RESUMEN

Schwann cells promote nerve regeneration by adaptation of a regenerative phenotype referred to as repair mediating Schwann cell. Down-regulation of myelin proteins, myelin clearance, formation of Bungner's bands, and secretion of trophic factors characterize this cell type. We have previously shown that the sphingosine-1-phosphate receptor agonist Fingolimod/FTY720P promotes the generation of this particular Schwann cell phenotype by activation of dedifferentiation markers and concomitant release of trophic factors resulting in enhanced neurite growth of dorsal root ganglion neurons. Despite its biomedical relevance, a detailed characterization of the corresponding Schwann cell secretome is lacking, and the impact of FTY720P on enhancing neurite growth is not defined. Here, we applied a label-free quantitative mass spectrometry approach to characterize the secretomes derived from primary neonatal and adult rat Schwann cells in response to FTY720P. We identified a large proportion of secreted proteins with a high overlap between the neonatal and adult Schwann cells, which can be associated with biologic processes such as development, axon growth, and regeneration. Moreover, FTY720P-treated Schwann cells release proteins downstream of Smad signaling known to support neurite growth. Our results therefore uncover a network of trophic factors involved in glial-mediated repair of the peripheral nervous system.-Schira, J., Heinen, A., Poschmann, G., Ziegler, B., Hartung, H.-P., Stühler, K., Küry, P. Secretome analysis of nerve repair mediating Schwann cells reveals Smad-dependent trophism.

Asunto(s)

Regeneración Nerviosa/fisiología , Células de Schwann/metabolismo , Proteínas Smad/metabolismo , Animales , Células Cultivadas , Cromatografía Liquida , Biología Computacional , Clorhidrato de Fingolimod/farmacología , Organofosfatos/farmacología , Ratas , Células de Schwann/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Smad/genética , Esfingosina/análogos & derivados , Esfingosina/farmacología , Espectrometría de Masas en Tándem , Ácido Tricloroacético/química

RESUMEN

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis-related genes RUNX-2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up-regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP-9. However, quantitative RT-PCR analysis of BMP-2, ALP, FGF-2, CYCLIN-D1, MMP-13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate-resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals.

Asunto(s)

Envejecimiento/genética , Hueso Esponjoso/metabolismo , Regulación del Desarrollo de la Expresión Génica , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Radio (Anatomía)/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Densidad Ósea/genética , Hueso Esponjoso/anatomía & histología , Hueso Esponjoso/crecimiento & desarrollo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoblastos/citología , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoclastos/citología , Osteogénesis/genética , Osteopontina/genética , Osteopontina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , Radio (Anatomía)/anatomía & histología , Radio (Anatomía)/crecimiento & desarrollo , Transducción de Señal , Factor de Transcripción Sp7/genética , Factor de Transcripción Sp7/metabolismo , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo

RESUMEN

INTRODUCTION: Bone infections after trauma, i.e. posttraumatic osteomyelitis, pose one of the biggest problems of orthopedic surgery. Even after sufficient clinical therapy including vast debridement of infected bone and antibiotic treatment, regeneration of postinfectious bone seems to be restricted. One explanation includes the large sized defects resulting from sufficient debridement. Furthermore, it remains unclear if inflammatory processes after bone infection do affect bone regeneration. For continuing studies in this field, an animal model is needed where bone regeneration after sufficient treatment can be studied in detail. METHODS: For this purpose we created a stable infection in murine tibiae by Staphylococcus aureus inoculation. Thereafter, osteomyelitic bones were debrided thoroughly and animals were subsequently treated with antibiotics. Controls included debrided, non-infected, as well as infected animals exclusively treated with antibiotics. To verify sufficient treatment of infected bone, different assessments detecting S. aureus were utilized: agar plates, histology and RT-qPCR. RESULTS: All three detection methods revealed massive reduction or eradication of S. aureus within debrided bones 1 and 2 weeks postoperatively, whereas sole antibiotic therapy could not provide sufficient treatment of osteomyelitic bones. Debrided, previously infected bones showed significantly decreased bone formation, compared to debrided, non-infected controls. DISCUSSION: Thus, the animal model presented herein provides a reliable and fascinating tool to study posttraumatic osteomyelitis for clinical therapies.

Asunto(s)

Antibacterianos/uso terapéutico , Osteomielitis/tratamiento farmacológico , Osteomielitis/cirugía , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/cirugía , Tibia/lesiones , Tibia/cirugía , Animales , Desbridamiento , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones Endogámicos C57BL , Osteomielitis/complicaciones , Infecciones Estafilocócicas/complicaciones , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Tibia/microbiología

RESUMEN

Scaphoid bones have a high prevalence for non-union. Even with adequate treatment, bone regeneration may not occur in certain instances. Although this condition is well described, the molecular pathology of scaphoid non-unions is still poorly defined. In this study, gene expression of osteogenic and angiogenic growth and transcription factors as well as inflammatory mediators were analysed in human scaphoid non-unions and intraindividually compared to adjacent autologous cancellous bone from the distal radius. In addition, histology and immunohistochemical stainings were performed to verify qRT-PCR data. Gene expression analysis revealed a significant up-regulation of RANKL, ALP, CYCLIN D1, MMP-13, OPG, NFATc1, TGF-ß and WNT5A in scaphoid non-unions. Interestingly, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in non-unions. Moreover, WNT5A was highly up-regulated in all non-union samples. TRAP staining confirmed the observation of induced osteoclastogenesis in non-unions. With respect to genes related to osteogenesis, alkaline phosphatase was significantly up-regulated in scaphoid non-unions. No differences were detectable for other osteogenic genes such as RUNX-2 or BMP-2. Importantly, we did not detect differences in angiogenesis between scaphoid non-unions and controls in both gene expression and immunohistochemistry. Summarized, our data indicate increased osteoclast activity in scaphoid non-unions possibly as a result of the alterations in RANKL, TGF-ß and WNT5A expression levels. These data increase our understanding for the reduced bone regeneration capacity present in scaphoid non-unions and may translate into the identification of new therapeutic targets to avoid secondary damages and prevent occurrence of non-unions to scaphoid bones.

Asunto(s)

Huesos/metabolismo , Fracturas Óseas/genética , Perfilación de la Expresión Génica/métodos , Osteoclastos/metabolismo , Osteogénesis/genética , Adolescente , Adulto , Anciano , Huesos/lesiones , Femenino , Fracturas Óseas/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Modelos Genéticos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Adulto Joven

RESUMEN

Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes.

Asunto(s)

Sangre Fetal/citología , Factores de Crecimiento Nervioso/metabolismo , Células Madre/metabolismo , Axones/fisiología , Células Cultivadas , Humanos , Neuronas/metabolismo , Fenotipo , Regeneración , Trasplante de Células Madre

RESUMEN

Although bone regeneration is typically a reliable process, type 2 diabetes is associated with impaired or delayed healing processes. In addition, angiogenesis, a crucial step in bone regeneration, is often altered in the diabetic state. In this study, different stages of bone regeneration were characterized in an unicortical bone defect model comparing transgenic type 2 diabetic (db-/db-) and wild type (WT) mice in vivo. We investigated angiogenesis, callus formation and bone remodeling at early, intermediate and late time points by means of histomorphometry as well as protein level analyses. In order to enhance bone regeneration, defects were locally treated with recombinant FGF-9 or VEGFA. Histomorphometry of aniline blue stained sections indicated that bone regeneration is significantly decreased in db-/db- as opposed to WT mice at intermediate (5 days post operation) and late stages (7 days post operation) of bone regeneration. Moreover, immunohistochemical analysis revealed significantly decreased levels of RUNX-2, PCNA, Osteocalcin and PECAM-1 in db-/db- defects. In addition, osteoclastogenesis is impaired in db-/db- indicating altered bone remodeling. These results indicate significant impairments in angiogenesis and osteogenesis in type 2 diabetic bones. Importantly, angiogenesis, osteogenesis and bone remodeling could be reconstituted by application of recombinant FGF-9 and, in part, by VEGFA application. In conclusion, our study demonstrates that type 2 diabetes affects angiogenesis, osteogenesis and subsequently bone remodeling, which in turn leads to decreased bone regeneration. These effects could be reversed by local application of FGF-9 and to a lesser degree VEGFA. These data could serve as a basis for future therapeutic applications aiming at improving bone regeneration in the type 2 diabetic patient population.

Asunto(s)

Regeneración Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Diabetes Mellitus Tipo 2/fisiopatología , Factor 9 de Crecimiento de Fibroblastos/farmacología , Neovascularización Patológica , Osteogénesis/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Ratones , Ratones Endogámicos C57BL , Osteoblastos/patología

RESUMEN

Transplanted stem cells provide beneficial effects on regeneration/recovery after spinal cord injury (SCI) by the release of growth-promoting factors, increased tissue preservation, and provision of a permissive environment for axon regeneration. A rise in chemokine stromal cell-derived factor 1 (SDF-1/CXCL12) expression levels in central nervous system (CNS) injury sites has been shown to play a central role in recruiting transplanted stem cells. Although technically more challenging, it has been shown that after SCI few endogenous stem cells are recruited via SDF-1/CXCR4 signaling. Evidence is accumulating that increasing SDF-1 levels at the injury site (e.g., by exogenous application or transfection methods) further enhances stem cell recruitment. Moreover, SDF-1 might, in addition to migration, also influence survival, proliferation, differentiation, and cytokine secretion of stem cells. Here, we discuss the experimental data available on the role of SDF-1 in stem and progenitor cell biology following CNS injury and suggest strategies for how manipulation of the SDF-1 system could facilitate stem cell-based therapeutic approaches in SCI. In addition, we discuss challenges such as how to circumvent off-target effects in order to facilitate the transfer of SDF-1 to the clinic.

Asunto(s)

Quimiocina CXCL12/metabolismo , Receptores CXCR4/metabolismo , Regeneración de la Medula Espinal , Células Madre/metabolismo , Animales , Carcinogénesis/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , Trasplante de Células Madre Mesenquimatosas , Neovascularización Patológica/metabolismo , Transducción de Señal , Traumatismos de la Médula Espinal , Células Madre/citología , Cicatrización de Heridas

RESUMEN

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.

Asunto(s)

Trasplante de Células Madre de Sangre del Cordón Umbilical , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/terapia , Animales , Axones/fisiología , Células Cultivadas , Femenino , Humanos , Actividad Motora/fisiología , Ratas , Ratas Wistar , Traumatismos de la Médula Espinal/fisiopatología

RESUMEN

BACKGROUND: MicroRNAs are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Here the functional impact of microRNAs on cell cycle arrest during neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood (USSC) was analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b in USSC differentiated into neuronal lineage but not in USSC differentiated into osteogenic lineage. Transfection experiments followed by Ki67 immunostainings demonstrated that each of these microRNAs was able to promote proliferation of native USSC and to prevent in part cell cycle arrest during neuronal lineage differentiation of USSC. Bioinformatic target gene predictions followed by experimental target gene validations revealed that miR-17, -20a, and -106b act in a common manner by downregulating an overlapping set of target genes mostly involved in regulation and execution of G(1)/S transition. Pro-proliferative target genes cyclinD1 (CCND1) and E2F1 as well as anti-proliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. Furthermore, these microRNAs also downregulate WEE1 which is involved in G(2)/M transition. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription factors, despite the fact that miR-17, -20a, and -106b directly target the transcripts that encode for this protein family. CONCLUSIONS/SIGNIFICANCE: Mir-17, -20a, and -106b downregulate a common set of pro- and anti-proliferative target genes to impact cell cycle progression of USSC and increase intracellular activity of E2F transcription factors to govern G(1)/S transition.

Asunto(s)

Ciclo Celular , Linaje de la Célula , Factores de Transcripción E2F/metabolismo , Sangre Fetal/citología , MicroARNs/fisiología , Neuronas/citología , Diferenciación Celular , Linaje de la Célula/genética , Proliferación Celular , Regulación hacia Abajo , Fase G1/genética , Humanos , Células Madre/citología

RESUMEN

Unrestricted somatic stem cells (USSCs) represent an intrinsically multipotent CD45-negative fetal population from human cord blood. They show differentiation into neuronal cells of a dopaminergic phenotype, which express neuronal markers such as synaptophysin, neuronal-specific nuclear protein, and neurofilament and release the neurotransmitter dopamine accompanied by expression of dopaminergic key factors tyrosine hydroxylase and Nurr1 (NR4A2). MicroRNA expression analysis highlighted their importance in neural development but their specific functions remain poorly understood. Here, downregulation of a set of 18 microRNAs during neuronal lineage differentiation of unrestricted somatic stem cells, including members of the miR-17-92 family and additional microRNAs such as miR-130a, -138, -218, and -335 as well as their target genes, is described. In silico target gene predictions for this microRNA group uncovered a large set of proteins involved in neuronal differentiation and having a strong impact on differentiation-related pathways such as axon guidance and TGFß, WNT, and MAPK signaling. Experimental target validations confirmed approximately 35% of predictions tested and revealed a group of proteins with specific impact on neuronal differentiation and function including neurobeachin, neurogenic differentiation 1, cysteine-rich motor neuron protein 1, neuropentraxin 1, and others. These proteins are combined targets for several subgroups from the set of 18 downregulated microRNAs. This finding was further supported by the observed upregulation of a significant amount of predicted and validated target genes based on Illumina Beadstudio microarray data. Confirming the functional relationship of a limited panel of microRNAs and predicted target proteins reveals a clear network-like impact of the group of 18 downregulated microRNAs on proteins involved in neuronal development and function.

Asunto(s)

Linaje de la Célula/genética , MicroARNs/metabolismo , Neurogénesis/genética , Células Madre/citología , Línea Celular , Dopamina/biosíntesis , Sangre Fetal/citología , Sangre Fetal/metabolismo , Humanos , MicroARNs/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neurofilamentos/biosíntesis , Neuronas/metabolismo , Proteínas Nucleares/biosíntesis , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Células Madre/metabolismo , Sinaptofisina/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Tirosina 3-Monooxigenasa/biosíntesis , Proteínas Wnt/metabolismo

RESUMEN

Recently, it has been shown that human unrestricted somatic stem cells (USSCs) from umbilical cord blood represent pluripotent, neonatal, nonhematopoietic stem cells with the potential to differentiate into the neural lineage. However, molecular and functional characterization of the neural phenotype and evaluation of the degree of maturity of the resulting cells are still lacking. In this study, we addressed the question of neuronal differentiation and maturation induced by a defined composition of growth and differentiation factors (XXL medium). We demonstrated the expression of different neuronal markers and their enrichment in USSC cultures during XXL medium incubation. Furthermore, we showed enrichment of USSCs expressing tyrosine hydroxylase (TH), an enzyme specific for dopaminergic neurons and other catecholamine-producing neurons, accompanied by induction of Nurr1, a factor regulating dopaminergic neurogenesis. The functionality of USSCs has been analyzed by patch-clamp recordings and high-performance liquid chromatography (HPLC). Voltage-gated sodium-channels could be identified in laminin-predifferentiated USSCs. In addition, HPLC analysis revealed synthesis and release of the neurotransmitter dopamine by USSC-derived cells, thus correlating well with the detection of TH transcripts and protein. This study provides novel insight into the potential of unrestricted somatic stem cells from human umbilical cord blood to acquire a neuronal phenotype and function.

Asunto(s)

Diferenciación Celular , Dopamina/metabolismo , Sangre Fetal/citología , Neuronas/fisiología , Células Madre/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Humanos , Neuronas/metabolismo , Fenotipo , Ratas , Canales de Sodio/metabolismo , Canales de Sodio/fisiología , Células Madre/metabolismo