RESUMO
This study aims to evaluate and compare cellular therapy with human Wharton's jelly (WJ) mesenchymal stem cells (MSCs) and neural precursors (NPs) in experimental autoimmune encephalomyelitis (EAE), a preclinical model of Multiple Sclerosis. MSCs were isolated from WJ by an explant technique, differentiated to NPs, and characterized by cytometry and immunocytochemistry analysis after ethical approval. Forty-eight rats were EAE-induced by myelin basic protein and Freund's complete adjuvant. Forty-eight hours later, the animals received intraperitoneal injections of 250 ng/dose of Bordetella pertussis toxin. Fourteen days later, the animals were divided into the following groups: a. non-induced, induced: b. Sham, c. WJ-MSCs, d. NPs, and e. WJ-MSCs plus NPs. 1 × 105. Moreover, the cells were placed in a 10 µL solution and injected via a stereotaxic intracerebral ventricular injection. After ten days, the histopathological analysis for H&E, Luxol, interleukins, and CD4/CD8 was carried out. Statistical analyses demonstrated a higher frequency of clinical manifestation in the Sham group (15.66%) than in the other groups; less demyelination was seen in the treated groups than the Sham group (WJ-MSCs, p = 0.016; NPs, p = 0.010; WJ-MSCs + NPs, p = 0.000), and a lower cellular death rate was seen in the treated groups compared with the Sham group. A CD4/CD8 ratio of <1 showed no association with microglial activation (p = 0.366), astrocytes (p = 0.247), and cell death (p = 0.577) in WJ-MSCs. WJ-MSCs and NPs were immunomodulatory and neuroprotective in cellular therapy, which would be translated as an adjunct in demyelinating diseases.
Assuntos
Encefalomielite Autoimune Experimental , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Esclerose Múltipla , Animais , Encefalomielite Autoimune Experimental/terapia , Encefalomielite Autoimune Experimental/patologia , Ratos , Esclerose Múltipla/terapia , Esclerose Múltipla/patologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Feminino , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Neurais , Modelos Animais de Doenças , Geleia de Wharton/citologiaRESUMO
Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas Recombinantes/farmacologia , Cicatrização/efeitos dos fármacos , Angiopoietina-1/metabolismo , Angiopoietina-1/farmacologia , Angiopoietina-2/metabolismo , Angiopoietina-2/farmacologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/química , Diabetes Mellitus Tipo 2/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Geleia de Wharton/citologia , Geleia de Wharton/metabolismoRESUMO
Manufacturing of mesenchymal stromal cell (MSC)-based therapies for regenerative medicine requires the use of suitable supply of growth factors that enhance proliferation, cell stability and potency during cell expansion. Human blood derivatives such as human platelet lysate (hPL) have emerged as a feasible alternative for cell growth supplement. Nevertheless, composition and functional characterization of hPL in the context of cell manufacturing is still under investigation, particularly regarding the content and function of pro-survival and pro-regenerative factors. We performed comparative analyses of hPL, human serum (hS) and fetal bovine serum (FBS) stability and potency to support Wharton's jelly (WJ) MSC production. We demonstrated that hPL displayed low inter-batch variation and unique secretome profile that was not present in hS and FBS. Importantly, hPL-derived factors including PDGF family, EGF, TGF-alpha, angiogenin and RANTES were actively taken up by WJ-MSC to support efficient expansion. Moreover, hPL but not hS or FBS induced secretion of osteoprotegerin, HGF, IL-6 and GRO-alpha by WJ-MSC during the expansion phase. Thus, hPL is a suitable source of factors supporting viability, stability and potency of WJ-MSC and therefore constitutes an essential raw material that in combination with WJ-MSC introduces a great opportunity for the generation of potent regenerative medicine products.
Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/citologia , Medicina Regenerativa , Cordão Umbilical/citologia , Geleia de Wharton/citologia , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical/metabolismo , Geleia de Wharton/metabolismoRESUMO
Intracerebral hemorrhage (ICH) is as a life-threatening condition that can occur in young adults, often causing long-term disability. Recent preclinical data suggest mesenchymal stromal cell (MSC)-based therapies as promising options to minimize brain damage after ICH. However, therapeutic evidence and mechanistic insights are still limited, particularly when compared with other disorders such as ischemic stroke. Herein, we employed a model of collagenase-induced ICH in young adult rats to investigate the potential therapeutic effects of an intravenous injection of human umbilical cord Wharton's jelly-derived MSCs (hUC-MSCs). Two doses of collagenase were used to cause moderate or severe hemorrhages. Magnetic resonance imaging showed that animals treated with hUC-MSCs after moderate ICH had smaller residual hematoma volumes than vehicle-treated rats, whereas the cell therapy failed to decrease the hematoma volume in animals with a severe ICH. Functional assessments (rotarod and elevated body swing tests) were performed for up to 21 days after ICH. Enduring neurological impairments were seen only in animals subjected to severe ICH, but the cell therapy did not induce statistically significant improvements in the functional recovery. The biodistribution of Technetium-99m-labeled hUC-MSCs was also evaluated, showing that most cells were found in organs such as the spleen and lungs 24 h after transplantation. Nevertheless, it was possible to detect a weak signal in the brain, which was higher in the ipsilateral hemisphere of rats subjected to a severe ICH. These data indicate that hUC-MSCs have moderately beneficial effects in cases of less severe brain hemorrhages in rats by decreasing the residual hematoma volume, and that optimization of the therapy is still necessary.
Assuntos
Hemorragia Cerebral/terapia , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Animais , Encéfalo/citologia , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Ratos , Recuperação de Função Fisiológica/fisiologia , Distribuição Tecidual/fisiologia , Geleia de Wharton/citologiaRESUMO
Sepsis induces organ dysfunction due to overexpression of the inflammatory host response, resulting in cardiopulmonary and autonomic dysfunction, thus increasing the associated morbidity and mortality. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) express genes and secrete factors with anti-inflammatory properties, neurological and immunological protection, as well as improve survival in experimental sepsis. The cholinergic anti-inflammatory pathway (CAP) is mediated by α7-nicotinic acetylcholine receptors (α7nAChRs), which play an important role in the control of systemic inflammation. We hypothesized that WJ-MSCs attenuate sepsis-induced organ injury in the presence of an activated CAP pathway. To confirm our hypothesis, we evaluated the effects of WJ-MSCs as a treatment for cardiopulmonary injury and on neuroimmunomodulation. Male Wistar rats were randomly divided into four groups: control (sham-operated); cecal ligation and puncture (CLP) alone; CLP+WJ-MSCs (1 × 106 cells, at 6 h post-CLP); and CLP+methyllycaconitine (MLA)+WJ-MSCs (5 mg/kg body wt, at 5.5 h post-CLP, and 1 × 106 cells, at 6 h post-CLP, respectively). All experiments, including the assessment of echocardiographic parameters and heart rate variability, were performed 24 h after CLP. WJ-MSC treatment attenuated diastolic dysfunction and restored baroreflex sensitivity. WJ-MSCs also increased cardiac sympathetic and cardiovagal activity. WJ-MSCs reduced leukocyte infiltration and proinflammatory cytokines, effects that were abolished by administration of a selective α7nAChR antagonist (MLA). In addition, WJ-MSC treatment also diminished apoptosis in the lungs and spleen. In cardiac and splenic tissue, WJ-MSCs downregulated α7nAChR expression, as well as reduced the phospho-STAT3-to-total STAT3 ratio in the spleen. WJ-MSCs appear to protect against sepsis-induced organ injury by reducing systemic inflammation, at least in part, via a mechanism that is dependent on an activated CAP.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Neuroimunomodulação , Sepse/terapia , Geleia de Wharton/citologia , Animais , Citocinas , Humanos , Masculino , Miocárdio/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Baço/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton's jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer's disease-linked amyloid beta oligomers (AßOs). METHODS: We isolated and characterized EVs released by human Wharton's jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AßOs. RESULTS: hMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AßOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AßOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole. CONCLUSIONS: hMSC-EVs protected hippocampal neurons from damage induced by AßOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/citologia , Neurônios/patologia , Neuroproteção , Estresse Oxidativo , Sinapses/patologia , Geleia de Wharton/citologia , Animais , Biomarcadores/metabolismo , Catalase/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/ultraestrutura , Hipocampo/patologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Multimerização Proteica , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sinapses/efeitos dos fármacosRESUMO
BACKGROUND: Procedures to remove adiposities and skin, such as dermolipectomy, can develop wounds that are difficult to heal by conventional therapies. Mesenchymal stem cells are indicated as potential candidates for regenerative therapy in wounds, due to their multipotentiality, low immunogenicity, modulating capacity of inflammation and tissue modeling processes. CASE REPORT: Patient with dehiscent chronic ulcer secondary to dermolipectomy, who received cutaneous treatment with mesenchymal stem cells. The therapy induced scar formation and neovascularization, as well as the decrease of infiltrated leukocytes and proinflammatory cytokines. Mesenchymal cells are proposed as an interesting alternative for the treatment of postoperative lesions.
INTRODUCCIÓN: Los procedimientos para retirar adiposidades y piel, como la dermolipectomía, pueden desarrollar heridas difíciles de sanar mediante tratamientos convencionales. Se ha señalado que es posible utilizar las células madre mesenquimales en el tratamiento regenerativo en heridas, en virtud de su multipotencialidad, baja inmunogenicidad, capacidad moduladora de inflamación y procesos modeladores de tejidos. CASO CLÍNICO: Paciente con dehiscencia en úlcera crónica secundaria a dermolipectomía, sometida a tratamiento cutáneo con células madre mesenquimales. Se indujo formación de cicatriz y neovascularización, así como la disminución de leucocitos infiltrados y citocinas proinflamatorias. Se propone a las células mesenquimales como una alternativa interesante para el tratamiento de lesiones postoperatorias.
Assuntos
Contorno Corporal/efeitos adversos , Lipectomia/efeitos adversos , Transplante de Células-Tronco Mesenquimais , Medicina Regenerativa/métodos , Úlcera Cutânea/terapia , Deiscência da Ferida Operatória/terapia , Geleia de Wharton/citologia , Adipogenia , Adulto , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Separação Celular , Doença Crônica , Cicatriz/etiologia , Feminino , Expressão Gênica , Humanos , Inflamação , Células-Tronco Mesenquimais , Neovascularização Fisiológica , Osteogênese , Úlcera Cutânea/etiologia , Deiscência da Ferida Operatória/etiologia , CicatrizaçãoRESUMO
The aim of this study was to evaluate the effect of the conditioned medium of ovine Wharton's jelly-derived mesenchymal stem cells (oWJ-MSCs) on the morphology, growth, reactive oxygen species (ROS) and glutathione (GSH) intracellular levels, active mitochondria, and meiotic resumption of isolated ovine secondary follicles in vitro. The oWJ-MSCs were isolated and the medium where they were cultured was recovered (conditioned medium). Isolated ovine secondary follicles were cultured for 6 days in 1) supplemented α-MEM+ (control); 2) 50% α-MEM+ + 50% conditioned medium (α-MEM + CM group) or 3) conditioned medium only (CM group). The parameters analyzed were morphology, antrum formation, follicle and oocyte growth, ROS and GSH levels, mitochondrial activity and meiotic resumption. The percentage of normal follicles, antrum formation, and fully grown oocytes did not differ (P > 0.05) among treatments. Follicles cultured in α-MEM + CM group had greater (P < 0.05) diameter than other treatments after culture. Moreover, the diameter of the follicles cultured in CM alone was higher (P < 0.05) than in the α-MEM+. In addition, α-MEM + CM and CM treatments increased the growth rate compared to the α-MEM+. Treatments containing conditioned medium (α-MEM + CM or CM) significantly reduced ROS levels compared to the control medium. Moreover, mitochondrial activity was higher in α-MEM+ and α-MEM + CM than in CM alone. All treatments showed oocytes in GV, GVBD and MI. In conclusion, oWJ-MSCs conditioned medium, especially when associated with α-MEM, improves the growth of secondary follicles and reduces ROS generation after short-term culture.
Assuntos
Meios de Cultivo Condicionados , Células-Tronco Mesenquimais/fisiologia , Folículo Ovariano/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Ovinos/fisiologia , Geleia de Wharton/citologia , Animais , Feminino , Técnicas de Cultura de TecidosRESUMO
Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell-substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.
Assuntos
Diferenciação Celular , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteômica , Linhagem Celular , Vesículas Extracelulares/ultraestrutura , Humanos , Análise de Componente Principal , Células Estromais/metabolismo , Espectrometria de Massas em Tandem , Geleia de Wharton/citologiaRESUMO
BACKGROUND AIMS: Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. METHODS: We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. RESULTS: After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). DISCUSSION: 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.