Kishore Bhakoo

National University of Singapore, Singapore, Singapore

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Publications (53)187.93 Total impact

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    ABSTRACT: Introduction. To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell tracking for years, but knowledge regarding possible cytotoxic ultrastructural changes subsequent to iron-oxide particle labeling is limited. Hence, the purpose of this study was to label MSCs with dextran-coated ultrasmall super-paramagnetic iron-oxide (USPIO) particles conjugated with the transduction sequence of trans-activator of transcription (TAT) (IODEX-TAT) and evaluate the effect of labeling on ultrastructure, viability, phenotype and proliferative capacity of the cells. Materials and methods. MSCs were labeled with 5 and 10 μg IODEX-TAT/10(5) cells for 2, 6 and 21 hours. IODEX-TAT uptake and cellular ultrastructure were determined by electron microscopy. Cell viability was determined by propidium iodide staining and cell proliferation capacity by 5-bromo-2-deoxyuridine (BrdU) incorporation. Maintenance of stem cell surface markers was determined by flow cytometry. Results. IODEX-TAT labeling for 2, 6 and 21 h did not influence cellular ultrastructure or viability. Moreover, neither stem cell surface markers nor cell proliferation capacity was affected by labeling with IODEX-TAT. Conclusion. Our results demonstrate that labeling of MSCs for 21 h with a clinically relevant dose of 10 μg IODEX-TAT/10(5) cells is feasible and does not affect MSC ultrastructure, viability, phenotype or proliferation capacity.
    Scandinavian journal of clinical and laboratory investigation 04/2014; · 1.38 Impact Factor
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    ABSTRACT: Aptamer-based biosensors (aptasensor) are powerful tools for rapid and sensitive biomarker detection. In this study, we report a DNA aptamer probe evolved from cell-SELEX that can recognize thrombospondin-1 protein in human plasma samples. The KD value of the aptamer M55 binding to thrombospondin-1 was determined as 0.5±0.2μM with an R(2) of 0.9144. A horseradish peroxidase-linked short oligo was complementarily bound onto the 3' end of the aptamer sequence to facilitate the 'smart' design of an M55-aptasensor for quantifying thrombospondin-1 protein in plasma samples. The limit of detection was 6.96fM. Thrombospondin-1 is a glycoprotein with multiple biological functions, including inflammation, platelet aggregation and endothelial cell apoptosis, and is involved in the pathology of atherosclerosis. In total, 118 plasma subjects were analyzed by using the aptasensor measurement with 1μL sample volume and 5min incubation time. The thrombospondin-1 concentrations in ST-Elevation Myocardial Infarction patients with severe atherosclerotic plaque burden were statistically significantly higher than in the healthy volunteers without atherosclerosis conditions, suggesting that thromboposnidn-1 is a potential plasma biomarker for atherosclerosis progression.
    Biosensors & bioelectronics 12/2013; 55C:405-411. · 5.43 Impact Factor
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    ABSTRACT: Aptamers are single-stranded oligonucleotides that are capable of binding wide classes of targets with high affinity and specificity. Their unique three-dimensional structures present numerous possibilities for recognizing virtually any class of target molecules, making them a promising alternative to antibodies used as molecular probes in biomedical analysis and clinical diagnosis. In recent years, cell-systematic evolution of ligands by exponential enrichment (SELEX) has been used extensively to select aptamers for various cell targets. However, aptamers that have evolved from cell-SELEX to distinguish the "stimulus-response cell" have not previously been reported. Moreover, a number of cumbersome and time-consuming steps involved in conventional cell-SELEX reduce the efficiency and efficacy of the aptamer selection. Here, we report a "two-step" methodology of cell-SELEX that successfully selected DNA aptamers specifically against "inflamed" endothelial cells. This has been termed as stimulus-response cell-SELEX (SRC-SELEX). The SRC-SELEX enables the selection of aptamers to distinguish the cells activated by stimulus of healthy cells or cells isolated from diseased tissue. We report a promising aptamer, N55, selected by SRC-SELEX, which can bind specifically to inflamed endothelial cells both in cell culture and atherosclerotic plaque tissue. This aptamer probe was demonstrated as a potential molecular probe for magnetic resonance imaging to target inflamed endothelial cells and atherosclerotic plaque detection.
    Analytical and Bioanalytical Chemistry 07/2013; · 3.66 Impact Factor
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    Jon Golding, Jo Jackson, Kishore Bhakoo
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    ABSTRACT: Neural crest cells (NCCs) constitute a population of neuroepithelial, migratory, multipotent stem cells that give rise to the peripheral nervous system, pigment cell, and much of the cranio-facial skeleton. Until recently, NCCs were thought to be a transient cell population that only existed during development. However, recent work has demonstrated that multipotent NCCs persist into adulthood within the stem cell niche of hair follicles. This raises the possibility that a patient's own NCCs could be harvested and used autologously to repair their injured nervous system. This work begins to explore the therapeutic potential of adult NCCs.
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    ABSTRACT: The G-protein coupled C-X-C chemokine receptor type 4 (CXCR4) is highly overexpressed in a range of cancers and is therefore an excellent biomarker for cancer imaging. To this end targeted iron oxide nanoparticles were developed and utilised for in vitro imaging of MDA-MB-231 breast cancer cells overexpressing the CXCR4 receptor. Nanoparticles comprising an iron oxide core, encapsulated in a stabilising epichlorohydrin crossed-linked dextran polymer, were conjugated to a cyclopentapeptide with affinity to the CXCR4 receptor. The particles were characterized for their size, surface charge and r2 relaxivity at 4.7 T. MR imaging of the CXCR4 receptor with targeted iron oxide nanoparticles revealed an approximately 3-fold increase in T2 signal enhancement of MDA-MB-231 cells compared to non-targeted controls. Prussian blue staining of labeled MDA-MB-231 cells revealed darker and more intense staining of the cellular membrane. This study demonstrates the potential of targeted iron oxide nanoparticles for the imaging of the CXCR4 receptor by magnetic resonance imaging (MRI).
    American Journal of Nuclear Medicine and Molecular Imaging 01/2013; 3(4):372-83.
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    ABSTRACT: Background. Regenerative therapy is an emerging treatment modality. To determine migration and retention of implanted cells, it is crucial to develop noninvasive tracking methods. The aim was to determine ex vivo magnetic resonance imaging (MRI) detection limits of ultrasmall superparamagnetic iron-oxide (USPIO) labeled mesenchymal stromal cells (MSCs). Materials and Methods. 248 gel-phantoms were constructed and scanned on a 1.5T MRI-scanner. Phantoms contained human MSCs preincubated with USPIO nanoparticles for 2, 6, or 21 hours using 5 or 10 μ g USPIO/10(5) MSCs. In addition, porcine hearts were scanned after injection of USPIO labeled MSCs. Results. Using 21 h incubation time and 10 μ g USPIO/10(5) MSCs, labeled cells were clearly separated from unlabeled cells on MRI using 250.000 (P < 0.001), 500.000 (P = 0.007), and 1.000.000 MSCs (P = 0.008). At lower incubation times and doses, neither labeled nor unlabeled cells could be separated. In porcine hearts labeled, but not unlabeled, MSCs were identified on MRI. Conclusions. As few as 250.000 MSCs can be detected on MRI using 21 h incubation time and 10 μ g USPIO/10(5) MSCs. At lower incubation times and doses, several million cells are needed for MRI detection. USPIO labeled cells can be visualized by MRI in porcine myocardial tissue.
    Stem cells international. 01/2013; 2013:353105.
  • Kishore Bhakoo
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    ABSTRACT: INTRODUCTION: Cell transplants to replace cells lost due to injury or degenerative diseases, for which there are currently no cures, are being pursued in a wide range of experimental models. Thus, the application of stem cell-based therapies to treat neurodegenerative and traumatic injuries is now a clinical reality. However, the monitoring of cellular grafts, non-invasively, is an important aspect of the ongoing efficacy and safety assessment of cell-based therapies. Hence, there is a need for non-invasive imaging techniques to ensure that transplants are not only administered to the relevant site, but also allow the monitoring of inappropriate cellular migration to improve our understanding of stem cell migration in the context of the whole organism. AREAS COVERED: This review provides an up to date overview of molecular imaging approaches that have been used for visualizing and tracking transplanted stem cells, in vivo. EXPERT OPINION: It's important to emphasize that the application of molecular imaging to interrogate transplanted cells may require one or even two imaging modalities to provide a reasonable assessment of transplanted cells in specific organs.
    Expert opinion on biological therapy 07/2011; 11(7):911-20. · 3.22 Impact Factor
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    ABSTRACT: As ultrasmall superparamagnetic particles of iron oxides (USPIO) have been widely used in clinical medicine as MRI contrast agents, hence their potential toxicity and adverse effects following administration have attracted particular attention. In the present study, high resolution magic-angle-spinning (1)H NMR spectroscopy coupled with multivariate statistical analysis was used to directly determine the metabolic consequences of specific-tissues, including kidney, liver and spleen following the intravenous administration of USPIO. Alterations of renal, hepatic and splenic function were reflected by changes in a number of metabolic pathways including small molecules involved in energy, lipid, glucose, and amino acids metabolism. The toxicological potential and metabolic fate of USPIO seems to be linked to their surface chemistry and particle size. Hierarchical principal component analysis was used to explore the multidimensional metabolic relationships between various biological matrices such as kidney, liver, spleen, plasma and urine. Information on the involvement of USPIO in transportation, absorption, biotransformation, biodistribution and secretion was derived from metabolic correlation analysis between different organs and biofluids. Such a metabonomic strategy provides methodology for investigating the potential adverse biological effects of similar nanoparticles on the environmental and human health and assessing the drug interventions on the targeted organ.
    Biomaterials 06/2011; 32(27):6558-69. · 8.31 Impact Factor
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    ABSTRACT: The metabonomic changes in murine RAW264.7 macrophage-like cell line induced by ultrasmall superparamagnetic particles of iron oxides (USPIO) have been investigated, by analyzing both the cells and culture media, using high-resolution NMR in conjunction with multivariate statistical methods. Upon treatment with USPIO, macrophage cells showed a significant decrease in the levels of triglycerides, essential amino acids such as valine, isoleucine, and choline metabolites together with an increase of glycerophospholipids, tyrosine, phenylalanine, lysine, glycine, and glutamate. Such cellular responses to USPIO were also detectable in compositional changes of cell media, showing an obvious depletion of the primary nutrition molecules, such as glucose and amino acids and the production of end-products of glycolysis, such as pyruvate, acetate, and lactate and intermediates of TCA cycle such as succinate and citrate. At 48 h treatment, there was a differential response to incubation with USPIO in both cell metabonome and medium components, indicating that USPIO are phagocytosed and released by macrophages. Furthermore, information on cell membrane modification can be derived from the changes in choline-like metabolites. These results not only suggest that NMR-based metabonomic methods have sufficient sensitivity to identify the metabolic consequences of murine RAW264.7 macrophage-like cell line response to USPIO in vitro, but also provide useful information on the effects of USPIO on cellular metabolism.
    Journal of Nanoparticle Research 01/2011; 13(5):2049-2062. · 2.18 Impact Factor
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    ABSTRACT: Ultra-small superparamagnetic particles of iron oxides (USPIO) have been developed as intravenous organ/tissue-targeted contrast agents to improve magnetic resonance imaging (MRI) in vivo. However, their potential toxicity and effects on metabolism have attracted particular attention. In the present study, uncoated and dextran-coated USPIO were investigated by analyzing both rat urine and plasma metabonomes using high-resolution NMR-based metabonomic analysis in combination with multivariate statistical analysis. The wealth of information gathered on the metabolic profiles from rat urine and plasma has revealed subtle metabolic changes in response to USPIO administration. The metabolic changes include the elevation of urinary alpha-hydroxy-n-valerate, o- and p-HPA, PAG, nicotinate and hippurate accompanied by decreases in the levels of urinary alpha-ketoglutarate, succinate, citrate, N-methylnicotinamide, NAG, DMA, allantoin and acetate following USPIO administration. The changes associated with USPIO administration included a gradual increase in plasma glucose, N-acetyl glycoprotein, saturated fatty acid, citrate, succinate, acetate, GPC, ketone bodies (beta-hydroxybutyrate, acetone and acetoacetate) and individual amino acids, such as phenylalanine, lysine, isoleucine, glycine, glutamine and glutamate and a gradual decrease of myo-inositol, unsaturated fatty acid and triacylglycerol. Hence USPIO administration effects are reflected in changes in a number of metabolic pathways including energy, lipid, glucose and amino acid metabolism. The size- and surface chemistry-dependent metabolic responses and possible toxicity were observed using NMR analysis of biofluids. These changes may be attributed to the disturbances of hepatic, renal and cardiac functions following USPIO administrations. The potential biotoxicity can be derived from metabonomic analysis and serum biochemistry analysis. Metabonomic strategy offers a promising approach for the detection of subtle physiological responses on mammalian metabolism, and can be employed to investigate the potential adverse effects of other nanoparticles and nanomaterials on the environment and human health.
    Nanotechnology 10/2010; 21(39):395101. · 3.84 Impact Factor
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    ABSTRACT: Metabolic syndrome is a fast growing public health burden for almost all the developed countries and many developing nations. Despite intense efforts from both biomedical and clinical scientists, many fundamental questions regarding its aetiology and development remain unclear, partly due to the lack of suitable imaging technologies to visualize lipid composition and distribution, insulin secretion, beta-cell mass and functions in vivo. Such technologies would not only impact on our understanding of the complexity of metabolic disorders such as obesity and diabetes, but also aid in their diagnosis, drug development and assessment of treatment efficacy. In this article we discuss and propose several strategies for visualization of physiological and pathological changes that affect pancreas and adipose tissue as a result of the development of metabolic diseases.
    EMBO Molecular Medicine 06/2010; 2(6):196-210. · 7.80 Impact Factor
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    ABSTRACT: This study aimed to determine the homing potential and fate of epidermal neural crest stem cells (eNCSCs) derived from hair follicles, and bone marrow-derived stem cells (BMSCs) of mesenchymal origin, in a lipopolysaccharide (LPS)-induced inflammatory lesion model in the rat brain. Both eNCSCs and BMSCs are easily accessible from adult tissues by using minimally invasive procedures and can differentiate into a variety of neuroglial lineages. Thus, these cells have the potential to be used in autologous cell-replacement therapies, minimizing immune rejection, and engineered to secrete a variety of molecules. Both eNCSCs and BMSCs were prelabeled with iron-oxide nanoparticles (IO-TAT-FITC) and implanted either onto the corpus callosum in healthy or LPS-lesioned animals or intravenously into lesioned animals. Both cell types were tracked longitudinally in vivo by using magnetic resonance imaging (MRI) for up to 30 days and confirmed by postmortem immunohistochemistry. Transplanted cells in nonlesioned animals remained localized along the corpus callosum. Cells implanted distally from an LPS lesion (either intracerebrally or intravenously) migrated only toward the lesion, as seen by the localized MRI signal void. Fluorescence microscopy of the FITC tag on the nanoparticles confirmed the in vivo MRI data, This study demonstrated that both cell types can be tracked in vivo by using noninvasive MRI and have pathotropic properties toward an inflammatory lesion in the brain. As these cells differentiate into the glial phenotype and are derived from adult tissues, they offer a viable alternative autologous stem cell source and gene-targeting potential for neurodegenerative and demyelinating pathologies.
    Stem Cell Research & Therapy 01/2010; 1(2):17. · 3.65 Impact Factor
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    ABSTRACT: Stem cell therapy in the nervous system aims to replace the lost neurons and provide functional recovery. However, it is imperative that we understand the in vivo behaviour of these cells post-implantation. We report visualisation of iron oxide labelled bone marrow-derived stem cells (BMSCs) implanted into the striatum of hemi-parkinsonian rats by magnetic resonance imaging (MRI). Functional efficacy of the donor cells was monitored in vivo using the positron emission tomography (PET) radioligand [11C]raclopride. The cells were visible for 28 days by in vivo MRI. BMSCs provided functional recovery demonstrated by a decreased binding of [11C]raclopride. Although, histology confirmed the persistence of donor cells, no tyrosine hydroxylase positive cells were present. This suggests that BMSCs may have a limited paracrine effect and influence functional recovery. We demonstrate, using multimodal imaging, that we can not only track BMSCs but also establish their effects in a pre-clinical model of Parkinson's disease.
    Journal of neuroscience methods 07/2009; 183(2):141-8. · 2.30 Impact Factor
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    ABSTRACT: The purpose of the study is to track iron-oxide nanoparticle-labelled adult rat bone marrow-derived stem cells (IO-rBMSCs) by magnetic resonance imaging (MRI) and determine their effect in host cardiac tissue using 2-deoxy-2-[F-18]fluoro-D: -glucose-positron emission tomography (FDG-PET). Infarcted rats were randomised to receive (1) live IO-rBMSCs by direct local injection, or (2) dead IO-rBMSCs as controls; (3) sham-operated rats received live IO-rBMSCs. The rats were then imaged from 2 days to 6 weeks post-cell implantation using both MRI at 9.4T and FDG-PET. Implanted IO-rBMSCs were visible in the heart by MRI for the duration of the study. Histological analysis confirmed that the implanted IO-rBMSCs were present for up to 6 weeks post-implantation. At 1 week post-IO-rBMSC transplantation, PET studies demonstrated an increase in FDG uptake in infarcted regions implanted with live IO-rBMSC compared to controls. Noninvasive multimodality imaging allowed us to visualise IO-rBMSCs and establish their affect on cardiac function in a rat model of myocardial infarction (MI).
    Molecular Imaging & Biology 10/2008; 11(1):31-8. · 3.10 Impact Factor
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    Catherine Chapon, Amy H Herlihy, Kishore K Bhakoo
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    ABSTRACT: To demonstrate the feasibility of using an inversion recovery pulse sequence and to define the optimal inversion time (TI) to assess myocardial infarction in mice by late gadolinium enhancement (LGE) MRI at 9.4T, and to obtain the maximal contrast between the infarcted and the viable myocardium. MRI was performed at 9.4T in mice, two days after induction of myocardial infarction (n = 4). For cardiovascular MR imaging, a segmented magnetization-prepared fast low angle shot (MP-FLASH) sequence was used with varied TIs ranging from 40 to 420 ms following administration of gadolinium-DTPA at 0.6 mmol/kg. Contrast-to-noise (CNR) and signal-to-noise ratio (SNR) were measured and compared for each myocardial region of interest (ROI). The optimal TI, which corresponded to a minimum SNR in the normal myocardium, was 268 ms +/- 27.3. The SNR in the viable myocardium was significantly different from that found in the infarcted myocardium (17.2 +/- 2.4 vs 82.1 +/- 10.8; p = 0.006) leading to a maximal relative SI (Signal Intensity) between those two areas (344.9 +/- 60.4). Despite the rapid heart rate in mice, our study demonstrates that LGE MRI can be performed at 9.4T using a protocol similar to the one used for clinical MR diagnosis of myocardial infarction.
    Journal of Cardiovascular Magnetic Resonance 02/2008; 10:6. · 4.44 Impact Factor
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    ABSTRACT: The aim of the study was to examine the association of arachidonic acid-related signal transduction with cerebral metabolism in patients with schizophrenia who have violently and dangerously offended while psychotic. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 11 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic. Spectra were obtained from 70 x 70 x 70 mm(3) voxels using an image-selected in vivo spectroscopy pulse sequence. Niacin flush testing results were quantified as the volumetric niacin response. There was a strong, and negative, correlation between the volumetric niacin response and the metabolite concentration of inorganic phosphate expressed as a ratio of the total 31-phosphorus signal (p<0.005). Our results suggest that patients with schizophrenia who have violently offended and have poor phospholipid-related signal transduction may have higher levels of cerebral energy metabolism.
    Prostaglandins Leukotrienes and Essential Fatty Acids 09/2007; 77(2):97-9. · 2.73 Impact Factor
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    Zarinah K Agnew, Kishore K Bhakoo, Basant K Puri
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    ABSTRACT: Electrophysiological data confirm the existence of neurons that respond to both motor and sensory events in the macaque brain. These mirror neurons respond to execution and observation of goal-orientated actions. It has been suggested that they comprise a neural basis for encoding an internal representation of action. In this paper the evidence for a parallel system in humans is reviewed and the implications for human theory of mind processing are discussed. Different components of theory of mind are discussed; the evidence for mirror activity within subtypes is addressed. While there is substantial evidence for a human mirror system, there are weaknesses in the attempts to localize such a system in the brain. Preliminary evidence indicates that mirror neurons may be involved in theory of mind; however, these data by their very nature are reliant on the presence, and precise characterization, of the human mirror system.
    Brain Research Reviews 07/2007; 54(2):286-93. · 7.82 Impact Factor
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    ABSTRACT: Imaging methods such as nuclear medicine (including positron emission tomography), magnetic resonance imaging, ultrasound, and optical imaging can be used to provide information about the expression of genes, and the location of molecules and cells in intact animals or patients. In the setting of transplantation, this will allow monitoring of inflammatory responses, as well as the state of the graft. In this review, the advantages and disadvantages of different approaches to imaging will be discussed, as well as their potential application to transplantation.
    Transplantation 12/2006; 82(9):1124-9. · 3.78 Impact Factor
  • Kishore K Bhakoo, Timothy Craig, Daniel Pearce
    Advances in experimental medicine and biology 02/2006; 576:27-47; discussion 361-3. · 1.83 Impact Factor

Publication Stats

1k Citations
187.93 Total Impact Points

Institutions

  • 2013
    • National University of Singapore
      • NUS Graduate School for Integrative Sciences and Engineering
      Singapore, Singapore
  • 2011
    • Agency for Science, Technology and Research (A*STAR)
      Tumasik, Singapore
  • 2010
    • Northeast Institute of Geography and Agroecology
      • Wuhan Institute of Physics and Mathematics
      Peping, Beijing, China
  • 2002–2010
    • Imperial College London
      • • Department of Imaging Sciences
      • • Faculty of Medicine
      Londinium, England, United Kingdom
    • Nathan Kline Institute
      Orangeburg, New York, United States
    • University of Cambridge
      Cambridge, England, United Kingdom
  • 2002–2008
    • MRC Clinical Sciences Centre
      London Borough of Harrow, England, United Kingdom
  • 1997–2001
    • University of Oxford
      • Department of Biochemistry
      Oxford, ENG, United Kingdom
  • 1996
    • Royal College of Surgeons of England
      Londinium, England, United Kingdom