Neuroregeneration after spinal cord injury and in amyotrophic lateral sclerosis -possibilities for stem cell therapy

Mesenchymální kmenové buňky izolované z tukové tkáně mohou být alternativou mesenchymálních buněk z kostní dřeně (BMSC) v terapii akutního traumatu míchy. Pro zlepšení motorických funkcí po míšní balónkové kompresní lézi jsme použili potkaní mesenchymální buňky derivované z tuku, které jsme implantovali intraspinálně. Implantované buňky byly jak naivní, tak prediferencované. Transplantované buňky přežívaly sedm týdnů po transplantaci, zlepšily motorickou aktivitu a zapojily se do hostitelské tkáně. Exprimovaly oligodendrocytární marker NG2 a příležitostně astrocytární marker GFAP, ale nediferencovaly do neurálního fenotypu. Mesenchymální kmenové buňky z kostní dřeně mohou měnit postup choroby a prodloužit dobu přežití u potkaního modelu amyotrofické laterální sklerózy (ALS). Kombinovaná intraspinální a intravenózní transplantace BMSC byla provedena u symptomatických zvířat s overexpresí genu SOD1 G93A. Buňkami léčená zvířata přežívala déle ve srovnání s kontrolními zvířaty a vykazovala signifikantní zlepšení motorické aktivity a síly úchopu. Potkaní BMSC přežívaly do konečného stadia choroby a migrovaly bílou hmotou míšní. Transplantované buňky zvýšily počet hostitelských buněk pozitivních na neurofilamenta a signifikantně zvýšily počet a velikost zbývajících míšních motoneuronů 10 - 11 týdnů po podání, ve...Adipose-derived MSC could be used as an alternative for bone marrow MSC in the treatment of acute SCI. We used the intraspinal grafting of rat adipose-derived naïve and predifferentiated MSC to improve motor function after a balloon-induced compression lesion of the rat spinal cord. Grafted cells survived for seven weeks after transplantation, improved motor activity and integrated into the host tissue. They expressed the oligodenrocyte precursor marker NG2 and, occasionally, the astrocytic marker GFAP, but did not transdifferentiate into a neuronal phenotype. Bone marrow MSC may change the disease course and extend lifespan in a rat model of ALS. Combined intraspinal and intravenous transplantation of rat BMSC was performed in symptomatic rats overexpressing the SOD1 G93A gene. Cell-treated animals lived longer compared with sham-treated rats and displayed significantly improved motor activity and grip strength. Rat BMSC survived until the end stage of the disease and were migrating along the white matter of the spinal cord. Grafted cells increased the number of host cells displaying positive staining for neurofilaments and significantly increased the number and also the size of the remaining spinal motoneurons 10-11 weeks after delivery, compared with vehicle-injection. The defragmentation of DNA, a sign...Units out of CUMimofakultní pracoviště2. lékařská fakultaSecond Faculty of Medicin

Expression and cellular localization of hepcidin mRNA and protein in normal rat brain

Abstract Background Hepcidin is a peptide hormone belonging to the defensin family of cationic antimicrobial molecules that has an essential role in systemic iron homeostasis. The peptide is synthesised by hepatocytes and transported in the circulation to target tissues where it regulates the iron export function of the ferrous iron permease, ferroportin. In the brain hepcidin protein has been identified using immuno-histochemistry and mRNA by real-time PCR but not by in situ hybridisation raising the question of whether there is measurable transcription of the hepcidin gene in the central nervous system. Alternatively hepcidin could be transported as a hormone to the brain via the circulation. Results By RT-PCR hepcidin mRNA was present at low level throughout normal rat brain while in situ hybridisation to detect low-abundant mRNA revealed that transcripts were restricted to endothelium of blood vessels and choroid plexus. In contrast, hepcidin protein analysed by immuno-histochemistry was highly expressed in blood vessels, in endothelium and in pericytes. Hepcidin was also present in glial cells and in the olfactory bulb, sub-ventricular zone and dentate gyrus, areas where neurogenesis and synaptic plasticity are maintained throughout adult life. The hepcidin species identified by Western blotting in sub-ventricular zone, cortex and hippocampus migrated as a ~2.8 kDa band, identical in size to hepcidin present in normal rat serum suggesting that hepcidin in brain was the full-length biologically active 25 amino acid peptide. Hepcidin co-localised with ferroportin in ependymal cells of the sub-ventricular zone and in the corpus callosum consistent with a regulatory role in iron metabolism at these sites. Conclusions Hepcidin protein was widely expressed in brain parenchyma while levels of hepcidin gene transcription appeared to be below the limits of detection of the in situ hybridisation probes. This disparity suggests that not all hepcidin in the brain is transcribed in situ and may originate in part outside the brain. The properties of hepcidin as a cationic peptide hormone are reflected in the finding of hepcidin in the walls of blood vessels and in pericytes and glia, cells that may be involved in transporting the peptide into brain interstitium

Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs)

Expression and cellular localization of hepcidin mRNA and protein in normal rat brain.

BACKGROUND: Hepcidin is a peptide hormone belonging to the defensin family of cationic antimicrobial molecules that has an essential role in systemic iron homeostasis. The peptide is synthesised by hepatocytes and transported in the circulation to target tissues where it regulates the iron export function of the ferrous iron permease, ferroportin. In the brain hepcidin protein has been identified using immuno-histochemistry and mRNA by real-time PCR but not by in situ hybridisation raising the question of whether there is measurable transcription of the hepcidin gene in the central nervous system. Alternatively hepcidin could be transported as a hormone to the brain via the circulation. RESULTS: By RT-PCR hepcidin mRNA was present at low level throughout normal rat brain while in situ hybridisation to detect low-abundant mRNA revealed that transcripts were restricted to endothelium of blood vessels and choroid plexus. In contrast, hepcidin protein analysed by immuno-histochemistry was highly expressed in blood vessels, in endothelium and in pericytes. Hepcidin was also present in glial cells and in the olfactory bulb, sub-ventricular zone and dentate gyrus, areas where neurogenesis and synaptic plasticity are maintained throughout adult life. The hepcidin species identified by Western blotting in sub-ventricular zone, cortex and hippocampus migrated as a ~2.8 kDa band, identical in size to hepcidin present in normal rat serum suggesting that hepcidin in brain was the full-length biologically active 25 amino acid peptide. Hepcidin co-localised with ferroportin in ependymal cells of the sub-ventricular zone and in the corpus callosum consistent with a regulatory role in iron metabolism at these sites. CONCLUSIONS: Hepcidin protein was widely expressed in brain parenchyma while levels of hepcidin gene transcription appeared to be below the limits of detection of the in situ hybridisation probes. This disparity suggests that not all hepcidin in the brain is transcribed in situ and may originate in part outside the brain. The properties of hepcidin as a cationic peptide hormone are reflected in the finding of hepcidin in the walls of blood vessels and in pericytes and glia, cells that may be involved in transporting the peptide into brain interstitium

Neuroregeneration after spinal cord injury and in amyotrophic lateral sclerosis -possibilities for stem cell therapy

Adipose-derived MSC could be used as an alternative for bone marrow MSC in the treatment of acute SCI. We used the intraspinal grafting of rat adipose-derived naïve and predifferentiated MSC to improve motor function after a balloon-induced compression lesion of the rat spinal cord. Grafted cells survived for seven weeks after transplantation, improved motor activity and integrated into the host tissue. They expressed the oligodenrocyte precursor marker NG2 and, occasionally, the astrocytic marker GFAP, but did not transdifferentiate into a neuronal phenotype. Bone marrow MSC may change the disease course and extend lifespan in a rat model of ALS. Combined intraspinal and intravenous transplantation of rat BMSC was performed in symptomatic rats overexpressing the SOD1 G93A gene. Cell-treated animals lived longer compared with sham-treated rats and displayed significantly improved motor activity and grip strength. Rat BMSC survived until the end stage of the disease and were migrating along the white matter of the spinal cord. Grafted cells increased the number of host cells displaying positive staining for neurofilaments and significantly increased the number and also the size of the remaining spinal motoneurons 10-11 weeks after delivery, compared with vehicle-injection. The defragmentation of DNA, a sign..

Neuroprotective Potential of Cell-Based Therapies in ALS: From Bench to Bedside

Motor neurons (MN) degeneration is a main feature of amyotrophic lateral sclerosis (ALS), a neurological disorder with a progressive course. The diagnosis of ALS is essentially a clinical one. Most common symptoms include a gradual neurological deterioration that reflect the impairment and subsequent loss of muscle functions. Up-to-date ALS has no therapy that would prevent or cure a disease. Modern therapeutic strategies comprise of neuroprotective treatment focused on antiglutamatergic, antioxidant, antiapoptotic, and anti-inflammatory molecules. Stem cells application and gene therapy has provided researchers with a powerful tool for discovery of new mechanisms and therapeutic agents, as well as opened new perspectives for patients and family members. Here, we review latest progress made in basic, translational and clinical stem cell research related to the ALS. We overviewed results of preclinical and clinical studies employing cell-based therapy to treat neurodegenerative disorders. A special focus has been made on the neuroprotective properties of adult mesenchymal stromal cells (MSC) application into ALS patients. Finally, we overviewed latest progress in the field of embryonic and induced pluripotent stem cells used for the modeling and application during neurodegeneration in general and in ALS in particular

Vasopressin and oxytocin in sensory neurones: expression, exocytotic release and regulation by lactation

Abstract The neurohormones arginine-vasopressin (AVP) and oxytocin (OT) synthesised in supraoptic and paraventricular nuclei of neurohypophysis regulate lactation, systemic water homeostasis and nociception. Using transgenic rats expressing AVP and OT tagged with fluorescent proteins we demonstrate that both neurohormones are expressed in sensory neurones both in vitro, in primary cultures, and in situ, in the intact ganglia; this expression was further confirmed with immunocytochemistry. Both neurohormones were expressed in nociceptive neurones immunopositive to transient receptor potential vannilloid 1 (TRPV1) channel antibodies. The AVP and OT-expressing DRG neurones responded to AVP, OT, 50 mM K+ and capsaicin with [Ca2+]i transients; responses to AVP and OT were specifically blocked by the antagonists of V1 AVP and OT receptors. Probing the extracellular incubation saline with ELISA revealed AVP and OT secretion from isolated DRGs; this secretion was inhibited by tetanus toxin (TeNT) indicating the role for vesicular release. Expression of OT, but not AVP in DRG neurones significantly increased during lactation. Together, the results indicate novel physiological roles (possibly related to nociception and mood regulation) of AVP and OT in the sensory neurones

Physiology of Ca2+ signalling in stem cells of different origins and differentiation stages

International audienceStem cells (SCs) of different origins have brought hope as potential tools for the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Calcium signalling plays a key role in SC differentiation and proliferation, and dysregulation of Ca(2+) homeostasis may instigate pathological scenarios. Currently, the role of ion channels and receptors in SCs is not fully understood. In the recent years, we found that (i) the pre-differentiation of human embryonic SCs (hESCs) led to the activation of Ca(2+) signalling cascades and enhanced the functional activities of these cells, (ii) the Ca(2+) homeostasis and the physiological properties of hESC-derived neural precursors (NPs) changed during long term propagation in vitro, (iii) differentiation of NPs derived from human induced pluripotent SCs affects the expression of ion channels and receptors, (iv) these neuronal precursors exhibited spontaneous activity, indicating that their electrophysiological and Ca(2+) handling properties are similar to those of mature neurones, and (v) in mesenchymal SCs isolated from the adipose tissue and bone marrow of rats the expression profile of ion channels and receptors depends not only on the differentiation conditions but also on the source from which the cells were isolated, indicating that the fate and functional properties of the differentiated cells are driven by intrinsic mechanisms. Together, identification and assignment of a unique ion channel and a Ca(2+) handling footprint for each cell type would be necessary to qualify them as physiologically suitable for medical research, drug screening, and cell therapy