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PubMedDate not supplied

Characterization of the eyespot regions of "blind" Chlamydomonas mutants after restoration of photophobic responses.

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2-4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Magnetically labeled cells can be detected by MR imaging.

To determine the feasibility of MR imaging of magnetically labeled cells, different cell lines were labeled with monocrystalline iron oxide (MION) particles. Phantoms containing MION labeled cells were then assembled and imaged by MR at 1.5 T using T1-weighted and T2-weighted pulse sequences. MION uptake ranged from 8.5 x 10(4) to 2.9 x 10(5) particles/cell for tumor cells (9L and LX1, respectively) to 1.5 x 10(6) to 4.8 x 10(8) particles/cell for "professional phagocytes" (J774 and peritoneal macrophages, respectively). On the T1-weighted images, cell-internalized MION appeared hyperintense relative to agar and similar to MION in aqueous solution. On T2-weighted images, signal intensity varied according to concentration of MION within cells. Cell-internalized MION caused similar MR signal changes of cells as did free MION; however, at a dose that was an order of magnitude lower, depending on the pulse sequence used. The detectability of MION within cells was approximately 2 ng Fe, which corresponded to 10(5) tumor cells/well or 5 x 10(3) macrophages/well. We conclude that a variety of cells can be efficiently labeled with MION by simple incubation. Intracellular labeling may be used for MR imaging of in vivo cell tracking.

Contrast Media↗

PubMed2006

Imaging stem cells implanted in infarcted myocardium.

Stem cell-based cellular cardiomyoplasty represents a promising therapy for myocardial infarction. Noninvasive imaging techniques would allow the evaluation of survival, migration, and differentiation status of implanted stem cells in the same subject over time. This review describes methods for cell visualization using several corresponding noninvasive imaging modalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cell visualization is compared with direct cell labeling for short- and long-term cell tracking.

Animals↗

PubMed2001

Circulating macrophages as well as developing thymocytes are enclosed within thymic nurse cells.

Both thymic nurse cells (TNCs) and macrophages have been reported to function as antigen-presenting cells during the process of MHC restriction. Negative selection, which results in the apoptosis of potentially autoreactive thymocytes, is believed to be associated with both macrophages and TNCs in the cortex. Both cell types have also been reported to ingest thymocytes undergoing positive and negative selection. However, macrophages ingest apoptotic thymocytes, while TNCs have been shown to internalize viable cells. A subset of the TNC-engulfed population is allowed to mature and is released, while the remaining fraction becomes apoptotic and is absorbed within the TNC cytoplasm through lysosomal activity. A recent report described a subset of rat TNCs that contain macrophages as well as thymocytes within their cytoplasm. We examined freshly isolated TNCs from C57BL/6 mice and found that, of the TNC population recovered, 1.7% contained macrophages within its cytoplasm. There also were macrophages tightly bound but not internalized into the multicellular structure at a rate of 2.9%. The total association of macrophages with TNCs was approximately 4.6%. This unique association of macrophages with TNCs was also observed in vitro when freshly isolated thymocytes (containing macrophages) were added to cultures of cells from the TNC cell line tsTNC-1. The macrophage-TNC interaction was found to be dynamic, with macrophages moving rapidly into and out of TNCs containing cytoplasmic thymocytes. Macrophages within TNCs showed a close association with cytoplasmic thymocytes. We then labeled peritoneal macrophages with CFDA SE, a cell tracking dye, and returned them to the mouse peritoneum. Within 1 h, labeled macrophages were detectable in the thymus. This is the first investigation to show a direct interaction between peripheral macrophages and TNCs. These results suggest that TNCs and macrophages work together as antigen-presenting cells.

Animals↗

PubMed2006

Growth factor-stimulated generation of new cortical tissue and functional recovery after stroke damage to the motor cortex of rats.

Recent studies suggest that proliferation in the adult forebrain subventricular zone increases in response to a forebrain stroke and intraventricular infusions of growth factors enhance this response. The potential for growth factor infusions to regenerate the damaged motor cortex and promote recovery of motor function after stroke has not been examined. Here, we report that intraventricular infusions of epidermal growth factor and erythropoietin together, but not individually, promote substantial regeneration of the damaged cerebral cortex and reverse impairments in spontaneous and skilled motor tasks, in a rat model of stroke. Cortical regeneration and functional recovery occurred even when growth factor administration was delayed for up to 7 days after the stroke-induced lesion. Cell tracking demonstrated the contribution of neural precursors originating in the forebrain subventricular zone to the regenerated cortex. Strikingly, removal of the regenerated cortical tissue reversed the growth factor-induced functional recovery. These findings reveal that specific combinations of growth factors can mobilize endogenous adult neural stem cells to promote cortical tissue re-growth and functional recovery after stroke.

Animals↗

PubMed1995

Some haematological parameters in Malawian neonates.

Studies of haematological parameters were performed on 366 (177 male and 189 female) normal Malawian neonates with mean +/- s.d. birthweight of 2.99 +/- 0.37 (range 2.1-4.0) kg using a Nova Cell Track, Model Nova CT11. Cord anaemia (Cord Hb < 13.5g dl-1) was detected in 100 (27.3%) of the neonates. It was also shown that although the male babies had a significantly higher erythrocyte protoporphyrin level (p < 0.001) than the females, there were no significant differences (p > 0.05) in the red cell, white cell and platelet indices between the two sexes. When the haematological parameters of the 266 (72.7%) non-anaemic (Cord Hb > 13.5g dl-1) neonates were analysed, the mean +/- s.d. values which may serve as local reference standards were: Hb 16.0 +/- 1.7 (range 13.5-21.3) g dl-1, Hct 47.0 +/- 6.0 (range 36.5-67.5) percent, MCV 112.6 +/- 8.9 (range 72.2-131.0) fl, MCH 31.9 +/- 5.5 (range 24.4-48.5) pg, MCHC 33.5 +/- 2.8 (range 29.1-48.9) g dl-1 reticulocyte count 6.9 +/- 3.6 (range 1.2-25.0) percent, free erythrocyte protoporphyrin 3.3 +/- 0.9 (range 1.9-7.7) mgs ZPP gm-1 Hb, platelet count 269.9 +/- 57.7 (range 134.0-454.0) x 10(9) l-1 and total leucocyte count 12.3 +/- 4.8 (range 5.5-35.3) x 10(9) l-1. Further analysis of the differential wbc count disclosed normal levels of eosinophils and neutrophils similar to those given in standard haematology textbooks for Caucasian neonates; thus strengthening the belief that eosinophilia and relative neutropenia previously reported in adult Africans is not of genetic origin, but rather an acquired phenomena.

Africa↗

PubMed2004

Quantitative imaging of lymphocyte membrane protein reorganization and signaling.

Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4+ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture.

Algorithms↗

PubMed2007

Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labeling.

A superparamagnetic iron oxide (SPIO) nanoparticle is emerging as an ideal probe for noninvasive cell tracking. However, its low intracellular labeling efficiency has limited the potential usage and has evoked great interest in developing new labeling strategies. We have developed fluorescein isothiocyanate (FITC)-incorporated silica-coated core-shell SPIO nanoparticles, SPIO@SiO2(FITC), with diameters of 50 nm, as a bifunctionally magnetic vector that can efficiently label human mesenchymal stem cells (hMSCs), via clathrin- and actin-dependent endocytosis with subsequent intracellular localization in late endosomes/lysosomes. The uptake process displays a time- and dose-dependent behavior. In our system, SPIO@SiO2(FITC) nanoparticles induce sufficient cell MRI contrast at an incubation dosage as low as 0.5 microg of iron/mL of culture medium with 1.2x105 hMSCs, and the in vitro detection threshold of cell number is about 1x104 cells. Furthermore, 1.2x105 labeled cells can also be MRI-detected in a subcutaneous model in vivo. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes which can still be readily MRI detected. This is the first report that hMSCs can be efficiently labeled with MRI contrast nanoparticles and can be monitored in vitro and in vivo with a clinical 1.5-T MRI imager under low incubation concentration of iron oxide, short incubation time, and low detection cell numbers at the same time.

Fluorescein-5-isothiocyanate↗

PubMed2002

Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography.

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu-PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu-PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu-PTSM than with FDG. However, both 64Cu-PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu-PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu-PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu-PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu-PTSM-labeled cells. Given the longer t(1/2) of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24-36 h) should be possible now with PET.

Animals↗

PubMed2004

Inhibition of cell migration via G protein-coupled receptors to opioid peptides and angiotensin.

Neurotransmitters are stimulatory as well as inhibitory regulators of cell migration. Angiotensin is such an inhibitory regulator of the SDF-1-induced migration of cytotoxic T lymphocytes, as we have investigated by time-lapse videomicroscopy and computer-assisted cell tracking. For angiotensin II, the most effective form of angiotensin for the inhibition of migration, two G protein-coupled receptors are known, which both downregulate the activity of the adenylyl cyclase via activation of inhibitory G proteins. This downregulation of the enzymatic activity is a key signaling event for the inhibition of T lymphocyte and tumor cell migration, while stimulatory neurotransmitters--for example, norepinephrine--cause an activation of the adenylyl cyclase. Similar to angiotensin, the SDF-1-induced migration of cytotoxic T lymphocytes was inhibited by DAMGO, a specific agonist for the mu-opioid receptor, which is coupled to inhibitory G proteins, too. More interestingly, DAMGO downregulated the met-enkephalin-induced migration of MDA-MB-468 breast carcinoma cells. Met-enkephalin binds to the delta-opioid receptor and, with lower affinity, to the mu-opioid receptor. Since the delta-opioid receptor also activates inhibitory G proteins, the promigratory effect of met-enkephalin is caused by an intracellular signaling distinct from the engagement of each opioid receptor alone. In summary, the dual control of the adenylyl cyclase functions as an integrator of stimulatory and inhibitory signals for T lymphocyte and tumor cell migration, which are delivered by neurotransmitters and other signal substances that bind to G protein-coupled receptors.

Angiotensins↗

PubMed2005

Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration.

Model systems mimicking the extracellular matrix (ECM) have greatly helped in quantifying cell migration in three dimensions and elucidated the molecular determinants of cellular motility in morphogenesis, regeneration, and disease progression. Here we tested the suitability of proteolytically degradable synthetic poly(ethylene glycol) (PEG)-based hydrogels as an ECM model system for cell migration research and compared this designer matrix with the two well-established ECM mimetics fibrin and collagen. Three-dimensional migration of dermal fibroblasts was quantified by time-lapse microscopy and automated single-cell tracking. A broadband matrix metalloproteinase (MMP) inhibitor and tumor necrosis factor-alpha, a potent MMP-inducer in fibroblasts, were used to alter MMP regulation. We demonstrate a high sensitivity of migration in synthetic networks to both MMP modulators: inhibition led to an almost complete suppression of migration in PEG hydrogels, whereas MMP upregulation increased the fraction of migrating cells significantly. Conversely, migration in collagen and fibrin proved to be less sensitive to the above MMP modulators, as their fibrillar architecture allowed for MMP-independent migration through preexisting pores. The possibility of molecularly recapitulating key functions of the natural extracellular microenvironment and the improved protease sensitivity makes PEG hydrogels an interesting model system that allows correlation between protease activity and cell migration.

Biocompatible Materials↗

PubMed2003

MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain.

Stem cell transplantation is being explored as a new paradigm for the treatment of demyelinating diseases. Magnetically labeled multipotential neural precursor cells were transplanted into the ventricles of rats with acute experimental allergic encephalomyelitis (EAE) and high-resolution (microscopic) MR images were obtained ex vivo. Migration patterns of live cells into periventricular white matter structures could be easily visualized, with a good correlation of the corresponding histopathology. The present results confirm that MR cell tracking can be used to guide the development of successful transplantation protocols.

Animals↗

PubMed2004

Molecular imaging of gene therapy for cancer.

Gene therapy of cancer has been one of the most exciting and elusive areas of scientific and clinical research in the past decade. One of the most critical issues for ensuring success of this therapy is the development of technology for noninvasive monitoring of the location, magnitude and duration of vector-mediated gene expression, as well as the distribution and targeting of vector particles in vivo. In recent years many advances have been made in high-resolution, in vivo imaging methods, including: radionuclide imaging, such as positron emission tomography (PET) and single photon emission tomography (SPECT), magnetic resonance (MR) imaging and spectroscopy, bioluminescence imaging and various fluorescence imaging techniques, including fluorescence-mediated tomography (FMT) and near-infrared fluorescence (NIRF) reflectance imaging. A variety of factors determine the choice of specific imaging system, some of them are the imaging requirements (single or repeated), intended use (animal or human) and spatial requirements (organs versus cellular resolution and depth). This review provides descriptions of modalities applicable to imaging different parameters of vector-mediated gene expression in tumors and stem cell tracking in vivo.

Animals↗

PubMed2003

Experimental model for cartilage tissue engineering to regenerate the zonal organization of articular cartilage.

OBJECTIVE: Regeneration of the zonal organization of articular cartilage may be an important advancement for cartilage tissue engineering. The first goal of this study was to validate our surgical technique as a method to selectively isolate chondrocytes from different zones of bovine articular cartilage. The second goal was to confirm that chondrocytes from different zones would have different proliferative and metabolic activities in two-dimensional (2-D) and 3-D cultures. Finally, to regenerate the zonal organization, we sought to make multi-layered constructs by encapsulating chondrocytes from different zones of articular cartilage. DESIGN: Cartilage slices were removed from three (upper, middle, and lower) zones of articular cartilage of young bovine legs. Histology and biochemical composition of the cartilage slices were analyzed to confirm that they had been obtained from the proper zone. Growth kinetics and gene expression in monolayer culture and matrix formation in photopolymerizing hydrogels were evaluated. Multi-layered photopolymerizing hydrogels were constructed with chondrocytes from each zone of native cartilage encapsulated. Cell viability and maintenance of the cells in the respective layer were evaluated using the Live/Dead Viability kit and cell tracking protocols, respectively. After 3 weeks, the multi-layered constructs were harvested for histologic examination including immunohistochemistry for type II collagen. RESULTS: Analysis of histology and biochemical composition confirmed that the cartilage slices had been obtained from the specific zone. Chondrocytes from different zones differed in growth kinetics and gene expression in monolayer and in matrix synthesis in 3-D culture. Cells encapsulated in each of the three layers of the hydrogel remained viable and remained in the respective layer in which they were encapsulated. After 3-week culture, each zone of multi-layered constructs had similar histologic findings to that of native articular cartilage. CONCLUSION: We present this as an experimental model to regenerate zonal organization of articular cartilage by encapsulating chondrocytes from different layers in multi-layered photopolymerizing gels.

Animals↗

PubMed2006

Species specificity of ectopic bone formation using periosteum-derived mesenchymal progenitor cells.

To investigate novel cell-based bone-engineering approaches using rabbit as a preclinical animal model, we compared the osteogenic potential of rabbit periosteum-derived cells (RPDCs) and human periosteum-derived cells (HPDCs) in vitro and in vivo. Adherent periosteal cells from both species were expanded in vitro and subsequently treated with osteogenic medium or bone morphogenetic protein 6 (BMP6). Alkaline phosphatase (ALP) activity was measured, and alizarin red staining was performed to evaluate osteogenic differentiation. In vivo ectopic bone formation was assessed by seeding 5x10(6) periosteal cells, grown in osteogenic conditions, in a Collagraft carrier and subsequent implantation subcutaneously in athymic mice. In vitro, growth analysis indicated that RPDCs expanded faster and were smaller than HPDCs under the same culture conditions. Osteogenic medium did not affect the ALP activity of HPDCs or RPDCs. In contrast, BMP6 stimulated ALP activity in cultured RPDCs and HPDCs but at different rates. In vivo, HPDCs gave rise to extensive bone formation, whereas RPDCs failed to make bone. In vivo, cell tracking revealed that engraftment and survival of HPDCs and RPDCs after 8 weeks in the implant were limited. Some HPDCs were incorporated into the newly formed bone. RPDCs and HPDCs displayed distinct growth characteristics and osteogenic differentiation capacity in vitro and in vivo under the culture conditions used. Our data indicate potential limitations of use of the rabbit as a preclinical model for cell-based treatments for bone repair.

Animals↗

PubMed2005

Modulation of homing properties of primitive progenitor cells generated by ex vivo expansion.

BACKGROUND AND OBJECTIVES: The maintenance of adequate interactions with the bone marrow (BM) microenvironment is critical to ensure efficient homing of ex vivo-expanded hematopoietic cells. This study was intended to assess adhesion and migration properties of long-term culture-initiating cells (LTC-IC) harvested after self-renewal division in ex vivo culture and to determine their susceptibility to growth-inhibitory signals mediated by adhesion to BM stromal ligands. DESIGN AND METHODS: We used cell tracking to isolate primitive LTC-IC that had accomplished 1 or 2 divisions ex vivo. Adhesion, migration and growth inhibition of divided LTC-IC were determined in the presence of purified BM ligands, and compared to the properties of uncultured LTC-IC. RESULTS: As compared to undivided LTC-IC, adhesion and migration mediated by very late antigen (VLA)-4 integrin across both vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (Fn) were downregulated in post-mitotic LTC-IC. Conversely, binding and motility via VLA-5 across Fn were stimulated. No changes occurred in LTC-IC interactions with intercellular adhesion molecule-1 (ICAM-1) or with E- or P-selectin. Proliferation of uncultured LTC-IC was inhibited by VLA-4-mediated binding to VCAM-1 and the CS-1 domain of Fn, as well as binding to P-selectin. Growth of ex vivo-generated LTC-IC became unresponsive to these 3 ligands but was suppressed through VLA-5 engagement by the cell binding domain of Fn. INTERPRETATION AND CONCLUSIONS: The generation of LTC-IC in expansion culture is associated with functional alterations of adhesion receptors, modulating not only binding and migration in the BM but also responsiveness to adhesion-mediated growth inhibitory signals. Such changes may limit homing and engraftment of expanded primitive stem/progenitor cells.

AC133 Antigen↗

PubMed2003

Blockade of GABA(B) receptors alters the tangential migration of cortical neurons.

To better understand the role of neurotransmitter receptors in neuronal differentiation and maturation a detailed knowledge of their identity, location and function in the plasma membrane of specific neuronal populations during development is required. Combining pre-embedding immunocytochemistry with cell tracking in embryonic brain slice cultures we show that virtually all neurons (approximately 98%) migrating through the lower intermediate zone (LIZ) on their way from the medial ganglionic eminence to the cerebral cortex, express GABA(B)R1. Blockade of GABA(B)Rs with a specific antagonist, CGP52432, resulted in a concentration-dependent accumulation of these tangentially migrating neurons in the ventricular/sub-ventricular zones (VZ/SVZ) of the cortex and fewer cells were observed in the cortical plate/marginal zone (CP/MZ) and LIZ. Moreover, they had significantly shorter leading processes compared with similar migrating cells in control slices. Electrophysiological recording in LIZ and CP cells revealed no direct effect of either CGP52432 or the GABA(B)R agonist, baclofen, on resting membrane properties suggesting that the effect of CGP52432 on migration might be mediated through a metabotropic action or the regulation of release of factors controlling migration. These results suggest that GABA(B)Rs have an important modulatory role in the migration of cortical interneurons.

Animals↗

PubMed2003

Integrin-mediated preadipocyte adhesion and migration on laminin-1.

Cell adhesion and migration are key events in many biological processes and depend on extracellular matrix proteins. Understanding these cellular events in adipogenesis is paramount to elucidating the biological mechanisms underlying preadipocyte-specific aspects of obesity, diabetes, and adipose tissue development for the design of pharmaceutical screening and tissue engineering strategies. We quantitatively investigated preadipocyte adhesion and migration on laminin-1 surfaces and identified candidate cognate preadipocyte receptors for laminin-1. In adhesion studies, we found that preadipocytes readily adhered to laminin-1 as compared with other extracellular matrix proteins. In addition, immunocytochemical analysis demonstrated that an array of integrin molecules was present on the surface of preadipocytes. Preadipocyte adhesion on laminin-1 was quantitatively assessed using a sedimentation adhesion assay, and results suggested that preadipocyte adhesion to laminin-1 was mediated by the alpha1beta1 integrin. In addition, digital time-lapse microscopy and quantitative cell tracking revealed that inhibition of the alpha1beta1 integrin resulted in abrogation of preadipocyte migration on laminin-1 surfaces. These results strongly support the hypothesis that preadipocyte adhesion to and migration on laminin-1 substrata are regulated, in part, by integrins.

Adipocytes↗