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Background: Cardiovascular surgery is confronted by a lack of suitable materials for patch repair. Acellular animal tissues serve as an abundant source of promising biomaterials. The aim of our study was to explore the bio-integration of decellularized or recellularized pericardial matrices in vivo. Methods: Porcine (allograft) and ovine (heterograft, xenograft) pericardia were decellularized using 1% sodium dodecyl sulfate ((1) Allo-decel and (2) Xeno-decel). We used two cell types for pressure-stimulated recellularization in a bioreactor: autologous adipose tissue-derived stromal cells (ASCs) isolated from subcutaneous fat of pigs ((3) Allo-ASC and (4) Xeno-ASC) and allogeneic Wharton's jelly mesenchymal stem cells (WJCs) ((5) Allo-WJC and (6) Xeno-WJC). These six experimental patches were implanted in porcine carotid arteries for one month. For comparison, we also implanted six types of control patches, namely, arterial or venous autografts, expanded polytetrafluoroethylene (ePTFE Propaten® Gore®), polyethylene terephthalate (PET Vascutek®), chemically stabilized bovine pericardium (XenoSure®), and detoxified porcine pericardium (BioIntegral® NoReact®). The grafts were evaluated through the use of flowmetry, angiography, and histological examination. Results: All grafts were well-integrated and patent with no signs of thrombosis, stenosis, or aneurysm. A histological analysis revealed that the arterial autograft resembled a native artery. All other control and experimental patches developed neo-adventitial inflammation (NAI) and neo-intimal hyperplasia (NIH), and the endothelial lining was present. NAI and NIH were most prominent on XenoSure® and Xeno-decel and least prominent on NoReact®. In xenografts, the degree of NIH developed in the following order: Xeno-decel > Xeno-ASC > Xeno-WJC. NAI and patch resorption increased in Allo-ASC and Xeno-ASC and decreased in Allo-WJC and Xeno-WJC. Conclusions: In our setting, pre-implant seeding with ASC or WJC had a modest impact on vascular patch remodeling. However, ASC increased the neo-adventitial inflammatory reaction and patch resorption, suggesting accelerated remodeling. WJC mitigated this response, as well as neo-intimal hyperplasia on xenografts, suggesting immunomodulatory properties.
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Trasplante de Células Madre Hematopoyéticas , Remodelación Vascular , Células Alogénicas , Animales , Prótesis Vascular , Arterias Carótidas , Bovinos , Humanos , Hiperplasia , Pericardio , Ovinos , Porcinos , Ingeniería de TejidosRESUMEN
Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.
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Reactores Biológicos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Enfermedades del Sistema Nervioso/terapia , Cordón Umbilical/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Trasplante de Células Madre Mesenquimatosas/tendencias , Enfermedades del Sistema Nervioso/patología , Cordón Umbilical/citología , Cordón Umbilical/trasplante , Gelatina de Wharton/citología , Gelatina de Wharton/fisiología , Gelatina de Wharton/trasplanteRESUMEN
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).
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Esclerosis Amiotrófica Lateral/terapia , Células-Madre Neurales/trasplante , Plasticidad Neuronal , Trasplante de Células Madre/métodos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Nervios Periféricos/fisiología , Ratas , Ratas Sprague-Dawley , Tenascina/genética , Tenascina/metabolismo , Versicanos/genética , Versicanos/metabolismoRESUMEN
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.
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Encéfalo/metabolismo , Hepcidinas/metabolismo , Adolescente , Adulto , Anciano , Animales , Análisis Químico de la Sangre , Encéfalo/irrigación sanguínea , Niño , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Persona de Mediana Edad , Neuroglía/metabolismo , Pericitos/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder resulting in a lethal outcome. We studied changes in ventral horn perineuronal nets (PNNs) of superoxide dismutase 1 (SOD1) rats during the normal disease course and after the intrathecal application (5 × 10(5) cells) of human bone marrow mesenchymal stromal cells (MSCs) postsymptom manifestation. We found that MSCs ameliorated disease progression, significantly improved motor activity, and prolonged survival. For the first time, we report that SOD1 rats have an abnormal disorganized PNN structure around the spinal motoneurons and give different expression profiles of chondroitin sulfate proteoglycans (CSPGs), such as versican, aggrecan, and phosphacan, but not link protein-1. Additionally, SOD1 rats had different profiles for CSPG gene expression (Versican, Hapln1, Neurocan, and Tenascin-R), whereas Aggrecan and Brevican profiles remained unchanged. The application of MSCs preserved PNN structure, accompanied by better survival of motorneurons. We measured the concentration of cytokines (IL-1α, MCP-1, TNF-α, GM-CSF, IL-4, and IFN-γ) in the rats' cerebrospinal fluid and found significantly higher concentrations of IL-1α and MCP-1. Our results show that PNN and cytokine homeostasis are altered in the SOD1 rat model of ALS. These changes could potentially serve as biological markers for the diagnosis, assessment of treatment efficacy, and prognosis of ALS. We also show that the administration of human MSCs is a safe procedure that delays the loss of motor function and increases the overall survival of symptomatic ALS animals, by remodeling the recipients' pattern of gene expression and having neuroprotective and immunomodulatory effects.
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Esclerosis Amiotrófica Lateral/metabolismo , Células Madre Mesenquimatosas/citología , Neuronas/citología , Médula Espinal/metabolismo , Animales , Diferenciación Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Matriz Extracelular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Masculino , Red Nerviosa/citología , Ratas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.
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BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals' lifespan. METHODS: At the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP(+) MSC) on the same day, intraspinally (10(5) cells) and intravenously (2 × 10(6) cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis. RESULTS: The combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP(+) MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site. CONCLUSIONS: Intraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.
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Esclerosis Amiotrófica Lateral/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Movimiento Celular , Supervivencia Celular , Modelos Animales de Enfermedad , Humanos , Células Madre Mesenquimatosas/patología , Actividad Motora , Neuronas Motoras/inmunología , Neuronas Motoras/patología , Ratas , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Adipose-derived stromal cells (ASCs) are an alternative source of stem cells for cell-based therapies of neurological disorders such as spinal cord injury (SCI). In the present study, we predifferentiated ASCs (pASCs) and compared their behavior with naïve ASCs in vitro and after transplantation into rats with a balloon-induced compression lesion. ASCs were predifferentiated into spheres before transplantation, then pASCs or ASCs were injected intraspinally 1 week after SCI. The cells' fate and the rats' functional outcome were assessed using behavioral, histological, and electrophysiological methods. Immunohistological analysis of pASCs in vitro revealed the expression of NCAM, NG2, S100, and p75. Quantitative RT-PCR at different intervals after neural induction showed the up-regulated expression of the glial markers NG2 and p75 and the neural precursor markers NCAM and Nestin. Patch clamp analysis of pASCs revealed three different types of membrane currents; however, none were fast activating Na(+) currents indicating a mature neuronal phenotype. Significant improvement in both the pASC and ASC transplanted groups was observed in the BBB motor test. In vivo, pASCs survived better than ASCs did and interacted closely with the host tissue, wrapping host axons and oligodendrocytes. Some transplanted cells were NG2- or CD31-positive, but no neuronal markers were detected. The predifferentiation of ASCs plays a beneficial role in SCI repair by promoting the protection of denuded axons; however, functional improvements were comparable in both the groups, indicating that repair was induced mainly through paracrine mechanisms.
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Tejido Adiposo/fisiología , Diferenciación Celular/fisiología , Células Madre Multipotentes/fisiología , Traumatismos de la Médula Espinal/cirugía , Trasplante de Células Madre/métodos , Células del Estroma/trasplante , Tejido Adiposo/citología , Animales , Conducta Animal/fisiología , Células Cultivadas , Masculino , Actividad Motora/fisiología , Células Madre Multipotentes/citología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Ratas Wistar , Traumatismos de la Médula Espinal/patologíaRESUMEN
Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage link protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.
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Proteínas de la Matriz Extracelular/metabolismo , Espacio Extracelular/fisiología , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Proteoglicanos/metabolismo , Animales , Axones/fisiología , Encéfalo/crecimiento & desarrollo , Encéfalo/fisiología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Oscuridad , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Lateralidad Funcional , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteoglicanos/deficiencia , Proteoglicanos/genética , Ratas , Ratas Sprague-Dawley , Privación Sensorial/fisiología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/fisiología , Percepción Visual/fisiologíaRESUMEN
Background: Toxic epidermal necrolysis (TEN) is a rare life-threatening disease that mainly affects the skin and mucous membranes, resulting from a toxic delayed-type hypersensitivity (DTH) reaction (type IV reaction) to the presence of foreign antigens such as drugs. The clinical symptoms are caused by pathophysiological processes leading to massive apoptosis of keratinocytes in the dermo-epidermal junction. This results in the formation of a bulla and subsequent separation of the entire epidermis with the exposure of the dermis. The current approach in the local therapy of TEN prefers the use of biological dressings, which helps provide several critical requirements for defect healing; in particular, it helps in the acceleration of the spontaneous wound closure (re-epithelialization) of the skin defect and the reduction of the risk of development of various complications and infections, such as the risk of pathological scar maturation. This paper is a case report of the use of a lyophilized amniotic membrane (AM) for accelerating wound healing in a patient with TEN. Case Presentation: We report a case of an 8-year-old girl transferred to our center with a histologically confirmed diagnosis of TEN. Despite the application of immunosuppressive therapy consisting of corticosteroids and intravenous immunoglobulins, we have observed disease progression and exfoliation of up to 60% of the total body surface area (TBSA). In the facial area, which is cosmetically privileged, we decided to use the lyophilized amniotic membrane (Amnioderm®) to cover up approximately 2% of the TBSA. Within 2 days after the application, we observed accelerated reepithelialisation, with rapid wound closure. We have not observed any side effects nor infections during the subsequent phases of wound healing. Skin defects in non-facial areas of the body were treated with synthetic dressings. When compared to the areas covered with the lyophilized AM, the healing process was prolonged. Conclusions: To our knowledge, this is the first case study using a lyophilized amniotic membrane in the treatment of a patient with TEN. The AM application in the cosmetically-privileged area (face), proved to be very efficient in the treatment of TEN patients. The use of this allogeneic material demonstrated excellent biocompatibility and caused a unique acceleration of epithelialization and wound healing, yielding also excellent long-term results. The current study opens broad possibilities for clinical application of the used material, the improvement of current therapy of patients with TEN and better outcomes and recovery of patients.
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An inability of the human body to heal acute wounds under certain conditions results in the formation of chronic ulcers. Chronic wounds not only cause significant pain and discomfort for patients but also serve as an entry for microorganisms into the human body, which can result in serious life-threatening problems and become a significant burden for the patients and society. The current work present results of a multicentre prospective observational study demonstrating the use of a lyophilized amniotic membrane (AM) in the treatment of chronic wounds (various etiologies). Lyophilized AM produced under the commercial brand Amnioderm® was used as an allograft material for therapy of chronic wounds, in addition to chronic ulcer standard-of-care (SoC) protocols. The duration of wounds considered for the application of AM ranged between 2 months and 11 years. In total, 16 patients were enrolled to the study, of which eight were completely healed, six demonstrated a significantly reduced ulcer size, and two did not respond to the AM therapy. The current study unambiguously demonstrates an effective alternative to the standard of chronic wound care and confirms a significant effect of the AM application for chronic wound management as a new SoC.
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An increasing number of studies have demonstrated the beneficial effects of human mesenchymal stem cells (hMSC) in the treatment of amyotrophic lateral sclerosis (ALS). We compared the effect of repeated intrathecal applications of hMSC or their conditioned medium (CondM) using lumbar puncture or injection into the muscle (quadriceps femoris), or a combination of both applications in symptomatic SOD1G93A rats. We further assessed the effect of the treatment on three major cell death pathways (necroptosis, apoptosis, and autophagy) in the spinal cord tissue. All the animals were behaviorally tested (grip strength test, Basso Beattie Bresnahan (BBB) test, and rotarod), and the tissue was analyzed immunohistochemically, by qPCR and Western blot. All symptomatic SOD1 rats treated with hMSC had a significantly increased lifespan, improved motor activity and reduced number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells. Moreover, a combined hMSC delivery increased motor neuron survival, maintained neuromuscular junctions in quadriceps femoris and substantially reduced the levels of proteins involved in necroptosis (Rip1, mixed lineage kinase-like protein, cl-casp8), apoptosis (cl-casp 9) and autophagy (beclin 1). Furthermore, astrogliosis and elevated levels of Connexin 43 were decreased after combined hMSC treatment. The repeated application of CondM, or intramuscular injections alone, improved motor activity; however, this improvement was not supported by changes at the molecular level. Our results provide new evidence that a combination of repeated intrathecal and intramuscular hMSC applications protects motor neurons and neuromuscular junctions, not only through a reduction of apoptosis and autophagy but also through the necroptosis pathway, which is significantly involved in cell death in rodent SOD1G93A model of ALS. Stem Cells Translational Medicine 2019;8:535-547.
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Esclerosis Amiotrófica Lateral/terapia , Trasplante de Células Madre Mesenquimatosas , Necroptosis , Médula Espinal/metabolismo , Superóxido Dismutasa-1/genética , Animales , Beclina-1/metabolismo , Caspasa 9/metabolismo , Modelos Animales de Enfermedad , Humanos , Inyecciones Intramusculares , Inyecciones Espinales , Longevidad , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Neuronas Motoras/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Músculo Cuádriceps/citología , Músculo Cuádriceps/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Médula Espinal/citología , Superóxido Dismutasa-1/metabolismoRESUMEN
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.
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Exocitosis , Lactancia , Oxitocina/metabolismo , Células Receptoras Sensoriales/metabolismo , Vasopresinas/metabolismo , Animales , Deshidratación/metabolismo , Femenino , Fluorescencia , Ganglios Espinales/metabolismo , Masculino , Nocicepción , Neurohipófisis/metabolismo , Ratas Transgénicas , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/metabolismoRESUMEN
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.
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Amyotrophic lateral sclerosis (ALS) is a progressive untreatable neurodegenerative disorder, leading to the death of the cortical and spinal motoneurons (MNs). Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) may represent a new approach to slowing down the progression of ALS by providing neurotrophic support to host MNs and by having an anti-inflammatory effect. We have designed a prospective, nonrandomized, open-label clinical trial (phase I/IIa, EudraCT No. 2011-000362-35) to assess the safety and efficacy of autologous multipotent BM-MSCs in ALS treatment. Autologous BM-MSCs were isolated and expanded under GMP conditions. Patients received 15 ± 4.5 × 106 of BM-MSCs via lumbar puncture into the cerebrospinal fluid. Patients were monitored for 6 months before treatment and then for an 18-month follow-up period. Potential adverse reactions were assessed, and the clinical outcome was evaluated by the ALS functional rating scale (ALSFRS), forced vital capacity (FVC), and weakness scales (WSs) to assess muscle strength on the lower and upper extremities. In total, 26 patients were enrolled in the study and were assessed for safety; 23 patients were suitable for efficacy evaluation. After intrathecal BM-MSC application, about 30% of the patients experienced a mild to moderate headache, resembling the headaches after a standard lumbar puncture. No suspected serious adverse reactions (SUSAR) were observed. We found a reduction in ALSFRS decline at 3 months after application (p < 0.02) that, in some cases, persisted for 6 months ( p < 0.05). In about 80% of the patients, FVC values remained stable or above 70% for a time period of 9 months. Values of WS were stable in 75% of patients at 3 months after application. Our results demonstrate that the intrathecal application of BM-MSCs in ALS patients is a safe procedure and that it can slow down progression of the disease.
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Esclerosis Amiotrófica Lateral/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Adulto , Esclerosis Amiotrófica Lateral/fisiopatología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Persona de Mediana Edad , Análisis de Regresión , Resultado del Tratamiento , Capacidad VitalRESUMEN
Astrocyte responses to neuronal injury may be beneficial or detrimental to neuronal recovery, but the mechanisms that determine these different responses are poorly understood. Here we show that ephrin type-B receptor 1 (EphB1) is upregulated in injured motor neurons, which in turn can activate astrocytes through ephrin-B1-mediated stimulation of signal transducer and activator of transcription-3 (STAT3). Transcriptional analysis shows that EphB1 induces a protective and anti-inflammatory signature in astrocytes, partially linked to the STAT3 network. This is distinct from the response evoked by interleukin (IL)-6 that is known to induce both pro inflammatory and anti-inflammatory processes. Finally, we demonstrate that the EphB1-ephrin-B1 pathway is disrupted in human stem cell derived astrocyte and mouse models of amyotrophic lateral sclerosis (ALS). Our work identifies an early neuronal help-me signal that activates a neuroprotective astrocytic response, which fails in ALS, and therefore represents an attractive therapeutic target.
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Astrocitos/citología , Interleucina-6/metabolismo , Neuronas/metabolismo , Receptor EphB1/metabolismo , Factor de Transcripción STAT3/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Antiinflamatorios/farmacología , Astrocitos/metabolismo , Axones/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Neuroprotección , Nervio Ciático/metabolismo , Transducción de Señal , TranscriptomaRESUMEN
Stem 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.
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Señalización del Calcio , Calcio/metabolismo , Diferenciación Celular , Células Madre/citología , Células Madre/metabolismo , Animales , HumanosRESUMEN
Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca(2+) signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca(2+) channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca(2+) stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca(2+)]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca(2+)]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca(2+)]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from -40 to -50mV in cells in basal to -80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K(+)-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium formulation is associated with significant changes in their Ca(2+) signaling and membrane properties.
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Tejido Adiposo/citología , Células de la Médula Ósea/citología , Canales Iónicos/metabolismo , Células del Estroma/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Potenciales Evocados/efectos de los fármacos , Microscopía por Video , Oxitocina/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/metabolismo , Receptores Purinérgicos/metabolismo , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Vasopresinas/farmacologíaRESUMEN
Restoration of lost neuronal function after spinal cord injury (SCI) still remains a big challenge for current medicine. One important repair strategy is bridging the SCI lesion with a supportive and stimulatory milieu that would enable axonal rewiring. Injectable extracellular matrix (ECM)-derived hydrogels have been recently reported to have neurotrophic potential in vitro. In this study, we evaluated the presumed neuroregenerative properties of ECM hydrogels in vivo in the acute model of SCI. ECM hydrogels were prepared by decellularization of porcine spinal cord (SC) or porcine urinary bladder (UB), and injected into a spinal cord hemisection cavity. Histological analysis and real-time qPCR were performed at 2, 4, and 8 weeks postinjection. Both types of hydrogels integrated into the lesion and stimulated neovascularization and axonal ingrowth into the lesion. On the other hand, massive infiltration of macrophages into the lesion and rapid hydrogel degradation did not prevent cyst formation, which progressively developed over 8 weeks. No significant differences were found between SC-ECM and UB-ECM. Gene expression analysis revealed significant downregulation of genes related to immune response and inflammation in both hydrogel types at 2 weeks post SCI. A combination of human mesenchymal stem cells with SC-ECM did not further promote ingrowth of axons and blood vessels into the lesion, when compared with the SC-ECM hydrogel alone. In conclusion, both ECM hydrogels bridged the lesion cavity, modulated the innate immune response, and provided the benefit of a stimulatory substrate for in vivo neural tissue regeneration. However, fast hydrogel degradation might be a limiting factor for the use of native ECM hydrogels in the treatment of acute SCI.