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Verma V.K.,Immunology and Stem Cell Laboratory | Kamaraju S.R.,Immunology and Stem Cell Laboratory | Kancherla R.,Immunology and Stem Cell Laboratory | Kona L.K.,Immunology and Stem Cell Laboratory | And 6 more authors.
International Journal of Nanomedicine | Year: 2015

Fluorescent magnetic iron oxide nanoparticles have been used to label cells for imaging as well as for therapeutic purposes. The purpose of this study was to modify the approach to develop a nanoprobe for cell selection and imaging with a direct therapeutic translational focus. The approach involves physical coincubation and adsorption of superparamagnetic iron oxide nanoparticle-polyethylene glycol (SPION-PEG) complexes with a monoclonal antibody (mAb) or a set of antibodies. Flow cytometry, confocal laser scanning microscopy, transmission electron microscopy, iron staining, and magnetic resonance imaging were used to assess cell viability, function, and labeling efficiency. This process has been validated by selecting adipose tissue-derived cardiac progenitor cells from the stromal vascular fraction using signal regulatory protein alpha (SIRPA)/kinase domain receptor (KDR) mAbs. These markers were chosen because of their sustained expression during cardiomyocyte differentiation. Sorting of cells positive for SIRPA and KDR allowed the enrichment of cardiac progenitors with 90% troponin-I positivity in differentiation cultures. SPION labeled cardiac progenitor cells (1×105 cells) was mixed with gel and used for 3T magnetic resonance imaging at a concentration, as low as 12.5 µg of iron. The toxicity assays, at cellular and molecular levels, did not show any detrimental effects of SPION. Our study has the potential to achieve moderate to high specific cell selection for the dual purpose of imaging and therapy. © 2015 Verma et al. Source


Verma V.K.,Immunology and Stem Cell Laboratory | Beevi S.S.,Immunology and Stem Cell Laboratory | Debnath T.,Immunology and Stem Cell Laboratory | Shalini U.,Immunology and Stem Cell Laboratory | And 4 more authors.
New Horizons in Translational Medicine | Year: 2015

Background: Cardiomyocyte enrichment strategies so far have not yielded scalable cardiac specific cell type. More so, the current data is restricted to embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs), wherein the use of viral vectors is fraught with increased risk during clinical use. Herein, we profiled time-dependent gene/protein expression patterns across the cardiac ectoderm, endoderm, and mesoderm for isolating cardiac precursors from human adipose derived stem cells (hADSC). Methods: Direct cardiac differentiation of hADSCs was carried out with 5-azacytidine and basic fibroblast growth factor (bFGF) in a one month long culture. The cells were periodically harvested, analyzed for unique persistent markers and their inherent regulation using quantitative polymerase chain reaction (qPCR), flow cytometry, immunoblot and immunocytochemistry assays. The identified markers were super paramagnetic iron oxide nanoparticle (SPION) tagged for segregation by magnetic activated cell sorting (MACS) and further evaluated their differentiation potential and checked for the purity by flow cytometry. Results: The results demonstrated pronounced up-regulation of mesodermal and mature cardiac lineage markers at three weeks, while there was a down-regulation of pluripotent stem cell markers. This perhaps could be attributed to de-differentiation in maintaining the cardiac phenotype. However, signal regulatory protein alpha (SIRPA) and kinase domain receptor (KDR) persisted all through the culture period of one month, making them the most relevant and reliable cardiac specific markers. Dual labeling of these markers to SPION for cardiomyocyte enrichment by MACS column yielded cardiomyogenic-like cells in differentiation cultures with several functional positive markers. Conclusions: Thus, SIRPA and KDR together provide cues in the enhancement and up-scaling of cardiomyocyte production in the cell replacement therapy. Benchside: Identification of specific cell phenotypic markers to identify cardiac precursors in any tissue source with minimal cell manipulation is a novel process development tool in clinical translation. Bedside: A product developed in a closed system would minimize extraneous contaminants in long term cultures and development of such procedures minimizes culture failure rates from bench side. Industry: This unique identification of cell-specific marker would enable a tissue-specific translational plan and immensely help in the cardiac regeneration. Government: Financial investment and support from the government is vital in the optimization and validation for better health care and would contribute in reducing the disease burden. © 2015 European Society for Translational Medicine. Source


Verma V.K.,Immunology and Stem Cell Laboratory | Beevi S.S.,Immunology and Stem Cell Laboratory | Tabassum A.,KK Nuclear Scans | Kumaresan K.,KK Nuclear Scans | And 3 more authors.
Nuclear Medicine and Biology | Year: 2014

Introduction: Noninvasive radionuclide imaging of cells using technetium99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) is a potential diagnostic tool for several applications. Herein we aimed to evaluate the labeling efficiency and cellular toxicity of 99mTc-HMPAO with Stromal Vascular Fraction (SVF) of adipose tissue to develop a process tool for theranostic purposes, in particular imaging cardiac stem cell therapy. Methods: Ten million cells of SVF were labeled with 99mTc-HMPAO complex and excess radiolabel was cleared off through washing in PBS. The labeling efficiency of 99mTc-HMPAO was detected in labeled cells and their subsequent supernatant wash using isotope dose calibrator and gamma camera. The cytotoxicity was assessed for the comparative reactive oxygen species (ROS) by H2DCFDDA, apoptotic events by annexin-V and TUNEL assay and mitochondrial potential by JC-1. Results: An encouraging labeling efficiency of 33% was observed with 99mTc-HMPAO complex. The radionuclide labeling of SVF demonstrated significant safety profile as evaluated by apoptotic assays. Conclusion: 99mTc-HMPAO labeling efficiency of 33% of total SV fraction would produce sufficient radioactive signals that would enable for in vivo tracking of cells by SPECT-CT. The radionuclide did not demonstrate any significant impact on the structural or functional organization of the labeled cells. Our study indicates that SVF can be safely labeled with 99mTc-HMPAO without adverse cytotoxic events and for its potential role in imaging cardiac stem cell therapy. © 2014 Elsevier Inc. Source

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