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.

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.

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.

Insulin-like growth factor 1 increases apical fibronectin in blastocysts to increase blastocyst attachment to endometrial epithelial cells in vitro.

STUDY QUESTION: Does insulin-like growth factor 1 (IGF1) increase adhesion competency of blastocysts to increase attachment to uterine epithelial cells in vitro? SUMMARY ANSWER: IGF1 increases apical fibronectin on blastocysts to increase attachment and invasion in an in vitro model of implantation. WHAT IS KNOWN ALREADY: Fibronectin integrin interactions are important in attachment of blastocysts to uterine epithelial cells at implantation. STUDY DESIGN, SIZE, DURATION: Mouse blastocysts (hatched or near completion of hatching) were cultured in serum starved (SS) medium with varying treatments for 24, 48 or 72 h. Treatments included 10 ng/ml IGF1 in the presence or absence of the PI3 kinase inhibitor LY294002, an IGF1 receptor (IGF1R) neutralizing antibody or fibronectin. Effects of treatments on blastocysts were measured by attachment of blastocysts to Ishikawa cells, blastocyst outgrowth and fibronectin and focal adhesion kinase (FAK) localization and expression. Blastocysts were randomly allocated into control and treatment groups and experiments were repeated a minimum of three times with varying numbers of blastocysts used in each experiment. FAK and integrin protein expression on Ishikawa cells was quantified in the presence or absence of IGF1. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fibronectin expression and localization in blastocysts was studied using immunofluorescence and confocal microscopy. Global surface expression of integrin αvß3, ß3 and ß1 was measured in Ishikawa cells using flow cytometry. Expression levels of phosphorylated FAK and total FAK were measured in Ishikawa cells and blastocysts by western blot and image J analysis. Blastocyst outgrowth was quantified using image J analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The presence of IGF1 significantly increased mouse blastocyst attachment to Ishikawa cells compared with SS conditions (P < 0.01). IGF1 treatment resulted in distinct apical fibronectin staining on blastocysts, which was reduced by the PI3 kinase inhibitor LY294002. This coincided with a significant increase in blastocyst outgrowth in the presence of IGF1 (P < 0.01) or fibronectin (P < 0.001), which was abolished by LY294002 (P < 0.001). Apical expression of integrin αvß3, ß3 and ß1 in Ishikawa cells was unaltered by IGF1. However, IGF1 increased phosphorylated FAK (P < 0.05) and total FAK expression in Ishikawa cells. FAK signalling is linked to integrin activation and can affect the integrins' ability to bind and recognize extracellular matrix proteins such as fibronectin. Treatment of blastocysts with IGF1 before co-culture with Ishikawa cells increased their attachment (P < 0.05). This effect was abolished in the presence of LY294002 (P < 0.001) or an IGF1R neutralizing antibody (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study uses an in vitro model of attachment that uses mouse blastocysts and human endometrial cells. This involves a species crossover and although this use has been well documented as a model for attachment (as human embryo numbers are limited) the results should be interpreted carefully. WIDER IMPLICATIONS OF THE FINDINGS: This study presents mechanisms by which IGF1 improves attachment of blastocysts to Ishikawa cells and documents for the first time how IGF1 can increase adhesion competency in blastocysts. Failure of the blastocyst to implant is the major cause of human assisted reproductive technology (ART) failure. As growth factors are absent during embryo culture, their addition to embryo culture medium is a potential avenue to improve IVF success. In particular, IGF1 could prove to be a potential treatment for blastocysts before transfer to the uterus in an ART setting.

Expression of podocalyxin separates the hematopoietic and vascular potentials of mouse embryonic stem cell-derived mesoderm.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages.

Heme oxygenase-1 deficiency alters erythroblastic island formation, steady-state erythropoiesis and red blood cell lifespan in mice.

Heme oxygenase-1 is critical for iron recycling during red blood cell turnover, whereas its impact on steady-state erythropoiesis and red blood cell lifespan is not known. We show here that in 8- to 14-week old mice, heme oxygenase-1 deficiency adversely affects steady-state erythropoiesis in the bone marrow. This is manifested by a decrease in Ter-119(+)-erythroid cells, abnormal adhesion molecule expression on macrophages and erythroid cells, and a greatly diminished ability to form erythroblastic islands. Compared with wild-type animals, red blood cell size and hemoglobin content are decreased, while the number of circulating red blood cells is increased in heme oxygenase-1 deficient mice, overall leading to microcytic anemia. Heme oxygenase-1 deficiency increases oxidative stress in circulating red blood cells and greatly decreases the frequency of macrophages expressing the phosphatidylserine receptor Tim4 in bone marrow, spleen and liver. Heme oxygenase-1 deficiency increases spleen weight and Ter119(+)-erythroid cells in the spleen, although α4ß1-integrin expression by these cells and splenic macrophages positive for vascular cell adhesion molecule 1 are both decreased. Red blood cell lifespan is prolonged in heme oxygenase-1 deficient mice compared with wild-type mice. Our findings suggest that while macrophages and relevant receptors required for red blood cell formation and removal are substantially depleted in heme oxygenase-1 deficient mice, the extent of anemia in these mice may be ameliorated by the prolonged lifespan of their oxidatively stressed erythrocytes.

Mitochondrially targeted redox probe reveals the variations in oxidative capacity of the haematopoietic cells.

Both oxidative stress and mitochondrial dysfunction play roles in a myriad of pathological conditions. There is therefore a need for tools that possess the ability to measure the dynamics of oxidative capacity within the mitochondria, particularly those that can measure reversible changes. Here, we report a mitochondrially-targeted fluorescent redox sensor NpFR2, which can reversibly measure changes in the mitochondrial redox environment. The probe has been used to report on variations in oxidative capacity of the haematopoietic cells in bone marrow, thymus and spleen.

Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts.

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3-6.6) of feline hosts or the reproductive tract (pH 7.4-7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro. Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity.

The embryonic origins of erythropoiesis in mammals.

Erythroid (red blood) cells are the first cell type to be specified in the postimplantation mammalian embryo and serve highly specialized, essential functions throughout gestation and postnatal life. The existence of 2 developmentally and morphologically distinct erythroid lineages, primitive (embryonic) and definitive (adult), was described for the mammalian embryo more than a century ago. Cells of the primitive erythroid lineage support the transition from rapidly growing embryo to fetus, whereas definitive erythrocytes function during the transition from fetal life to birth and continue to be crucial for a variety of normal physiologic processes. Over the past few years, it has become apparent that the ontogeny and maturation of these lineages are more complex than previously appreciated. In this review, we highlight some common and distinguishing features of the red blood cell lineages and summarize advances in our understanding of how these cells develop and differentiate throughout mammalian ontogeny.

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice.

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5-E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo.

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-ß1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.

Dose-dependent regulation of primitive erythroid maturation and identity by the transcription factor Eklf.

The primitive erythroid (EryP) lineage is the first to differentiate during mammalian embryogenesis. Eklf/Klf1 is a transcriptional regulator that is essential for definitive erythropoiesis in the fetal liver. Dissection of the role(s) of Eklf within the EryP compartment has been confounded by the simultaneous presence of EryP and fetal liver-derived definitive erythroid (EryD) cells in the blood. To address this problem, we have distinguished EryP from their definitive counterparts by crossing Eklf(+/-) mutant and ε-globin::histone H2B-GFP transgenic mice. Eklf-deficient EryP exhibit membrane ruffling and a failure to acquire the typical discoidal erythroid shape but they can enucleate. Flow cytometric analyses of H2B-GFP(+) EryP revealed that Eklf heterozygosity results in the loss of Ter119 surface expression on EryP but not on EryD. Null mutation of Eklf resulted in abnormal expression of a range of surface proteins by EryP. In particular, several megakaryocyte markers were ectopically expressed by maturing Eklf-null EryP. Unexpectedly, the platelet tetraspanin CD9 was detected on nucleated wild-type EryP but not on mature EryD and thus provides a useful marker for purifying circulating EryP. We conclude that Eklf gene dosage is crucial for regulating the surface phenotype and molecular identity of maturing primitive erythroid cells.

The fetal liver is a niche for maturation of primitive erythroid cells.

Primitive erythroid cells (EryP) are the earliest differentiated cell type of the mammalian embryo. They appear in the yolk sac by embryonic day 7.5, begin to enter the embryonic circulation 2 days later and continue to mature in a stepwise and synchronous fashion. Like their adult counterparts, EryP enucleate. However, EryP circulate throughout the embryo for several days before the first enucleated forms can be identified in the blood. We have used transgenic mouse lines in which GFP marks EryP to investigate this seemingly long lag and have identified a previously unrecognized developmental niche for EryP maturation. After exiting the yolk sac, EryP begin to express cell adhesion proteins, including alpha4, alpha5, and beta1 integrins, on their surface and migrate into the fetal liver (FL), where they interact with macrophages within erythroblastic islands. Binding of EryP to FL macrophages in vitro is stage-specific and partly depends on VCAM-1. The ability to tag and track EryP nuclei using a transgenic mouse line expressing an H2B-EGFP fusion allowed us to identify and characterize extruded EryP nuclei and to demonstrate that molecules such as alpha4, alpha5, and beta1 integrins are redistributed onto the plasma membrane surrounding the extruding nucleus. FL macrophages engulf extruded EryP nuclei in cocultures and in the native FL in vivo. We conclude that EryP home to, complete their maturation, and enucleate within the FL, a tissue that is just developing as EryP begin to circulate. Our observations suggest a simple solution for a puzzling aspect of the development of the primitive erythroid lineage.

Embryonic regulation of the mouse hematopoietic niche.

Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells (HCs) (such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages) and also undergo self-renewal to sustain hematopoiesis throughout an organism's lifetime. HSCs are currently used clinically as transplantation therapy in regenerative medicine and are typically obtained from healthy donors or cord blood. However, problems remain in HSC transplantation, such as shortage of cells, donor risks, rejection, and graft-versus-host disease (GVHD). Thus, increased understanding of HSC regulation should enable us to improve HSC therapy and develop novel regenerative medicine techniques. HSC regulation is governed by two types of activity: intrinsic regulation, programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from so-called "niche cells" surrounding HSCs. Here, we focus on the latter and discuss HSC regulation with special emphasis on the role played by niche cells.

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.

Developmental niches for embryonic erythroid cells.

Primitive erythroid cells (EryP) are the first differentiated cell type to be specified during mammalian embryogenesis. EryP arise from a pool of lineage-restricted progenitors in the yolk sac (YS) and then enter the newly formed embryonic circulation to mature in a stepwise, synchronous fashion. Numbering in the millions in the mid-gestation mouse embryo, EryP are the dominant circulating blood cell prior to the rapid generation of adult-type definitive erythroid (EryD) cells in the fetal liver. The identification of maturational events in this lineage presented a significant challenge, as EryD begin to outnumber EryP in the bloodstream from approximately E14.5 onwards. We used human epsilon-globin gene regulatory elements to drive lineage-specific expression of a histone-H2B::EGFP fusion protein, allowing us to label the chromatin of EryP during their development and to track and quantify EryP nuclei following their expulsion from the cell. Using this transgenic fluorescent reporter mouse line, we have monitored primitive erythropoiesis in three distinct niches: the YS, where EryP progenitors arise; the circulation, where EryP continue to divide and mature; and the fetal liver, where EryP complete the terminal stages of their differentiation.

Transcriptional activation by the Mixl1 homeodomain protein in differentiating mouse embryonic stem cells.

Members of the Mix/Bix family of paired class homeobox genes play important roles in the development of vertebrate mesoderm and endoderm. The single Mix/Bix family member identified in the mouse, Mix-like 1 (Mixl1), is required for mesendoderm patterning during gastrulation and promotes mesoderm formation and hematopoiesis in embryonic stem cell (ESC)-derived embryoid bodies. Despite its crucial functions the transcriptional activity and targets of Mixl1 have not been well described. To investigate the molecular mechanisms of Mixl1-mediated transcriptional regulation, we have characterized the DNA-binding specificity and transcriptional properties of this homeodomain protein in differentiating ESCs. Mixl1 binds preferentially as a dimer to an 11-base pair (bp) Mixl1 binding sequence (MBS) that contains two inverted repeats separated by a 3-bp spacer. The MBS mediates transcriptional activation by Mixl1 in both NIH 3T3 cells and in a new application of an inducible ESC differentiation system. Consistent with our previous observation that early induction of Mixl1 expression in ESCs results in premature activation of Goosecoid (Gsc), we have found that Mixl1 occupies two variant MBSs within and activates transcription from the Gsc promoter in vitro and in vivo. These results strongly suggest that Gsc is a direct target gene of Mixl1 during embryogenesis. STEM CELLS 2009;27:2884-2895.

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.

Embryonic fates for extraembryonic lineages: New perspectives.

The prevailing view of the functions of the extraembryonic lineages of the mammalian embryo has been that they serve solely to support its intrauterine development. In recent years, a number of studies have suggested that the extraembryonic mesoderm and visceral endoderm in fact contribute cells to tissues of the developing animal. In this mini-review, we discuss evidence that the yolk sac is an early source of hematopoietic stem and progenitor cells and that the cells of the visceral endoderm, once thought to be segregated solely to the yolk sac, constitute a subpopulation of cells within the developing gut tube and perhaps other endodermal structures. Fascinating questions remain to be addressed and are likely to establish a new paradigm for studying early mammalian development. Understanding the processes that give rise to stem cell populations in development may lead to advances in stem cell therapies and regenerative medicine.

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.