Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Thermally drawn biodegradable fibers with tailored topography for biomedical applications.

There is a growing demand for polymer fiber scaffolds for biomedical applications and tissue engineering. Biodegradable polymers such as polycaprolactone have attracted particular attention due to their applicability to tissue engineering and optical neural interfacing. Here we report on a scalable and inexpensive fiber fabrication technique, which enables the drawing of PCL fibers in a single process without the use of auxiliary cladding. We demonstrate the possibility of drawing PCL fibers of different geometries and cross-sections, including solid-core, hollow-core, and grooved fibers. The solid-core fibers of different geometries are shown to support cell growth, through successful MCF-7 breast cancer cell attachment and proliferation. We also show that the hollow-core fibers exhibit a relatively stable optical propagation loss after submersion into a biological fluid for up to 21 days with potential to be used as waveguides in optical neural interfacing. The capacity to tailor the surface morphology of biodegradable PCL fibers and their non-cytotoxicity make the proposed approach an attractive platform for biomedical applications and tissue engineering.

Detection of cell-surface phosphatidylserine using the fluorogenic probe P-IID.

The fluorogenic probe P-IID enables the detection of cell-surface phosphatidylserine (PS) using both fluorescence imaging and flow cytometry. Here we provide a detailed protocol for the use of P-IID for the qualitative detection of externalized PS in apoptotic cells using confocal microscopy, including the real-time imaging of apoptosis upon drug treatment. We also provide a detailed method for the quantitative analysis of cell death by flow cytometry, using P-IID in conjunction with the nuclear stain propidium iodide. P-IID is superior to commonly used Annexin-V fluorophore conjugates for PS detection as it provides a "turn-on" fluorescence response, displays rapid binding kinetics and can be used at low temperature (4°C), without washing and in the absence of Ca2+ ions.

Cellular dynamics of mammalian red blood cell production in the erythroblastic island niche.

Red blood cells, or erythrocytes, make up approximately a quarter of all cells in the human body with over 2 billion new erythrocytes made each day in a healthy adult human. This massive cellular production system is coupled with a set of cell biological processes unique to mammals, in particular, the elimination of all organelles, and the expulsion and destruction of the condensed erythroid nucleus. Erythrocytes from birds, reptiles, amphibians and fish possess nuclei, mitochondria and other organelles: erythrocytes from mammals lack all of these intracellular components. This review will focus on the dynamic changes that take place in developing erythroid cells that are interacting with specialized macrophages in multicellular clusters termed erythroblastic islands. Proerythroblasts enter the erythroblastic niche as large cells with active nuclei, mitochondria producing heme and energy, and attach to the central macrophage via a range of adhesion molecules. Proerythroblasts then mature into erythroblasts and, following enucleation, in reticulocytes. When reticulocytes exit the erythroblastic island, they are smaller cells, without nuclei and with few mitochondria, possess some polyribosomes and have a profoundly different surface molecule phenotype. Here, we will review, step-by-step, the biophysical mechanisms that regulate the remarkable process of erythropoiesis with a particular focus on the events taking place in the erythroblastic island niche. This is presented from the biological perspective to offer insight into the elements of red blood cell development in the erythroblastic island niche which could be further explored with biophysical modelling systems.

Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.

The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution of biologically pertinent questions. Here a method for differentiating urothelium from mouse embryonic stem cells in chemically defined conditions is described. The method includes a description of flow cytometry and RT-PCR analysis of definitive endoderm markers Cxcr4, c-Kit, and FoxA2, and of terminally differentiated urothelial cell markers Upk1b and Upk2.

Identification and Analysis of Mouse Erythroid Progenitor Cells.

The most common cell type in the human body, the red blood cell or erythrocyte, has a life span of approximately 3 months. To compensate for this massive cellular requirement and short life span, the major blood producing tissues contain vast numbers of erythroid progenitor cells. Erythroid progenitors differentiate progressively from hematopoietic stem cells to committed erythroid progenitors to reticulocytes lacking a nucleus and finally to functionally mature erythrocytes in the circulation. Different erythroid progenitor activity, representative of distinct stages of erythropoiesis, can be observed using semisolid colony assays. Distinct stages of erythroid maturation can also be monitored by flow cytometry. Here, we discuss the range of different technical approaches that are used to identify and quantify erythroid progenitors, with particular focus on the mouse as a model system.

The iron islands: Erythroblastic islands and iron metabolism.

BACKGROUND: A healthy human can produce over 1 × 1015 blood cells throughout their life. This remarkable amount of biomass requires a concomitantly vast amount of iron to generate functional haemoglobin and functional erythrocytes. SCOPE OF THE REVIEW: Erythroblasts form multicellular clusters with macrophages in the foetal liver, bone marrow and spleen termed erythroblastic islands. How the central erythroblastic island macrophage co-ordinates the supply of iron to the developing erythroblasts will be a central focus of this review. MAJOR CONCLUSION: Despite being studied for over 60 years, the mechanisms by which the erythroblastic island niche serves to control erythroid cell iron metabolism are poorly resolved. GENERAL SIGNIFICANCE: Over 2 billion people suffer from some form of anaemia. Iron deficiency anaemia is the most prevalent form of anaemia. Therefore, understanding the processes by which iron is trafficked to, and metabolised in developing erythrocytes, is crucially important.

A Fluorogenic Probe for Cell Surface Phosphatidylserine Using an Intramolecular Indicator Displacement Sensing Mechanism.

The detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic, and necrotic cells. The tools of choice for many researchers to study apoptosis are annexin V-fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks, including temperature sensitivity, Ca2+ dependence, and slow binding kinetics. Herein, a fluorogenic probe for cell surface PS, P-IID, is described, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS, leading to a fluorescence "turn-on" response. P-IID demonstrates superior performance when compared to annexin V, for both fluorescence imaging and flow cytometry. This allows P-IID to be used in time-lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells.

Induced pluripotent stem cell technology and inherited arrhythmia syndromes.

Inherited arrhythmia syndromes, including familial long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and Brugada syndrome, can cause life-threatening arrhythmias and are responsible for a significant proportion of sudden deaths in the young. Identification of genetic mutations and pathophysiological changes that underlie disease development can inform clinical practice and guide novel drug development. However, disease mechanisms in a large number of patients remain elusive and pharmacologic treatment is suboptimal, so many patients rely on implantable cardioverter-defibrillator therapy. Induced pluripotent stem cell models of disease facilitate analysis of disease mechanisms in patient-specific cardiomyocytes, overcoming limitations of animal models and human tissue restrictions. This review outlines how studies using induced pluripotent stem cell-derived cardiomyocytes are contributing to our understanding of the mechanisms that underpin disease pathogenesis and their potential to facilitate new pharmacologic therapies and personalized medicine.

Analyzing the Formation, Morphology, and Integrity of Erythroblastic Islands.

The bone marrow is the primary site of erythropoiesis in healthy adult mammals. In the bone marrow, erythroid cells mature within specialized microenvironments termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters comprised of a central macrophage surrounded by red blood cell (erythroid) progenitors. It has been proposed that the central macrophage functions as a "nurse-cell" providing iron, cytokines, and growth factors for the developing erythroid cells. The central macrophage also engulfs and destroys extruded erythroid nuclei. EBIs have recently been shown to play clinically important roles during human hematological disease. The molecular mechanisms regulating this hematopoietic niche are largely unknown. In this chapter, we detail protocols to study isolated EBIs using multiple microscopy platforms. Adhesion molecules regulate cell-cell interactions within the EBI and maintain the integrity of the niche. To improve our understanding of the molecular regulation of erythroid cells in EBIs, we have developed protocols for immuno-gold labeling of erythroid surface antigens to combine with scanning electron microscopy. These protocols have allowed imaging of EBIs at the nanometer scale, offering novel insights into the processes regulating red blood cell production.

Splenomegaly: Pathophysiological bases and therapeutic options.

The spleen is the largest immune organ in the human body and is also essential for red blood cell homeostasis and iron recycling. An average human spleen is approximately 10 centimetres in length and weighs 150g. Pathological conditions can result in the spleen weighing in excess of 2000g and extending over 30 centrimetres in length. This remarkable property of the spleen to expand is termed splenomegaly. Splenomegaly can occur as a physiological response to stress or as a chronic process that is often detrimental to the wellbeing of the individual. Here, we will discuss the normal function and physiology of the spleen, the pathophysiological bases of splenomegaly and the commonly available therapeutic options. Additionally we will address experimental systems to determine the regulatory mechanisms underlying splenomegaly.

Investigation of the Tissue Distribution and Physiological Roles of Indoleamine 2,3-Dioxygenase-2.

Indoleamine 2,3-dioxygenase-2 (IDO2) is 1 of the 3 enzymes that can catalyze the first step in the kynurenine pathway of tryptophan metabolism. Of the 2 other enzymes, tryptophan 2,3-dioxygenase is highly expressed in the liver and has a role in tryptophan homeostasis, whereas indoleamine 2,3-dioxygenase-1 (IDO1) expression is induced by inflammatory stimuli. Indoleamine 2,3-dioxygenase-2 is reportedly expressed comparatively narrow, including in liver, kidney, brain, and in certain immune cell types, and it does not appear to contribute significantly to systemic tryptophan catabolism under normal physiological conditions. Here, we report the identification of an alternative splicing pattern, including the use of an alternative first exon, that is conserved in the mouse Ido1 and Ido2 genes. These findings prompted us to assess IDO2 protein expression and enzymatic activity in tissues. Our analysis, undertaken in Ido2 +/+ and Ido2-/- mice using immunohistochemistry and measurement of tryptophan and kynurenine levels, suggested an even more restricted pattern of tissue expression than previously reported. We found IDO2 protein to be expressed in the liver with a perinuclear/nuclear, rather than cytoplasmic, distribution. Consistent with earlier reports, we found Ido2 -/- mice to be phenotypically similar to their Ido2+/+ counterparts regarding levels of tryptophan and kynurenine in the plasma and liver. Our findings suggest a specialized function or regulatory role for IDO2 associated with its particular subcellular localization.

A Carborane-Containing Fluorophore as a Stain of Cellular Lipid Droplets.

The use of fluorescent markers and probes greatly enhances biological investigations but relies on the provision of an array of fluorophores with diverse properties. Herein we report a novel carborane-containing coumarin, 5, which is sufficiently lipophilic to localise in cellular lipid droplets. In non-polar solvents which show comparable polarities to those of a lipid environment, compound 5 exhibits a fluorescence quantum yield two orders of magnitude greater than found in aqueous solvents, adding a further degree of selectivity to lipid droplet imaging. Compound 5 can stain lipid droplets in ex vivo adipocytes as well as in cultured cells, and can be utilised in flow cytometry as well as confocal microscopy.

Regulation of the embryonic erythropoietic niche: a future perspective.

The production of red blood cells, termed erythropoiesis, occurs in two waves in the developing mouse embryo: first primitive erythropoiesis followed by definitive erythropoiesis. In the mouse embryo, both primitive and definitive erythropoiesis originates in the extra-embryonic yolk sac. The definitive wave then migrates to the fetal liver, fetal spleen and fetal bone marrow as these organs form. The fetal liver serves as the major organ for hematopoietic cell expansion and erythroid maturation after mid-gestation. The erythropoietic niche, which expresses critical cytokines such as stem cell factor (SCF), thrombopoietin (TPO) and the insulin-like growth factors IGF1 and IGF2, supports hematopoietic expansion in the fetal liver. Previously, our group demonstrated that DLK1+ hepatoblasts support fetal liver hematopoiesis through erythropoietin and SCF release as well as extracellular matrix deposition. Loss of DLK1+ hepatoblasts in Map2k4-/- mouse embryos resulted in decreased numbers of hematopoietic cells in fetal liver. Genes encoding proteinases and peptidases were found to be highly expressed in DLK1+ hepatoblasts. Capitalizing on this knowledge, and working on the assumption that these proteinases and peptidases are generating small, potentially biologically active peptides, we assessed a range of peptides for their ability to support erythropoiesis in vitro. We identified KS-13 (PCT/JP2010/067011) as an erythropoietic peptide-a peptide which enhances the production of red blood cells from progenitor cells. Here, we discuss the elements regulating embryonic erythropoiesis with special attention to niche cells, and demonstrate how this knowledge can be applied in the identification of niche-derived peptides with potential therapeutic capability.

Flow cytometric single cell analysis reveals heterogeneity between adipose depots.

Understanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level.

Peripheral blood derived induced pluripotent stem cells (iPSCs) from a female with familial hypertrophic cardiomyopathy.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) obtained from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM). PBMCs were reprogrammed to a pluripotent state following transfection with non-integrative episomal vectors carrying reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotency markers, possess trilineage differentiation potential, carry rare variants identified in DNA isolated directly from the patient's whole blood, have a normal karyotype and no longer carry episomal vectors for reprogramming. This line is a useful resource for identifying unknown genetic causes of HCM.

Generation of induced pluripotent stem cells (iPSCs) from a hypertrophic cardiomyopathy patient with the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7) gene.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM) who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7). Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4, SOX2, LIN28, KLF4 and L-MYC. iPSCs were shown to express pluripotent markers, have trilineage differentiation potential, carry the pathogenic MYH7 variant p.Val698Ala, have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM.

Induced pluripotent stem cells in the inherited cardiomyopathies: From disease mechanisms to novel therapies.

Inherited cardiomyopathies lead to diverse clinical outcomes including heart failure, arrhythmias, and sudden death. Mutations in over 100 genes have been implicated in the pathogenesis of genetic heart diseases, including the main inherited cardiomyopathies, such as hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathies. Understanding how these gene mutations lead to clinical disease and the various secondary genetic and environmental factors, which may modify the clinical phenotype, are key areas of research ultimately influencing diagnosis and management of patients. The emergence of patient-derived induced pluripotent stem cells (iPSCs), which can be differentiated into functional cardiomyocytes (CMs) in vitro, may provide an exciting new approach to understand disease mechanisms underpinning inherited heart diseases. This review will focus specifically on the key role of iPSC-based studies in the inherited cardiomyopathies, both in their potential utility as well as the significant challenges they present.

Scanning Electron Microscopy Reveals Two Distinct Classes of Erythroblastic Island Isolated from Adult Mammalian Bone Marrow.

Erythroblastic islands are multicellular clusters in which a central macrophage supports the development and maturation of red blood cell (erythroid) progenitors. These clusters play crucial roles in the pathogenesis observed in animal models of hematological disorders. The precise structure and function of erythroblastic islands is poorly understood. Here, we have combined scanning electron microscopy and immuno-gold labeling of surface proteins to develop a better understanding of the ultrastructure of these multicellular clusters. The erythroid-specific surface antigen Ter-119 and the transferrin receptor CD71 exhibited distinct patterns of protein sorting during erythroid cell maturation as detected by immuno-gold labeling. During electron microscopy analysis we observed two distinct classes of erythroblastic islands. The islands varied in size and morphology, and the number and type of erythroid cells interacting with the central macrophage. Assessment of femoral marrow isolated from a cavid rodent species (guinea pig, Cavis porcellus) and a marsupial carnivore species (fat-tailed dunnarts, Sminthopsis crassicaudata) showed that while the morphology of the central macrophage varied, two different types of erythroblastic islands were consistently identifiable. Our findings suggest that these two classes of erythroblastic islands are conserved in mammalian evolution and may play distinct roles in red blood cell production.

Studies of Hematopoietic Cell Differentiation with a Ratiometric and Reversible Sensor of Mitochondrial Reactive Oxygen Species.

AIMS: Chronic elevations in cellular redox state are known to result in the onset of various pathological conditions, but transient increases in reactive oxygen species (ROS)/reactive nitrogen species (RNS) are necessary for signal transduction and various physiological functions. There is a distinct lack of reversible fluorescent tools that can aid in studying and unraveling the roles of ROS/RNS in physiology and pathology by monitoring the variations in cellular ROS levels over time. In this work, we report the development of ratiometric fluorescent sensors that reversibly respond to changes in mitochondrial redox state. RESULTS: Photophysical studies of the developed flavin-rhodamine redox sensors, flavin-rhodamine redox sensor 1 (FRR1) and flavin-rhodamine redox sensor 2 (FRR2), confirmed the reversible response of the probes upon reduction and re-oxidation over more than five cycles. The ratiometric output of FRR1 and FRR2 remained unaltered in the presence of other possible cellular interferants (metals and pH). Microscopy studies indicated clear mitochondrial localization of both probes, and FRR2 was shown to report the time-dependent increase of mitochondrial ROS levels after lipopolysaccharide stimulation in macrophages. Moreover, it was used to study the variations in mitochondrial redox state in mouse hematopoietic cells at different stages of embryonic development and maturation. INNOVATION: This study provides the first ratiometric and reversible probes for ROS, targeted to the mitochondria, which reveal variations in mitochondrial ROS levels at different stages of embryonic and adult blood cell production. CONCLUSIONS: Our results suggest that with their ratiometric and reversible outputs, FRR1 and FRR2 are valuable tools for the future study of oxidative stress and its implications in physiology and pathology. Antioxid. Redox Signal. 24, 667-679.