1,720,970 research outputs found
Ineffective Erythropoiesis in beta thalassemia : a new model characterized by enhanced survival mechanisms that reduce p21 mediated cell death
No full textnul
Alpha Hemoglobin Stabilizing Protein (AHSP) gene : molecular analysis in caucasian beta thalassemic subjects
No full textIn vivo imaging of human skeletal muscle cells (HSkMC) biodistribution : comparison between SPET, MRI and BLI
No full textIntroduction and aim: The interest about cell-mediated therapy for tissues regeneration is increased in the last few years. Numerous protocols which include implantation of healthy stem cells in diseased models were set up but cell distribution, localization, survival, proliferation and differentiation cannot be evaluated in vivo. Here we compared SPET, MRI and BLI visualization of a human muscle cell line biodistribution proposing the application of these procedures for a better evaluation of muscle stem cell mediated treatments. Methods: Human Skeletal Muscle Cells (HSkMC) were labelled with with Endorem® (0-100-200 μg Fe/mL) for 24 or 48h in presence or not of Poly-L-Lysine (PLL), Protamine Sulfate (PrS), Polybrene (PB) or infected with a lentiviral vector carrying Luciferase gene under control of the Myogenin promoter (pGZ.Myo.L) and then labelled with 111Indium-Oxine (60 μCi/106 cells). Labelled HSkMC were analyzed for viability, iron content, morphology and intrarterially (i.a.) injected into NUDE mice for in-vivo SPET, MRI or BLI. Gamma counting of explanted organ and IHC was performed to validate imaging data. Results: HSkMC incubated for 24h or 48h with 0-100-200μg Fe/mL didn’t show differences, in terms of viability, between labelled/non-labelled cells in the presence or absence of carriers (n=3). The percentage of Iron+ cells increased in proportion to the iron content in the medium; in particular 200ug/mL endorem+PLL was deemed as the ideal cell labelling condition for in vivo MRI giving more than 90% Iron+ cells. Iron loaded or 111Indium- Oxine-luciferase expressing HSkMC were i.a. injected into a murine model of muscle inflammation. MRI permitted to follow over time HSkMC distribution into the injured muscle. SPET and BLI of HSkMC confirmed cell distribution to muscle and revealed an early localisation into the lung. HSkMC infected with pGZ.Myo.L, i.a. injected were detectable in muscle up to 2 months after injection. Interestingly, at this time point, the signal is still present only near the lesion area while disappeared in the rest of the body. Conclusions: Human muscle cells precursors visualization by MRI, SPET or in vivo BLI confirmed the same biodistribution. The different specific protocols will be useful to study the fate and the in vivo behaviour over time of stem cells once injected into recipient animals in a more complete way. It will be possible to use these instruments for the in vivo study of muscular stem cells in restoring skeletal muscles after damage. Acknowledgement: This work was supported by the FP6 Hi-CAM project (LSHC-CT-2006-037737)
Methods and techniques for preclinical in vivo evaluation of radiosensitivity and radioresponsivity
No full textOver the years, radiotherapy (RT) has considerably broadened the possibility for treating tumors and led to an overall improvement in the quality of life of cancer patients. Cancer-cell radiosensitivity and radioresponsivity, being related to patient-specific tumor features and conditions, such as proliferation rate, metabolism, percentage of apoptosis, new vessel formation and level of hypoxia, influence the efficacy of RT treatment. Radiosensitivity and radioresponsivity can now be studied in the clinical setting using in vivo molecular imaging techniques, such as positron emission tomography, single photon emission tomography and magnetic resonance imaging. Furthermore, the availability of many new radiotracers and imaging probes is improving the approach to the exploration of tumor response to RT. However, to be successful in clinical, in vivo imaging investigations require an in-depth understanding of the molecular basis of tumor response to RT. Small-animal models of neoplasia are now an essential tool for the preclinical evaluation of novel anti-neoplastic therapies, also at molecular level. However, the accuracy of RT techniques for small animal needs to be improved. Indeed, human conformal RT devices are less accurate when used in small-animal irradiation, which involves relatively small radiation fields and demands more precise beam localisation than is typical of clinical irradiators. The aim of this paper is to review current preclinical protocols and small-animal models for the in vivo study of radiosensitivity and radioresponsivity. More-over, the new solutions for dedicated small-animal conformal RT devices are also analyzed
AHSP (Alpha Hemoglobin Stabilizing Protein) Gene Expression during Normal and ß-Thalassemic Erythroid Differentiation
No full textAHSP (Alpha Hemoglobin Stabilizing Protein) gene expression during normal and beta-Thalassemic erythroid differentiation
No full textReliable protocol to track mesenchymal stem cells using a lentiviral vector expressing luciferase protein
No full textAdult mesenchymal stem cells (MSC) isolated from adipose tissue represent a useful tool to regenerate damaged tissues in regenerative medicine protocols due to their high proliferative and differentiation potential and easily accessible, ethically-approved source. Nevertheless, interactions between transplanted cells and host tissues, is not completely understood.
Imaging is a new in vivo approach that allows to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing inter-individual variability and the number of experimental animals needed.
In this study the feasibility of luciferase lentiviral infection has been evaluated in order to achieve the direct visualization of mouse apidose tissue-derived adult mesenchymal stem cells by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models.
Adipose –derived MSCs from CD1 mice were infected with different concentrations of a lentiviral vector carrying the luciferase gene under the control of Phospho Glicerate Kinase promoter (PLW vector).
Labeled MSCs were analyzed for viability, morphology, and osteogenic differentiation capability along with maintenance of luminescence labeling. Moreover, intracellular calcium dynamics following 1mM ATP stimulation was analysed by means of single cell calcium imaging recordings.
Our analysis showed that MSCs can be efficiently transduced with PLW vector maintaining the proportion with the amount of virus used.
The infected cells showed that biological features of luciferase-positive MSC were not altered. Moreover, cells maintained their physiological differentiation potential, quantitatively assayed by analyzing calcium deposits via Alizarin Red staining. Moreover, just like non-infected cells, they showed responsiveness to stimulation by extracellular ATP.
Our protocol efficiently labeled Adipose–derived MSCs without altering their biological properties and could allow direct cell detection ex vivo and in vivo by optical imaging. Insertion of luciferase probe appears as a reliable technique to follow the fate Adipose–derived MSCs upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies
Ex vivo visualization of transfected human mesenchymal stem cells after transplantation: a reliable cell-labeling protocol for optical imaging
No full textStem cells, due to their high proliferative and differentiation potential, have been transplanted into different animal models of neurodegenerative diseases with uneven results since an exhaustive comprehension of the interactions between transplanted cells and host tissues is still missing. Imaging is a new approach that allows to investigate overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) in vivo, thereby reducing interindividual variability and the number of experimental animals needed. Here we evaluate the feasibility of m-cherry lentiviral infection (transduction) as specific labelling protocol for the real time visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC) in vivo by Optical Imaging as a proof of principle for their long-term tracking in pre-clinical models. Commercial hMSC were infected with different concentrations of a lentiviral vector carrying the m-cherry gene under the control of Phospho Glycerate Kinase promoter (PGK). Labeled hMSC were analyzed for viability, morphology, and differentiation capability along with maintenance of fluorescent labeling after extensive culture in vitro. Thereafter, we transplanted them in a rodent model of Parkinson’s disease based on the unilateral intrastriatal injection of 6-hydroxydopamine, a procedure that causes a progressive and retrograde degeneration of the nigrostriatal pathway.
Our FACS analysis showed that a high percentage of hMSCs expressed the reporter gene (87,2% and 92.4% with a MOI equal respectively to 2,5 and 5; non infected control cells: less than 1%) indicating that the cells can be efficiently transduced with the lentiviral vector bearing the m-cherry. Infected cells showed a high level of vector copy number inserted in their DNA able to stably express the corresponding mRNA, as estimated by real-time PCR. Biological features of m-cherry-positive hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates were comparable to control cells and no morphological alterations were detected by confocal analysis. Upon striatal transplantation, it was possible to visualize hMSC ex vivo in the whole brain by a sensitive CCD camera for fluorescent imaging. Finally, the presence of the m-cherry-positive cells at the injection site was also confirmed using human specific antibodies on frozen microscope slides.
Our protocol efficiently labeled hMSC without altering their biological properties and allowed direct cell detection ex vivo by optical imaging. Insertion of m-cherry fluorescent probe appears as a reliable technique to follow the fate hMSC upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies promptly applicable to patients
Application to an experimental model of Parkinson’s disease of a reliable protocol to track human mesenchymal stem cells using a lentiviral vector expressing mCherry fluorescent protein
No full textStem cells, due to their high proliferative and differentiation potential, have been transplanted into different animal models of neurodegenerative diseases with uneven results since an exhaustive comprehension of the interactions between transplanted cells and host tissues is still missing. Imaging is a new approach that allows in vivo, to investigated overtime some important parameters (such as cell distribution, survival, localization and fate of injected cells) thereby reducing interindividual variability and the number of experimental animals needed. The purpose of the study was to evaluate the feasibility of mcherry lentiviral infection for the direct visualization of bone marrow-derived human Mesenchymal Stem Cells (hMSC), by optical imaging, as a proof of principle for their long-term tracking in pre-clinical models.
Commercial hMSC were infected with different concentrations of a lentiviral vector carrying the mcherry gene under the control of Phospho Glicerate Kinase promoter. Labeled hMSC were analyzed for viability, morphology, and differentiation capability along with maintenance of fluorescent labeling after extensive culture in vitro. Thereafter, we transplanted them in a rodent model of Parkinson’s disease based on the unilateral intrastriatal injection of 6-hydroxydopamine, a procedure that causes a progressive and retrograde degeneration of the nigrostriatal pathway.
Our FACS analysis showed that hMSC can be efficiently transduced with the lentiviral vector bearing the mcherry protein since a high percentage of cells expressed the reporter gene (around 80-90%). The infected cells showed a high level of vector copy number inserted in their DNA able to stably express mcherry mRNA, as estimated by real-time PCR. Biological features of mcherry+ hMSC were not altered, even in long term cultures, since their doubling time and metabolic rates appeared comparable to control cells and no morphological alterations were retrieved by confocal analysis. Upon striatal transplantation, it was possible to visualize hMSC ex vivo in the whole brain by a sensitive CCD camera for fluorescent imaging. Finally, the presence of the mcherry+ cells at the injection site was also confirmed using human specific antibodies on frozen microscope slides.
Our protocol efficiently labeled hMSC without altering their biological properties and allowed direct cell detection ex vivo by optical imaging. Insertion of mcherry fluorescent probe appears as a reliable technique to follow the fate hMSC upon transplantation and for studying their behaviour in vivo and ex vivo in order to establish efficient therapeutic strategies promptly applicable to patients
- …
