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Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer’s Disease
(2021)
Laura Gómez-Virgilio
;
Alejandro Luarte
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Daniela P. Ponce
;
Bárbara A. Bruna
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BEHRENS PELLEGRINO, MARIA ISABEL
<jats:p>Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer’s disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.</jats:p>
7Scopus© Citations 9
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Proteomic Analysis of Niemann-Pick Type C Hepatocytes Reveals Potential Therapeutic Targets for Liver Damage
(2021)
Elisa Balboa
;
Tamara Marín
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Juan Esteban Oyarzún
;
Pablo S. Contreras
;
Robert Hardt
<jats:p>Niemann-Pick type C disease (NPCD) is a lysosomal storage disorder caused by mutations in the NPC1 gene. The most affected tissues are the central nervous system and liver, and while significant efforts have been made to understand its neurological component, the pathophysiology of the liver damage remains unclear. In this study, hepatocytes derived from wild type and Npc1−/− mice were analyzed by mass spectrometry (MS)-based proteomics in conjunction with bioinformatic analysis. We identified 3832 proteins: 416 proteins had a p-value smaller than 0.05, of which 37% (n = 155) were considered differentially expressed proteins (DEPs), 149 of them were considered upregulated, and 6 were considered downregulated. We focused the analysis on pathways related to NPC pathogenic mechanisms, finding that the most significant changes in expression levels occur in proteins that function in the pathways of liver damage, lipid metabolism, and inflammation. Moreover, in the group of DEPs, 30% (n = 47) were identified as lysosomal proteins and 7% (n = 10) were identified as mitochondrial proteins. Importantly, we found that lysosomal DEPs, including CTSB/D/Z, LIPA, DPP7 and GLMP, and mitocondrial DEPs, AKR1B10, and VAT1 had been connected with liver fibrosis, damage, and steatosis in previous studies, validiting our dataset. Our study found potential therapeutic targets for the treatment of liver damage in NPCD.</jats:p>
Scopus© Citations 9 1
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Sensory neuron cultures derived from adult db/db mice as a simplified model to study type-2 diabetes-associated axonal regeneration defects
(2021)
DE GREGORIO CONCHA, CRISTIAN ALEJANDRO
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EZQUER, EDUARDO FERNANDO
<jats:title>ABSTRACT</jats:title> <jats:p>Diabetic neuropathy (DN) is an early common complication of diabetes mellitus (DM), leading to chronic pain, sensory loss and muscle atrophy. Owing to its multifactorial etiology, neuron in vitro cultures have been proposed as simplified systems for DN studies. However, the most used models currently available do not recreate the chronic and systemic damage suffered by peripheral neurons of type-2 DM (T2DM) individuals. Here, we cultured neurons derived from dorsal root ganglia from 6-month-old diabetic db/db-mice, and evaluated their morphology by the Sholl method as an easy-to-analyze readout of neuronal function. We showed that neurons obtained from diabetic mice exhibited neuritic regeneration defects in basal culture conditions, compared to neurons from non-diabetic mice. Next, we evaluated the morphological response to common neuritogenic factors, including nerve growth factor NGF and Laminin-1 (also called Laminin-111). Neurons derived from diabetic mice exhibited reduced regenerative responses to these factors compared to neurons from non-diabetic mice. Finally, we analyzed the neuronal response to a putative DN therapy based on the secretome of mesenchymal stem cells (MSC). Neurons from diabetic mice treated with the MSC secretome displayed a significant improvement in neuritic regeneration, but still reduced when compared to neurons derived from non-diabetic mice. This in vitro model recapitulates many alterations observed in sensory neurons of T2DM individuals, suggesting the possibility of studying neuronal functions without the need of adding additional toxic factors to culture plates. This model may be useful for evaluating intrinsic neuronal responses in a cell-autonomous manner, and as a throughput screening for the pre-evaluation of new therapies for DN.</jats:p>
2 1Scopus© Citations 3
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Spheroids derived from the stromal vascular fraction of adipose tissue self-organize in complex adipose organoids and secrete leptin
(2023)
Fermín Robledo
;
Lila González-Hodar
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Pablo Tapia
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Ana-María Figueroa
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EZQUER, EDUARDO FERNANDO
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Adipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation. </jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>SVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses. </jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Cultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater <jats:italic>Pparg</jats:italic>, <jats:italic>Plin1,</jats:italic> and <jats:italic>Lep</jats:italic> expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Newborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation.</jats:p> </jats:sec>
Scopus© Citations 9 1
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Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro
(2020)
Carolina Inostroza
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Ana María Vega-Letter
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Claudia Brizuela
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Luis Castrillón
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Nicole Saint Jean
22Scopus© Citations 20
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The Autophagy Protein Pacer Positively Regulates the Therapeutic Potential of Mesenchymal Stem Cells in a Mouse Model of DSS-Induced Colitis
(2022)
Cristian A. Bergmann
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Sebastian Beltran
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Ana Maria Vega-Letter
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Paola Murgas
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Maria Fernanda Hernandez
<jats:p>Mesenchymal stem cells (MSC) have emerged as a promising tool to treat inflammatory diseases, such as inflammatory bowel disease (IBD), due to their immunoregulatory properties. Frequently, IBD is modeled in mice by using dextran sulfate sodium (DSS)-induced colitis. Recently, the modulation of autophagy in MSC has been suggested as a novel strategy to improve MSC-based immunotherapy. Hence, we investigated a possible role of Pacer, a novel autophagy enhancer, in regulating the immunosuppressive function of MSC in the context of DSS-induced colitis. We found that Pacer is upregulated upon stimulation with the pro-inflammatory cytokine TNFα, the main cytokine released in the inflammatory environment of IBD. By modulating Pacer expression in MSC, we found that Pacer plays an important role in regulating the autophagy pathway in this cell type in response to TNFα stimulation, as well as in regulating the immunosuppressive ability of MSC toward T-cell proliferation. Furthermore, increased expression of Pacer in MSC enhanced their ability to ameliorate the symptoms of DSS-induced colitis in mice. Our results support previous findings that autophagy regulates the therapeutic potential of MSC and suggest that the augmentation of autophagic capacity in MSC by increasing Pacer levels may have therapeutic implications for IBD.</jats:p>
4Scopus© Citations 12
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Betaine-urea deep eutectic solvent improves imipenem antibiotic activity
(2022)
OLIVARES, MARIA BELEN
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Fabián A. Martínez
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Bernardo J. Morales
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EZQUER, EDUARDO MARCELO
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Margarita Calvo
Scopus© Citations 23 4
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Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24
(2022)
Sophia Davidson
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Chien-Hsiung Yu
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Annemarie Steiner
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Frédéric Ebstein
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Paul J. Baker
<jats:p>Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor–induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum–associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.</jats:p>
Scopus© Citations 55 3
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Ski Is Required for Tri-Methylation of H3K9 in Major Satellite and for Repression of Pericentromeric Genes: Mmp3, Mmp10 and Mmp13, in Mouse Fibroblasts
(2020)
Claudio Cappelli
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Hugo Sepulveda
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Solange Rivas
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Víctor Pola
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Ulises Urzúa
Scopus© Citations 2 5
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Microencapsulation of cellular aggregates composed of differentiated insulin and glucagon-producing cells from human mesenchymal stem cells derived from adipose tissue
(2020)
Claudia Jara
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Felipe Oyarzun-Ampuero
;
Flavio Carrión
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Esteban González-Echeverría
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Claudio Cappelli
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>In type I diabetes mellitus (T1DM) pancreatic β cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies.</jats:p> </jats:sec><jats:sec> <jats:title>Aims</jats:title> <jats:p>We postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>hASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>The results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of ~ 80 µm, and 65% of the aggregates reached 4000 µm<jats:sup>2</jats:sup> at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba<jats:sup>2+</jats:sup>, yielding average diameters of ~ 300 µm. Interestingly, Ba<jats:sup>2+</jats:sup>-microencapsulated aggregates respond to high external glucose with insulin secretion.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The IPC/GPC differentiation process from hASC, followed by the generation of cellular aggregates that are later microencapsulated, could represent a possible treatment for T1DM.</jats:p> </jats:sec>
16Scopus© Citations 9

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