Vibrational Spectroscopy for In Vitro Monitoring Stem Cell Differentiation

Stem cell technology has attracted considerable attention over recent decades due to its enormous potential in regenerative medicine and disease therapeutics. Studying the underlying mechanisms of stem cell differentiation and tissue generation is critical, and robust methodologies and different technologies are required. Towards establishing improved understanding and optimised triggering and control of differentiation processes, analytical techniques such as flow cytometry, immunohistochemistry, reverse transcription polymerase chain reaction, RNA in situ hybridisation analysis, and fluorescence-activated cell sorting have contributed much. However, progress in the field remains limited because such techniques provide only limited information, as they are only able to address specific, selected aspects of the process, and/or cannot visualise the process at the subcellular level. Additionally, many current analytical techniques involve the disruption of the investigation process (tissue sectioning, immunostaining) and cannot monitor the cellular differentiation process in situ, in real-time. Vibrational spectroscopy, as a label-free, non-invasive and non-destructive analytical technique, appears to be a promising candidate to potentially overcome many of these limitations as it can provide detailed biochemical fingerprint information for analysis of cells, tissues, and body fluids. The technique has been widely used in disease diagnosis and increasingly in stem cell technology. In this work, the efforts regarding the use of vibrational spectroscopy to identify mechanisms of stem cell differentiation at a single cell and tissue level are summarised. Both infrared absorption and Raman spectroscopic investigations are explored, and the relative merits, and future perspectives of the techniques are discussed

Monitoring Stem Cell Differentiation Using Raman Microspectroscopy: Chondrogenic Differentiation, Towards Cartilage Formation

Mesenchymal Stem Cells (MSCs) have the ability to differentiate into chondrocytes, the only cellular components of cartilage and are therefore ideal candidates for cartilage and tissue repair technologies. Chondrocytes are surrounded by cartilage-like extracellular matrix (ECM), a complex network rich in glycosaminoglycans, proteoglycans, and collagen, which, together with a multitude of intracellular signalling molecules, trigger the chondrogenesis and allow the chondroprogenitor to acquire the spherical morphology of the chondrocytes. However, although the mechanisms of the differentiation of MSCs have been extensively explored, it has been difficult to provide a holistic picture of the process, in situ. Raman Micro Spectroscopy (RMS) has been demonstrated to be a powerful analytical tool, which provides detailed label free biochemical fingerprint information in a non-invasive way, for analysis of cells, tissues and body fluids. In this work, RMS is explored to monitor the process of Mesenchymal Stem Cell (MSC) differentiation into chondrocytes in vitro, providing a holistic molecular picture of cellular events governing the differentiation. Spectral signatures of the subcellular compartments, nucleolus, nucleus and cytoplasm were initially probed and characteristic molecular changes between differentiated and undifferentiated were identified. Moreover, high density cell micromasses were cultured over a period of three weeks, and a systematic monitoring of cellular molecular components and the progress of the ECM formation, associated with the chondrogenic differentiation, was performed. This study shows the potential applicability of RMS as a powerful tool to monitor and better understand the differentiation pathways and process

Interfacial Properties of Colloidal Silica Dispersions in Contact with Solutions of Fatty Amines in Hexane

Many natural phenomena and technologies are concerned with the interactions between micro- or nano-metre sized particles and surfactant molecules at liquid interfaces. Highly stable emulsions are produced by using surfactants to modify the surfaces of nanoparticles. Particle attachment to bubbles is controlled by surfactant adsorption in flotation technologies. So far, however, few experimental studies have explored the properties of these complex interfacial layers

Evaluating the Impact of Hydrophobic Silicon Dioxide in the Interfacial Properties of Lung Surfactant Films

CRUE-CSIC (Acuerdos Transformativos 2022)The interaction of hydrophobic silicon dioxide particles (fumed silicon dioxide), as model air pollutants, and Langmuir monolayers of a porcine lung surfactant extract has been studied in order to try to shed light on the physicochemical bases underlying the potential adverse effects associated with pollutant inhalation. The surface pressure−area isotherms of lung surfactant (LS) films including increasing amounts of particles revealed that particle incorporation into LS monolayers modifies the organization of the molecules at the water/vapor interface, which alters the mechanical resistance of the interfacial films, hindering the ability of LS layers for reducing the surface tension, and reestablishing the interface upon compression. This influences the normal physiological function of LS as is inferred from the analysis of the response of the Langmuir films upon the incorporation of particles against harmonic changes of the interfacial area (successive compression−expansion cycles). These experiments evidenced that particles alter the relaxation mechanisms of LS films, which may be correlated to a modification of the transport of material within the interface and between the interface and the adjacent fluid during the respiratory cycle.Depto. de Química FísicaFac. de Ciencias QuímicasTRUEUnión Europea. Horizonte 2020Ministerio de Ciencia e Innovación (MICINN)pu

Effect of Silica Nanoparticles on Rheological and Structural Properties of DPPC Langmuir Monolayers

The study of the interaction between nanoparticles and model of lung surfactant is of great relevance to understand possible adverse effects of inhalable nanoparticles on the respiratory function [1,2]. We report therefore a study on the interfacial properties, structure and dilational rheology of monolayers of the major lipidic component of the lung surfactant, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in the presence of silica nanoparticles. Investigations are performed by a Langmuir trough equipped with Brewster Angle Microscopy

Incremental peritoneal dialysis: a 10 year single-centre experience

INTRODUCTION: Incremental dialysis consists in prescribing a dialysis dose aimed towards maintaining total solute clearance (renal + dialysis) near the targets set by guidelines. Incremental peritoneal dialysis (incrPD) is defined as one or two dwell-times per day on CAPD, whereas standard peritoneal dialysis (stPD) consists in three-four dwell-times per day. PATIENTS AND METHODS: Single-centre cohort study. Enrollement period: January 2002-December 2007; end of follow up (FU): December 2012. INCLUSION CRITERIA: incident patients with FU ≥6 months, initial residual renal function (RRF) 3-10 ml/min/1.73 sqm BSA, renal indication for PD. RESULTS: Median incrPD duration was 17 months (I-III Q: 10; 30). There were no statistically significant differences between 29 patients on incrPD and 76 on stPD regarding: clinical, demographic and anthropometric characteristics at the beginning of treatment, adequacy indices, peritonitis-free survival (peritonitis incidence: 1/135 months-patients in incrPD vs. 1/52 months-patients in stPD) and patient survival. During the first 6 months, RRF remained stable in incrPD (6.20 ± 2.02 vs. 6.08 ± 1.47 ml/min/1.73 sqm BSA; p = 0.792) whereas it decreased in stPD (4.48 ± 2.12 vs. 5.61 ± 1.49; p < 0.001). Patient survival was affected negatively by ischemic cardiopathy (HR: 4.269; p < 0.001), peripheral and cerebral vascular disease (H2.842; p = 0.006) and cirrhosis (2.982; p = 0.032) and positively by urine output (0.392; p = 0.034). Hospitalization rates were significantly lower in incrPD (p = 0.021). Eight of 29 incrPD patients were transplanted before reaching full dose treatment. CONCLUSIONS: IncrPD is a safe modality to start PD; compared to stPD, it shows similar survival rates, significantly less hospitalization, a trend towards lower peritonitis incidence and slower reduction of renal function

Altered glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops due to motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, while perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analysed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment. This article is protected by copyright. All rights reserved