7 postimplantation. Brain sections were examined using conventional and fluorescent double-labelling immunohistochemistry. Five days after implantation, clusters of GFP-positive cells undergoing partial differentiation along neuronal pathway, were detected at the implantation site. However, after 7 weeks, only a few GFP-positive cells were found, indicating an extensive loss of stem cells during this time period. For the first time, we proved the observed cell loss to be mediated via phagocytosis of implanted cells by activated macrophages. Cerebral trauma, induced 3 days prior to implantation, has activated the inflammatory potential of otherwise immunologically privileged tissue. Subsequent cell implantation was accompanied by reactive astrogliosis, activation of microglia, as well as a massive invasion of macrophages into transplantation sites even if the grafts were placed into contralateral healthy hemispheres, remote from the traumatic lesion. Our results demonstrate a significant post-traumatic inflammatory response, which impairs survival and integration of implanted stem cells and has generally not been taken into account in designs of previous transplantation studies.

Proteomics of experimental stroke in mice.

Multi-Western blots of more than 400 proteins were performed from brain extracts of mice submitted to transient focal ischemia induced by 1 h middle cerebral artery (MCA) thread occlusion. Measurements were carried out in groups of six animals in sham-operated controls, at the end of 1 h ischemia, and after 3 and 12 h recirculation. After MCA occlusion up to 45% of proteins were up- or downregulated in the ipsilateral hemisphere by a factor of 1.5 or more, as compared to sham-operated controls. The temporal regulation of several proteins in the ischemia-affected hemisphere after 1 h MCA thread occlusion is described. In the non-ischemic hemisphere the number of regulated proteins was close to 50%, indicating a hitherto unrecognized involvement of the opposite side. The proteomic approach of brain injury analysis goes beyond previous screenings of gene expression at the transcriptional level and although our study provides further evidence for the complexity of multiinjury pathways in the evolution of ischemic brain damage it may help to identify key mediators of ischemic injury.

Implementation of palliative care as a mandatory cross-disciplinary subject (QB13) at the Medical Faculty of the Heinrich-Heine-University Düsseldorf, Germany.

BACKGROUND: By means of the revision of the Medical Licensure Act for Physicians (ÄAppO) in 2009, undergraduate palliative care education (UPCE) was incorporated as a mandatory cross sectional examination subject (QB13) in medical education in Germany. Its implementation still constitutes a major challenge for German medical faculties. There is a discrepancy between limited university resources and limited patient availabilities and high numbers of medical students. Apart from teaching theoretical knowledge and skills, palliative care education is faced with the particular challenge of imparting a professional and adequate attitude towards incurably ill and dying patients and their relatives. PROJECT DESCRIPTION: Against this background, an evidence-based longitudinal UPCE curriculum was systematically developed following Kern's Cycle [1] and partly implemented and evaluated by the students participating in the pilot project. Innovative teaching methods (virtual standardised/simulated patient contacts, e-learning courses, interdisciplinary and interprofessional collaborative teaching, and group sessions for reflective self-development) aim at teaching palliative care-related core competencies within the clinical context and on an interdisciplinary and interprofessional basis. RESULTS: After almost five years of development and evaluation, the UPCE curriculum comprises 60 teaching units and is being fully implemented and taught for the first time in the winter semester 2014/15. The previous pilot phases were successfully concluded. To date, the pilot phases (n=26), the subproject "E-learning in palliative care" (n=518) and the blended-learning elective course "Communication with dying patients" (n=12) have been successfully evaluated. CONCLUSION: All conducted development steps and all developed programmes are available for other palliative care educators (Open Access). The integrated teaching formats and methods (video, e-learning module, interprofessional education, group sessions for reflexive self-development) and their evaluations are intended to make a contribution to an evidence-based development of palliative care curricula in Germany.

Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke.

The therapeutical potential of transplantation of undifferentiated and predifferentiated murine embryonic stem cells for the regeneration of the injured brain was investigated in two rodent stroke models. Undifferentiated embryonic stem cells xenotransplanted into the rat brain at the hemisphere opposite to the ischemic injury migrated along the corpus callosum towards the damaged tissue and differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. The authors demonstrated a hitherto unrecognized inverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells, which raises concerns about safety provisions when the therapeutical potential of human embryonic stem cells is tested in preclinical animal models.

[Embryonic stem cell therapy in experimental stroke: host-dependent malignant transformation]. / Embrionális ossejt terápia kísérletes stroke-ban: gazdafüggo malignus transzformáció.

INTRODUCTION: Therapeutic application of embryonic stem cells in neurodegenerative disorders like stroke is widely investigated in preclinical animal models. AIM: The authors studied the therapeutic potential of murine embryonic stem cells in two rodent models of stroke. METHODS: Undifferentiated and predifferentiated stem cells were implanted into the non-ischemic hemisphere of mice and rats following focal brain ischemia. The brains were analysed by immunohistochemistry and histology. The in vitro differentiation of the cells was checked by immunocytochemistry and Western-blot. RESULTS: After xenotransplantation in rats undifferentiated cells migrated along the corpus callosum towards the ischemic injury. Later stem cells differentiated into neurons in the border zone of the lesion. In the homologous mouse brain, the same murine embryonic stem cells did not migrate, but produced highly malignant teratocarcinomas at the site of implantation, independent of whether they were predifferentiated in vitro to neural progenitor cells. These experiments demonstrated a hitherto unrecognized adverse outcome after xenotransplantation and homologous transplantation of embryonic stem cells. CONCLUSION: This observation raises serious concerns about safety provisions when the therapeutic potential of human embryonic stem cells is tested in preclinical animal models. The clinical trials are based on the positive outcome of the xenologous experiments.

Lentiviral labeling reveals three germ layer differentiation potential of a single unrestricted somatic stem cell from human cord blood.

OBJECTIVE: Generation and expression of unrestricted somatic stem cells (USSC) from human cord blood as well as their in vitro functional characterization at the clonal level. MATERIALS AND METHODS: USSC generation was initiated from fresh cord blood followed by lentiviral transfection and clone generation via limiting dilution. Individual clones were analyzed for lentiviral genomic integration patterns by ligation-mediated polymerase chain reaction. In vitro differentiation of clonal USSC was performed into mesodermal, endodermal, and ectodermal lineages according to our published protocols. Respective osteogenic, hepatic, and neuronal lineage-specification was documented by immunohistochemistry and tissue-specific protein expression was analyzed by Western blotting. MicroRNA expression analysis was achieved using the TaqMan microRNA Megaplex array. RESULTS: Lentivirally labeled USSC cultures were successfully subjected to limiting dilution cloning. One clone containing a single lentiviral integration site was identified (clone 4) and used for further differentiation experiments. Ligation-mediated polymerase chain reaction results from mesodermally, endodermally, and ectodermally differentiated USSC clone 4 consistently showed only the primary single lentiviral integration site. Lineage-specific differentiation experiments were confirmed by morphology and cell-fate-specific monoclonal antibodies in immunocytochemistry. MicroRNA expression profiles did not reveal dramatic differences between clonal and nonclonal USSC. CONCLUSIONS: The proof of the clonal existence of USSC is important for the assessment of biological properties unique for these unrestricted human stem cell candidates. As clones they can be subjected to advanced methods that enable defining of the multilayer nature of regulatory mechanisms through single-cell analysis.

Hepatocyte growth factor/c-MET axis-mediated tropism of cord blood-derived unrestricted somatic stem cells for neuronal injury.

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.

Caffeine-mediated enhancement of glucocorticoid receptor activity in human osteoblastic cells.

Caffeine-containing beverage consumption has been reported to be associated with an increased risk for osteoporosis. Since the glucocorticoid receptor (GR) is a major factor in the induction of osteoporosis we analyzed whether caffeine may act via altering GR function. Applying a reporter gene assay we provide evidence that caffeine drastically amplifies GR transcriptional activity in human osteoblastic cells. Substances that increase the intracellular cyclic AMP-concentration also strengthen the transactivity of the GR and coincubation with caffeine further reinforces this potentiation, indicating that caffeine-mediated enhancement of GR transcriptional function is due to the inhibitory activity of caffeine on the cyclic AMP phosphodiesterase. Our data suggest evidence for a hitherto unrecognized crosstalk between caffeine-modulated signalling and GR-initiated gene expression in human osteoblastic cells and could provide the molecular basis for the role of caffeine in osteoporosis.

Chronic glucocorticoid receptor activation impairs CREB transcriptional activity in clonal neurons.

Excessive circulating levels of glucocorticoids are thought to be associated with cognitive impairment. We provide evidence that chronic activation of the glucocorticoid receptor (GR) in clonal neurons inhibits the transcriptional activity of the cyclic AMP response element-binding protein (CREB), which is believed to be involved in memory processes. To investigate the underlying mechanism we studied the phosphorylation of CREB and found altered phosphorylation kinetics in neurons chronically treated with glucocorticoids. Our results demonstrate a hitherto unrecognized crosstalk between the cyclic AMP and glucocorticoid pathway and may provide the molecular basis for the effects of long-term glucocorticoid exposure on cognitive function.

Immunohistochemical analysis of protein expression after middle cerebral artery occlusion in mice.

The effect of transient focal cerebral ischemia on protein regulation was studied in mice using multiparametric immunohistochemistry. Injury was characterized by measurements of blood flow, regional protein synthesis and terminal transferase biotinylated-dUTP nick end labeling (TUNEL). The proteins studied were selected from a previously established list of differentially regulated proteins and included the GTPases dynamin, RhoB, CAS and Ran BP-1, the transcription factors Nurr1 and p-Stat 6, the protein kinase MAPK p49, the splicing factors SRPK1 and hPrp16, the cell cycle control proteins cyclin B1 and Nek2, the inflammatory proteins FKBP12 and Rag2, the cell adhesion protein paxillin and the folding protein TCP-1. Regulation patterns were diverse and comprised ipsi- and/or contralateral up- and down-regulation with or without topical association to impeding cell death. Some proteins (SRPK1, TCP-1 and Nurr1) also exhibited post-ischemic translocation from the nucleus to the cytosol. Our observations stress the importance of regional analysis for the interpretation of proteomic data, and contribute to the identification of new pathways that may be involved in the evolution of post-ischemic brain injury.

Transgenic mice overexpressing XIAP in neurons show better outcome after transient cerebral ischemia.

X-chromosome linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein (IAP) family and known to inhibit death of various cells under different experimental conditions. Although present in brain tissue, little is known about the physiology of the IAPs in nerve cells. Here we report on the establishment of transgenic mice with overexpression of human XIAP in brain neurons. The mice developed normally, and were more resistant to brain injury caused by transient forebrain ischemia after occlusion of the middle cerebral artery compared to control mice. The XIAP transgenic animals exhibited significantly smaller brain damage, as shown by TUNEL labelling, less reduction in brain protein synthesis, and less active caspase-3 after ischemia compared with controls. Upregulation of RhoB, which is an early indicator of neurological damage, was markedly reduced in the XIAP-overexpressing mice, which had also a better neurological outcome than control animals. This together with the increase in XIAP in normal mouse brain in regions surviving the infarct demonstrates that XIAP is an important factor promoting neuronal survival after ischemia. The results suggest that interference with the levels and the activity of XIAP in neurons may provide targets for the development of drugs limiting neuronal death after ischemia, and possibly in other brain injuries.

Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat.

In vivo monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under in vivo conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-microm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.