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1.
Trials ; 22(1): 595, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488845

RESUMO

BACKGROUND: Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. METHODS: A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. DISCUSSION: Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. TRIAL REGISTRATION: ClinicalTrials.gov NCT04466007 . Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.


Assuntos
COVID-19 , Diabetes Mellitus Tipo 2 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Noma , Tecido Adiposo , Animais , Ensaios Clínicos Fase II como Assunto , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/terapia , Método Duplo-Cego , Humanos , Isquemia/diagnóstico , Isquemia/terapia , Estudos Multicêntricos como Assunto , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , SARS-CoV-2 , Resultado do Tratamento
2.
In Vitro Cell Dev Biol Anim ; 57(6): 587-597, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34212340

RESUMO

Conventional methods for obtaining pancreatic ß cells are based on simulating the embryonic development phase of endocrine cells via hierarchical differentiation of pluripotent stem cells (PSCs). Accordingly, we attempted to modify the protocols for obtaining insulin-secreting cells (ISCs) by sequential differentiation of a human embryonic stem cell (hESC), using the HS181 cell line. Furthermore, we hypothesize that actual pancreatic endocrine cells may arise from trans-differentiation of mature ductal cells after the embryonic developmental stage and throughout the rest of life. According to the hypothesis, ductal cells are trans-differentiated into endocrine and exocrine cells, undergoing a partial epithelial to mesenchymal transition (EMT). To address this issue, we developed two new protocols based on hESC differentiation to obtain ductal cells and then induce EMT in cells to obtain hormone-secreting islet-like cells (HSCs). The ductal (pre-EMT exocrine) cells were then induced to undergo partial EMT by treating with Wnt3a and activin A, in hypoxia. The cell derived from the latter method significantly expressed the main endocrine cell-specific markers and also ß cells, in particular. These experiments not only support our hypothetical model but also offer a promising approach to develop new methods to compensate ß cell depletion in patients with type 1 diabetes mellitus (T1DM). Although this protocol of generating islet-like cells from ductal cells has a potential to treat T1DM, this strategy may be exploited to optimize the function of these cells in an animal model and future clinical applications.

4.
ACS Omega ; 5(32): 20270-20282, 2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32832780

RESUMO

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3ß, GSK3ß, p-ß-catenin, and ß-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3ß, and ß-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3ß Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.

5.
EClinicalMedicine ; 25: 100454, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32838232

RESUMO

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None.

6.
Artigo em Inglês | MEDLINE | ID: mdl-32117924

RESUMO

Mesenchymal stem cells (MSCs) are among the most frequently used cell type for regenerative medicine. A large number of studies have shown the beneficial effects of MSC-based therapies to treat different pathologies, including neurological disorders, cardiac ischemia, diabetes, and bone and cartilage diseases. However, the therapeutic potential of MSCs in cancer is still controversial. While some studies indicate that MSCs may contribute to cancer pathogenesis, emerging data reported the suppressive effects of MSCs on cancer cells. Because of this reality, a sustained effort to understand when MSCs promote or suppress tumor development is needed before planning a MSC-based therapy for cancer. Herein, we provide an overview on the therapeutic application of MSCs for regenerative medicine and the processes that orchestrates tissue repair, with a special emphasis placed on cancer, including central nervous system tumors. Furthermore, we will discuss the current evidence regarding the double-edged sword of MSCs in oncological treatment and the latest advances in MSC-based anti-cancer agent delivery systems.

7.
Front Immunol ; 11: 609961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33633730

RESUMO

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/fisiopatologia , Regeneração , Mordeduras de Serpentes/cirurgia , Animais , Humanos , Mediadores da Inflamação/metabolismo , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Necrose , Fenótipo , Transdução de Sinais , Mordeduras de Serpentes/diagnóstico , Mordeduras de Serpentes/metabolismo , Mordeduras de Serpentes/fisiopatologia , Resultado do Tratamento
8.
Aging (Albany NY) ; 11(18): 7746-7779, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31518338

RESUMO

An inverse correlation between thyroid hormone levels and longevity has been reported in several species and reduced thyroid hormone levels have been proposed as a biomarker for healthy aging and metabolic fitness. However, hypothyroidism is a medical condition associated with compromised health and reduced life expectancy. Herein, we show, using wild-type and the Pax8 ablated model of hypothyroidism in mice, that hyperthyroidism and severe hypothyroidism are associated with an overall unhealthy status and shorter lifespan. Mild hypothyroid Pax8 +/- mice were heavier and displayed insulin resistance, hepatic steatosis and increased prevalence of liver cancer yet had normal lifespan. These pathophysiological conditions were precipitated by hepatic mitochondrial dysfunction and oxidative damage accumulation. These findings indicate that individuals carrying mutations on PAX8 may be susceptible to develop liver cancer and/or diabetes and raise concerns regarding the development of interventions aiming to modulate thyroid hormones to promote healthy aging or lifespan in mammals.


Assuntos
Envelhecimento/metabolismo , Fígado Gorduroso/patologia , Resistência à Insulina/fisiologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Hormônios Tireóideos/sangue , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/sangue , Masculino , Camundongos , Camundongos Knockout , Fator de Transcrição PAX8/genética , Fator de Transcrição PAX8/metabolismo
9.
Sci Rep ; 9(1): 11311, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383924

RESUMO

Dietary composition plays an important role in the pathophysiology of type 2 diabetes. Monounsaturated fatty acid consumption has been positively associated with improved insulin sensitivity and ß-cell function. We examined whether an extra virgin olive oil (EVOO) high fat diet (HFD) can improve glucose homeostasis. C57BL/6J mice were fed a standard diet or a lard-based HFD to induce type 2 diabetes. Then, HFD mice were fed with three different based HFD (lard, EVOO and EVOO rich in phenolic compounds) for 24 weeks. HFD-EVOO diets significantly improved glycemia, insulinemia, glucose tolerance, insulin sensitivity and insulin degradation. Moreover, EVOO diets reduced ß-cell apoptosis, increased ß-cell number and normalized islet glucose metabolism and glucose induced insulin secretion. No additional effects were observed by higher levels of phenolic compounds. Thus, EVOO intake regulated glucose homeostasis by improving insulin sensitivity and pancreatic ß-cell function, in a type 2 diabetes HFD animal model.


Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Dieta Hiperlipídica/métodos , Resistência à Insulina , Células Secretoras de Insulina/metabolismo , Azeite de Oliva/uso terapêutico , Animais , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos Endogâmicos C57BL
10.
Diabetologia ; 62(9): 1667-1680, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31250031

RESUMO

AIMS/HYPOTHESIS: Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical that has been associated with type 2 diabetes development. Low doses of BPA modify pancreatic beta cell function and induce insulin resistance; some of these effects are mediated via activation of oestrogen receptors α (ERα) and ß (ERß). Here we investigated whether low doses of BPA regulate the expression and function of ion channel subunits involved in beta cell function. METHODS: Microarray gene profiling of isolated islets from vehicle- and BPA-treated (100 µg/kg per day for 4 days) mice was performed using Affymetrix GeneChip Mouse Genome 430.2 Array. Expression level analysis was performed using the normalisation method based on the processing algorithm 'robust multi-array average'. Whole islets or dispersed islets from C57BL/6J or oestrogen receptor ß (ERß) knockout (Erß-/-) mice were treated with vehicle or BPA (1 nmol/l) for 48 h. Whole-cell patch-clamp recordings were used to measure Na+ and K+ currents. mRNA expression was evaluated by quantitative real-time PCR. RESULTS: Microarray analysis showed that BPA modulated the expression of 1440 probe sets (1192 upregulated and 248 downregulated genes). Of these, more than 50 genes, including Scn9a, Kcnb2, Kcnma1 and Kcnip1, encoded important Na+ and K+ channel subunits. These findings were confirmed by quantitative RT-PCR in islets from C57BL/6J BPA-treated mice or whole islets treated ex vivo. Electrophysiological measurements showed a decrease in both Na+ and K+ currents in BPA-treated islets. The pharmacological profile indicated that BPA reduced currents mediated by voltage-activated K+ channels (Kv2.1/2.2 channels) and large-conductance Ca2+-activated K+ channels (KCa1.1 channels), which agrees with BPA's effects on gene expression. Beta cells from ERß-/- mice did not present BPA-induced changes, suggesting that ERß mediates BPA's effects in pancreatic islets. Finally, BPA increased burst duration, reduced the amplitude of the action potential and enlarged the action potential half-width, leading to alteration in beta cell electrical activity. CONCLUSIONS/INTERPRETATION: Our data suggest that BPA modulates the expression and function of Na+ and K+ channels via ERß in mouse pancreatic islets. Furthermore, BPA alters beta cell electrical activity. Altogether, these BPA-induced changes in beta cells might play a role in the diabetogenic action of BPA described in animal models.


Assuntos
Compostos Benzidrílicos/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Receptor beta de Estrogênio/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Fenóis/farmacologia , Animais , Receptor alfa de Estrogênio/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Potássio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sódio/metabolismo
11.
Front Immunol ; 10: 1151, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31231366

RESUMO

Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.

12.
Front Cell Neurosci ; 13: 204, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156392

RESUMO

Radiotherapy is a highly effective tool for the treatment of brain cancer. However, radiation also causes detrimental effects in the healthy tissue, leading to neurocognitive sequelae that compromise the quality of life of brain cancer patients. Despite the recognition of this serious complication, no satisfactory solutions exist at present. Here we investigated the effects of intranasal administration of human mesenchymal stem cells (hMSCs) as a neuroprotective strategy for cranial radiation in mice. Our results demonstrated that intranasally delivered hMSCs promote radiation-induced brain injury repair, improving neurological function. This intervention confers protection against inflammation, oxidative stress, and neuronal loss. hMSC administration reduces persistent activation of damage-induced c-AMP response element-binding signaling in irradiated brains. Furthermore, hMSC treatment did not compromise the survival of glioma-bearing mice. Our findings encourage the therapeutic use of hMSCs as a non-invasive approach to prevent neurological complications of radiotherapy, improving the quality of life of brain tumor patients.

13.
Genes (Basel) ; 10(5)2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072002

RESUMO

The high prevalence of type 2 diabetes mellitus (T2DM), together with the fact that current treatments are only palliative and do not avoid major secondary complications, reveals the need for novel approaches to treat the cause of this disease. Efforts are currently underway to identify therapeutic targets implicated in either the regeneration or re-differentiation of a functional pancreatic islet ß-cell mass to restore insulin levels and normoglycemia. However, T2DM is not only caused by failures in ß-cells but also by dysfunctions in the central nervous system (CNS), especially in the hypothalamus and brainstem. Herein, we review the physiological contribution of hypothalamic neuronal and glial populations, particularly astrocytes, in the control of the systemic response that regulates blood glucose levels. The glucosensing capacity of hypothalamic astrocytes, together with their regulation by metabolic hormones, highlights the relevance of these cells in the control of glucose homeostasis. Moreover, the critical role of astrocytes in the response to inflammation, a process associated with obesity and T2DM, further emphasizes the importance of these cells as novel targets to stimulate the CNS in response to metabesity (over-nutrition-derived metabolic dysfunctions). We suggest that novel T2DM therapies should aim at stimulating the CNS astrocytic response, as well as recovering the functional pancreatic ß-cell mass. Whether or not a common factor expressed in both cell types can be feasibly targeted is also discussed.


Assuntos
Encéfalo/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Doenças Metabólicas/metabolismo , Animais , Astrócitos/metabolismo , Metabolismo Energético , Homeostase , Humanos
14.
Mol Ther ; 26(11): 2696-2709, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30195725

RESUMO

Diabetes is a chronic metabolic disorder that affects 415 million people worldwide. This pathology is often associated with long-term complications, such as critical limb ischemia (CLI), which increases the risk of limb loss and mortality. Mesenchymal stromal cells (MSCs) represent a promising option for the treatment of diabetes complications. Although MSCs are widely used in autologous cell-based therapy, their effects may be influenced by the constant crosstalk between the graft and the host, which could affect the MSC fate potential. In this context, we previously reported that MSCs derived from diabetic patients with CLI have a defective phenotype that manifests as reduced fibrinolytic activity, thereby enhancing the thrombotic risk and compromising patient safety. Here, we found that MSCs derived from diabetic patients with CLI not only exhibit a prothrombotic profile but also have altered multi-differentiation potential, reduced proliferation, and inhibited migration and homing to sites of inflammation. We further demonstrated that this aberrant cell phenotype is reversed by the platelet-derived growth factor (PDGF) BB, indicating that PDGF signaling is a key regulator of MSC functionality. These findings provide an attractive approach to improve the therapeutic efficacy of MSCs in autologous therapy for diabetic patients.


Assuntos
Diabetes Mellitus/genética , Inflamação/genética , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Complicações do Diabetes/genética , Complicações do Diabetes/patologia , Complicações do Diabetes/terapia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Humanos , Inflamação/patologia , Inflamação/terapia , Camundongos , Camundongos SCID , Osteogênese/genética , Fenótipo , Proteínas Proto-Oncogênicas c-sis/uso terapêutico , Transdução de Sinais , Cicatrização/genética
15.
Mol Ther Nucleic Acids ; 12: 463-477, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-30195784

RESUMO

Human pluripotent stem cells retain the extraordinary capacity to differentiate into pancreatic beta cells. For this particular lineage, more effort is still required to stress the importance of developing an efficient, reproducible, easy, and cost-effective differentiation protocol to obtain more mature, homogeneous, and functional insulin-secreting cells. In addition, microRNAs (miRNAs) have emerged as a class of small non-coding RNAs that regulate many cellular processes, including pancreatic differentiation. Some miRNAs are known to be preferentially expressed in islets. Of note, miR-375 and miR-7 are two of the most abundant pancreatic miRNAs, and they are necessary for proper pancreatic islet development. Here we provide new insight into specific miRNAs involved in pancreatic differentiation. We found that miR-7 is differentially expressed during the differentiation of human embryonic stem cells (hESCs) into a beta cell-like phenotype and that its modulation plays an important role in generating mature pancreatic beta cells. This strategy may be exploited to optimize the potential for in vitro differentiation of hESCs into insulin-producing beta-like cells for use in preclinical studies and future clinical applications as well as the prospective uses of miRNAs to improve this process.

16.
PLoS One ; 13(5): e0195891, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29768414

RESUMO

BACKGROUND: Currently available treatments for secondary progressive multiple sclerosis(SPMS) have limited efficacy and/or safety concerns. Adipose-mesenchymal derived stem cells(AdMSCs) represent a promising option and can be readily obtained using minimally invasive procedures. PATIENTS AND METHODS: In this triple-blind, placebo-controlled study, cell samples were obtained from consenting patients by lipectomy and subsequently expanded. Patients were randomized to a single infusion of placebo, low-dose(1x106cells/kg) or high-dose(4x106cells/kg) autologous AdMSC product and followed for 12 months. Safety was monitored recording adverse events, laboratory parameters, vital signs and spirometry. Expanded disability status score (EDSS), magnetic-resonance-imaging, and other measures of possible treatment effects were also recorded. RESULTS: Thirty-four patients underwent lipectomy for AdMSCs collection, were randomized and thirty were infused (11 placebo, 10 low-dose and 9 high-dose); 4 randomized patients were not infused because of karyotype abnormalities in the cell product. Only one serious adverse event was observed in the treatment arms (urinary infection, considered not related to study treatment). No other safety parameters showed changes. Measures of treatment effect showed an inconclusive trend of efficacy. CONCLUSION: Infusion of autologous AdMSCs is safe and feasible in patients with SPMS. Larger studies and probably treatment at earlier phases would be needed to investigate the potential therapeutic benefit of this technique.


Assuntos
Tecido Adiposo/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Esclerose Múltipla Crônica Progressiva/terapia , Adulto , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
17.
Nat Commun ; 9(1): 1488, 2018 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-29662071

RESUMO

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.


Assuntos
Comunicação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Fenalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Insulina/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/imunologia , Estreptozocina , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Transplante Heterólogo
18.
Cell Death Dis ; 9(3): 279, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29449530

RESUMO

HMG20A (also known as iBRAF) is a chromatin factor involved in neuronal differentiation and maturation. Recently small nucleotide polymorphisms (SNPs) in the HMG20A gene have been linked to type 2 diabetes mellitus (T2DM) yet neither expression nor function of this T2DM candidate gene in islets is known. Herein we demonstrate that HMG20A is expressed in both human and mouse islets and that levels are decreased in islets of T2DM donors as compared to islets from non-diabetic donors. In vitro studies in mouse and human islets demonstrated that glucose transiently increased HMG20A transcript levels, a result also observed in islets of gestating mice. In contrast, HMG20A expression was not altered in islets from diet-induced obese and pre-diabetic mice. The T2DM-associated rs7119 SNP, located in the 3' UTR of the HMG20A transcript reduced the luciferase activity of a reporter construct in the human beta 1.1E7 cell line. Depletion of Hmg20a in the rat INS-1E cell line resulted in decreased expression levels of its neuronal target gene NeuroD whereas Rest and Pax4 were increased. Chromatin immunoprecipitation confirmed the interaction of HMG20A with the Pax4 gene promoter. Expression levels of Mafa, Glucokinase, and Insulin were also inhibited. Furthermore, glucose-induced insulin secretion was blunted in HMG20A-depleted islets. In summary, our data demonstrate that HMG20A expression in islet is essential for metabolism-insulin secretion coupling via the coordinated regulation of key islet-enriched genes such as NeuroD and Mafa and that depletion induces expression of genes such as Pax4 and Rest implicated in beta cell de-differentiation. More importantly we assign to the T2DM-linked rs7119 SNP the functional consequence of reducing HMG20A expression likely translating to impaired beta cell mature function.


Assuntos
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Células Secretoras de Insulina/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões 3' não Traduzidas , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Feminino , Predisposição Genética para Doença , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Lipídeos/sangue , Masculino , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fenótipo , Ratos
19.
Diabetes ; 67(3): 448-460, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29263149

RESUMO

GATA4 and GATA6 play essential, but redundant, roles in pancreas formation in mice, and GATA6 mutations cause pancreatic agenesis in humans. GATA6 mutations have also recently been linked to adult-onset diabetes, with subclinical or no exocrine insufficiency, suggesting an important role for GATA6 in human ß-cell physiology. To investigate the role of GATA6 in the adult endocrine pancreas, we generated mice in which Gata6 is specifically inactivated in the pancreas. These mice develop glucose intolerance. Islets deficient in GATA6 activity display decreased insulin content and impaired insulin secretion. Gata6-deficient ß-cells exhibit ultrastructural abnormalities, including increased immature insulin granules, swollen mitochondria, and disorganized endoplasmic reticulum. We also demonstrate that Pdx1 expression in adult ß-cells depends on GATA sites in transgenic reporter mice and that loss of GATA6 greatly affects ß-cell-specific gene expression. These findings demonstrate the essential role of GATA6 in ß-cell function.


Assuntos
Estresse do Retículo Endoplasmático , Fator de Transcrição GATA6/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Mitocôndrias/metabolismo , Vesículas Secretórias/metabolismo , Animais , Glicemia/análise , Feminino , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Intolerância à Glucose/sangue , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Intolerância à Glucose/fisiopatologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/ultraestrutura , Masculino , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Mutação , Biogênese de Organelas , Vesículas Secretórias/patologia , Vesículas Secretórias/ultraestrutura , Técnicas de Cultura de Tecidos , Transativadores/genética , Transativadores/metabolismo
20.
PLoS One ; 12(11): e0187547, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29099856

RESUMO

ß-cells release hexameric Zn2+-insulin into the extracellular space, but monomeric Zn2+-free insulin appears to be the only biologically active form. The mechanisms implicated in dissociation of the hexamer remain unclear, but they seem to be Zn2+ concentration-dependent. In this study, we investigate the influence of albumin binding to Zn2+ on Zn2+-insulin dissociation into Zn2+-free insulin and its physiological, methodological and therapeutic relevance. Glucose and K+-induced insulin release were analyzed in isolated mouse islets by static incubation and perifusion experiments in the presence and absence of albumin and Zn2+ chelators. Insulin tolerance tests were performed in rats using different insulin solutions with and without Zn2+ and/or albumin. Albumin-free buffer does not alter quantification by RIA of Zn2+-free insulin but strongly affects RIA measurements of Zn2+-insulin. In contrast, accurate determination of Zn2+-insulin was obtained only when bovine serum albumin or Zn2+ chelators were present in the assay buffer solution. Albumin and Zn2+ chelators do not modify insulin release but do affect insulin determination. Preincubation with albumin or Zn2+ chelators promotes the conversion of "slow" Zn2+-insulin into "fast" insulin. Consequently, insulin diffusion from large islets is ameliorated in the presence of Zn2+ chelators. These observations support the notion that the Zn2+-binding properties of albumin improve the dissociation of Zn2+-insulin into subunits after exocytosis, which may be useful in insulin determination, insulin pharmacokinetic assays and islet transplantation.


Assuntos
Quelantes/química , Exocitose , Insulina/metabolismo , Albumina Sérica/metabolismo , Zinco/química , Animais , Glicemia/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Radioimunoensaio , Ratos , Ratos Wistar , Albumina Sérica/química , Zinco/metabolismo
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