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1.
Sci Rep ; 10(1): 20095, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208869

RESUMO

A pool of different types of neural progenitor cells resides in the adult hippocampus. Apart from doublecortin-expressing (DCX+) neuronal progenitor cells (NPCs), the hippocampal parenchyma also contains oligodendrocyte precursor cells (OPCs), which can differentiate into myelinating oligodendrocytes. It is not clear yet to what extent the functions of these different progenitor cell types overlap and how plastic these cells are in response to pathological processes. The aim of this study was to investigate whether hippocampal DCX+ NPCs can generate new oligodendrocytes under conditions in which myelin repair is required. For this, the cell fate of DCX-expressing NPCs was analyzed during cuprizone-induced demyelination and subsequent remyelination in two regions of the hippocampal dentate gyrus of DCX-CreERT2/Flox-EGFP transgenic mice. In this DCX reporter model, the number of GFP+ NPCs co-expressing Olig2 and CC1, a combination of markers typically found in mature oligodendrocytes, was significantly increased in the hippocampal DG during remyelination. In contrast, the numbers of GFP+PDGFRα+ cells, as well as their proliferation, were unaffected by de- or remyelination. During remyelination, a higher portion of newly generated BrdU-labeled cells were GFP+ NPCs and there was an increase in new oligodendrocytes derived from these proliferating cells (GFP+Olig2+BrdU+). These results suggest that DCX-expressing NPCs were able to contribute to the generation of mature oligodendrocytes during remyelination in the adult hippocampus.


Assuntos
Cuprizona/farmacologia , Hipocampo/citologia , Proteínas Associadas aos Microtúbulos/fisiologia , Células-Tronco Neurais/citologia , Neuropeptídeos/fisiologia , Oligodendroglia/citologia , Remielinização , Animais , Diferenciação Celular , Quelantes/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo
2.
Stem Cell Res Ther ; 11(1): 233, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532320

RESUMO

BACKGROUND: Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. METHODS: Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. RESULTS: MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. CONCLUSIONS: Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.


Assuntos
Desenvolvimento Muscular , Células-Tronco , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Músculo Esquelético , Mioblastos , Miócitos de Músculo Liso
3.
Cereb Cortex ; 30(3): 1499-1515, 2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-31647533

RESUMO

The extent of functional maturation and integration of nonproliferative neuronal precursors, becoming neurons in the adult murine piriform cortex, is largely unexplored. We thus questioned whether precursors eventually become equivalent to neighboring principal neurons or whether they represent a novel functional network element. Adult brain neuronal precursors and immature neurons (complex cells) were labeled in transgenic mice (DCX-DsRed and DCX-CreERT2 /flox-EGFP), and their cell fate was characterized with patch clamp experiments and morphometric analysis of axon initial segments. Young (DCX+) complex cells in the piriform cortex of 2- to 4-month-old mice received sparse synaptic input and fired action potentials at low maximal frequency, resembling neonatal principal neurons. Following maturation, the synaptic input detected on older (DCX-) complex cells was larger, but predominantly GABAergic, despite evidence of glutamatergic synaptic contacts. Furthermore, the rheobase current of old complex cells was larger and the maximal firing frequency was lower than those measured in neighboring age-matched principal neurons. The striking differences between principal neurons and complex cells suggest that the latter are a novel type of neuron and new coding element in the adult brain rather than simple addition or replacement for preexisting network components.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Córtex Piriforme/fisiologia , Animais , Diferenciação Celular/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Córtex Piriforme/metabolismo
4.
Front Neurol ; 10: 1225, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31849808

RESUMO

Spinal cord injury is characterized by initial neural tissue disruption that triggers secondary damage and extensive non-resolving inflammation, which aggravates loss of function and hinders recovery. The early onset of inflammation following traumatic spinal cord injury underscores the importance of acute intervention after the initial trauma. Injections of mesenchymal stromal cells (MSCs) can reduce inflammation following spinal cord injury. We asked if extracellular vesicles (EVs) can substitute the anti-inflammatory and anti-scarring activities of their parental MSCs in a rat model of contusion spinal cord injury. We report that MSC-EVs were as potent as the parental intact cells in reducing the level of neuroinflammation for up to 2 weeks post-injury. Acute application of EVs after spinal cord injury was shown to robustly decrease the expression of pro-inflammatory cytokines in the spinal cord parenchyma in the very early phase of secondary damage. Moreover, the anti-scarring impact of MSC-EVs was even more efficient than the parental cells. We therefore conclude that anti-inflammatory and anti-scarring activities of MSC application can be mediated by their secreted EVs. In light of their substantial safety and druggability advantages, EVs may have a high potential in early therapeutic treatment following traumatic spinal cord injury.

5.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
6.
Neurobiol Dis ; 124: 93-107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30445024

RESUMO

The development and characterization of new improved animal models is pivotal in Alzheimer's Disease (AD) research, since valid models enable the identification of early pathological processes, which are often not accessible in patients, as well as subsequent target discovery and evaluation. The TgF344-AD rat model of AD, bearing mutant human amyloid precursor protein (APPswe) and Presenilin 1 (PSEN1ΔE9) genes, has been described to manifest the full spectrum of AD pathology similar to human AD, i.e. progressive cerebral amyloidosis, tauopathy, neuronal loss and age-dependent cognitive decline. Here, AD-related pathology in female TgF344-AD rats was examined longitudinally between 6 and 18 months by means of complementary translational MRI techniques: resting state functional MRI (rsfMRI) to evaluate functional connectivity (FC) and diffusion tensor imaging (DTI) to assess the microstructural integrity. Additionally, an evaluation of macroscopic changes (3D anatomical MRI) and an image-guided validation of ex vivo pathology were performed. We identified slightly decreased FC at 6 months followed by severe and widespread hypoconnectivity at 10 months of age as the earliest detectable pathological MRI hallmark. This initial effect was followed by age-dependent progressive microstructural deficits in parallel with age-dependent ex vivo AD pathology, without signs of macroscopic alterations such as hippocampal atrophy. This longitudinal MRI study in the TgF344-AD rat model of AD revealed early rsfMRI and DTI abnormalities as seen in human AD patients. The characterization of AD pathology in this rat model using non-invasive MRI techniques further highlights the translational value of this model, as well as its use for potential treatment evaluation.


Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Doença de Alzheimer/diagnóstico por imagem , Precursor de Proteína beta-Amiloide/genética , Animais , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Modelos Animais de Doenças , Feminino , Estudos Longitudinais , Imageamento por Ressonância Magnética , Vias Neurais/diagnóstico por imagem , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Presenilina-1/genética , Ratos Endogâmicos F344 , Ratos Transgênicos
7.
Cereb Cortex ; 28(7): 2610-2621, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29688272

RESUMO

Neurogenesis in the healthy adult murine brain is based on proliferation and integration of stem/progenitor cells and is thought to be restricted to 2 neurogenic niches: the subventricular zone and the dentate gyrus. Intriguingly, cells expressing the immature neuronal marker doublecortin (DCX) and the polysialylated-neural cell adhesion molecule reside in layer II of the piriform cortex. Apparently, these cells progressively disappear along the course of ageing, while their fate and function remain unclear. Using DCX-CreERT2/Flox-EGFP transgenic mice, we demonstrate that these immature neurons located in the murine piriform cortex do not vanish in the course of aging, but progressively resume their maturation into glutamatergic (TBR1+, CaMKII+) neurons. We provide evidence for a putative functional integration of these newly differentiated neurons as indicated by the increase in perisomatic puncta expressing synaptic markers, the development of complex apical dendrites decorated with numerous spines and the appearance of an axonal initial segment. Since immature neurons found in layer II of the piriform cortex are generated prenatally and devoid of proliferative capacity in the postnatal cortex, the gradual maturation and integration of these cells outside of the canonical neurogenic niches implies that they represent a valuable, but nonrenewable reservoir for cortical plasticity.


Assuntos
Plasticidade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/fisiologia , Córtex Piriforme/citologia , Córtex Piriforme/embriologia , Células-Tronco/fisiologia , Animais , Bromodesoxiuridina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Embrião de Mamíferos , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ácidos Siálicos/metabolismo
8.
Heliyon ; 4(2): e00540, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29560455

RESUMO

Following spinal cord injury, severe deficits result from damages to ascending and descending tracts, such as the corticospinal tract (CST) which is highly relevant for the motor execution in humans. Unfortunately, no curative treatment is available and intensive efforts are deployed in animal models, such as the CST transection model, to identify interventions providing functional regeneration after spinal cord injury. The CatWalk XT is a system for multi-parameter gait analysis of voluntary locomotion. In this study, the performance of the CatWalk XT for monitoring of functional deficits associated with dorsal CST lesion in rats was compared to skilled locomotion tests. Motor deficits associated with dorsal CST transection could be reliably monitored over seven weeks based on skilled locomotion testing, i.e. Horizontal Ladder Walk and Grid Walk. The collateral lesion to the overlaying gracile and cuneate funiculi occurring during dorsal CST transection resulted in slight hyposensitivity and proprioceptive deficit, which likely contributed to the lowered performance in skilled locomotion. In contrast, parameters of voluntary locomotion were not significantly affected by dorsal CST transection. Finally, an abnormal adduction reflex was detected immediately after lesion of the CST and could be conveniently used to confirm successful CST lesion in rats of experimental groups. The functional relevance of the dorsal CST in locomotion of rats is not as prominent as compared to in humans and thus challenging the motor execution is mandatory to reliably investigate CST function. A detailed analysis of voluntary walking using the CatWalk XT is not adequate to detect deficits following dorsal CST lesion in rats.

9.
Front Neurosci ; 11: 27, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28203140

RESUMO

Adult neurogenesis is a tightly regulated process continuously taking place in the central nervous system of most mammalian species. In neuroscience research, transgenic animals bearing the tamoxifen-inducible CreERT2-Lox system are widely used. In this study, we made use of a Nestin-CreERT2/R26R-YFP transgenic mouse model in which the CreERT2 activates the expression of YFP in multipotent neural stem cells upon tamoxifen application. Humoral factors, such as the levels of estrogens, have been reported to affect the hippocampal neurogenesis. The application of tamoxifen, a mixed agonist/antagonist of the estrogen receptor that permeates the blood-brain-barrier, could thus influence adult neurogenesis. Although the functions of adult neurogenesis are yet to be fully deciphered, a reciprocal interaction between rates of neurogenesis on the one hand and learning and mood regulation on the other hand, has been suggested. The impact of tamoxifen on neurogenesis and behavior was therefore addressed following five daily applications according to the open field test, the elevated plus maze, and Morris water maze. In addition, the impact of short-term tamoxifen application on progenitor cell proliferation, morphology, and fate in the neurogenic niche of the dentate gyrus were investigated. Finally, the influence of the route of administration (oral vs. intra-peritoneal) and gender-specific response were scrutinized. The sub-acute analysis did neither reveal significant differences in behavior, such as voluntary motor activity, anxiety behavior, and spatial learning, nor in cell proliferation, cell survival, dendritic arborization or maturation rate within the dentate gyrus between saline solution-, corn oil-, and tamoxifen-treated groups. Finally, neither the route of application, nor the gender of treated mice influenced the response to tamoxifen. We conclude that short tamoxifen treatments used to activate the CreERT2 system in transgenic mouse models does not have a measurable impact on adult neurogenesis or the here tested behavior, and is therefore appropriate for most studies in the field.

10.
Neurobiol Dis ; 99: 47-57, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28007584

RESUMO

Stroke is a leading cause of death and disability worldwide with no treatment for the chronic phase available. Interestingly, an endogenous repair program comprising inflammation and neurogenesis is known to modulate stroke outcome. Several studies have shown that neurogenesis decreases with age but the therapeutic importance of endogenous neurogenesis for recovery from cerebral diseases has been indicated as its ablation leads to stroke aggravation and worsened outcome. A detailed characterization of the neurogenic response after stroke related to ageing would help to develop novel and targeted therapies. In an innovative approach, we used the DCX-Luc mouse, a transgenic model expressing luciferase in doublecortin-positive neuroblasts, to monitor the neurogenic response following middle cerebral artery occlusion over three weeks in three age groups (2, 6, 12months) by optical imaging while the stroke lesion was monitored by quantitative MRI. The individual longitudinal and noninvasive time profiles provided exclusive insight into age-dependent decrease in basal neurogenesis and neurogenic upregulation in response to stroke which are not accessible by conventional BrdU-based measures of cell proliferation. For cortico-striatal strokes the maximal upregulation occurred at 4days post stroke followed by a continuous decrease to basal levels by three weeks post stroke. Older animals effectively compensated for reduced basal neurogenesis by an enhanced sensitivity to the cerebral lesion, resulting in upregulated neurogenesis levels approaching those measured in young mice. In middle aged and older mice, but not in the youngest ones, additional upregulation of neurogenesis was observed in the contralateral healthy hemisphere. This further substantiates the increased propensity of older brains to respond to lesion situation. Our results clearly support the therapeutic relevance of endogenous neurogenesis for stroke recovery and particularly in older brains.


Assuntos
Envelhecimento/fisiologia , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/fisiopatologia , Corpo Estriado/fisiopatologia , Neurogênese/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Envelhecimento/patologia , Animais , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/patologia , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/patologia , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/patologia , Modelos Animais de Doenças , Progressão da Doença , Lateralidade Funcional , Imuno-Histoquímica , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Óptica , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologia
11.
Front Cell Neurosci ; 10: 23, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26903808

RESUMO

Under physiological conditions, lymphatic vessels are thought to be absent from the central nervous system (CNS), although they are widely distributed within the rest of the body. Recent work in the eye, i.e., another organ regarded as alymphatic, revealed numerous cells expressing lymphatic markers. As the latter can be involved in the response to pathological conditions, we addressed the presence of cells expressing lymphatic markers within the spinal cord by immunohistochemistry. Spinal cord of young adult Fisher rats was scrutinized for the co-expression of the lymphatic markers PROX1 and LYVE-1 with the cell type markers Iba1, CD68, PGP9.5, OLIG2. Rat skin served as positive control for the lymphatic markers. PROX1-immunoreactivity was detected in many nuclei throughout the spinal cord white and gray matter. These nuclei showed no association with LYVE-1. Expression of LYVE-1 could only be detected in cells at the spinal cord surface and in cells closely associated with blood vessels. These cells were found to co-express Iba1, a macrophage and microglia marker. Further, double labeling experiments using CD68, another marker found in microglia and macrophages, also displayed co-localization in the Iba1+ cells located at the spinal cord surface and those apposed to blood vessels. On the other hand, PROX1-expressing cells found in the parenchyma were lacking Iba1 or PGP9.5, but a significant fraction of those cells showed co-expression of the oligodendrocyte lineage marker OLIG2. Intriguingly, following spinal cord injury, LYVE-1-expressing cells assembled and reorganized into putative pre-vessel structures. As expected, the rat skin used as positive controls revealed classical lymphatic vessels, displaying PROX1+ nuclei surrounded by LYVE-1-immunoreactivity. Classical lymphatics were not detected in adult rat spinal cord. Nevertheless, numerous cells expressing either LYVE-1 or PROX1 were identified. Based on their localization and overlapping expression with Iba1, the LYVE-1+ cell population likely represents a macrophage subpopulation, while a significant fraction of PROX1+ cells belong to the oligodendrocytic lineage based on their distribution and the expression of OLIG2. The response of these LYVE-1+ and PROX1+ cell subpopulations to pathological conditions, especially in spinal cord inflammatory conditions, needs to be further elucidated.

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