High-precision in vivo tracking of stem cells using carbon-14 labeling: a novel quantitative approach for assessing therapeutic efficacy and safety

This study develops multimodal magnetic nanoclusters (M‐MNCs) for gene transfer, directed migration, and tracking of human mesenchymal stem cells (hMSCs). The M‐MNCs are designed with 5 nm iron oxide nanoparticles and a fluorescent dye (i.e., Rhodamine B) in the matrix of the Food and Drug Administration approved polymer poly(lactide‐co‐glycolide) using a nanoemulsion method. The synthesized M‐MNCs have a hydrodynamic diameter of ≈150 nm, are internalized by stem cells via endocytosis, and deliver genes with high efficiency. The cellular internalization and gene expression efficiency of the clustered nanoparticles are significantly higher than that of single nanoparticles. The M‐MNC‐labeled hMSCs migrate upon application of a magnetic force and can be visualized by both optical and magnetic resonance (MR) imaging. In animal models, the M‐MNC‐labeled hMSCs are also successfully tracked using optical and MR imaging. Thus, the M‐MNCs not only allow the efficient delivery of genes to stem cells but also the tracking of cells in animal models. Taken together, the results show that this new type of nanocomposite can be of great help in future stem cell research and in the development of cell‐based therapeutic agents.

Multi-modal transfection agent based on monodisperse magnetic nanoparticles for stem cell gene delivery and tracking

Event Abstract Back to Event A Novel Benchmark Data Set for Adult Stem Cell Tracking in Time-Lapse Microscopy Amir Madany-Mamlouk1*, Tim Becker2 and Daniel Rapoport3 1 University of Lubeck, Institute for Neuro and Bioinformatics, Germany 2 Graduate School at University of Lubeck, Germany 3 Fraunhofer EMB, Germany In modern stem cell therapies -- for cancer treatment as well as for cardiac operations -- typically both types of stem cells (autologous or allogenic) are immediately transplanted after isolation. Especially the potential of growing these cells in culture is neglected this way, even though clinical cell farming would significantly enhance the scope of stem cell treatment. There is just no profound diagnostic and documentation system for clinical cell farming.To gather such knowledge we established a robust and complete cell detection in time-lapse microscopy to have a highly effective tracking of several hundreds to thousands cells per image. Doing so, cell populations can be traced for days up to weeks. Unfortunately, the fully-automated cell tracking poses some challenging problems, especially as every new cell culture tends to vary strongly for several degrees of freedom, even within the same cell types.In this work, we will present the following three results of our ongoing project: 1. Cell Detection and Tracking on Phase-Contrast Microscopy Time-Lapse Data Essential for a robust and reliable cell tracking is a successful cell detection task, in which we find all cells in a given microscopy snapshot. We propose a preprocessing using a cascade of morphological filters, and a prototyping approach for unknown cell types using a supervised neural network. For certain cell types, the presented framework is capable of detecting up to 95% of all cells in real data scenarios and track most of them over all frames of the recording.(See the below picture, in which the red cell is of interest. Red dots mark the path this cell moved over time, yellow cells are sister cells and yellow dots their path over time) 2. Proposing a Benchmark Set for the Evaluation of Cell Tracking/Detection Methods. To evaluate our above approaches, we are currently building up a huge database of several thousand completely labeled cell culture images taken under different conditions and objectives. This data is collected in a semi-automatic fashion, with an initial fully-automated detection and an assisted manual correction and control step afterwards. The human operator has not to bother about all clear cases and can focus on the remaining 5% of cells, which is significant speaking of 500 instead of 10.000 cells to be hand-labeled. This benchmark database will be available to the public in the near future and might help to compare existing approaches in cell tracking.3. sensor fusion of phase contrast, light microscopy and immunofluorescence images.In a next step, we want to propose an approach to fuse all available information during cell tracking to gain insides of the cells behavior that is not visible observing a single measure alone. In detail, we will discuss the fusion of phase contrast and light microscopy images together with information about the optical flow in the image sequences and correlating this information to a standard measure, the immunofluorescence staining of the cells, for which cells typically have to be destroyed. INCF-09-106 Conference: Neuroinformatics 2009, Pilsen, Czechia, 6 Sep - 8 Sep, 2009. Presentation Type: Poster Presentation Topic: General neuroinformatics Citation: Madany-Mamlouk A, Becker T and Rapoport D (2019). A Novel Benchmark Data Set for Adult Stem Cell Tracking in Time-Lapse Microscopy. Front. Neuroinform. Conference Abstract: Neuroinformatics 2009. doi: 10.3389/conf.neuro.11.2009.08.081 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 May 2009; Published Online: 09 May 2019. * Correspondence: Amir Madany-Mamlouk, University of Lubeck, Institute for Neuro and Bioinformatics, Lubeck, Germany, madany@inb.uni-luebeck.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Amir Madany-Mamlouk Tim Becker Daniel Rapoport Google Amir Madany-Mamlouk Tim Becker Daniel Rapoport Google Scholar Amir Madany-Mamlouk Tim Becker Daniel Rapoport PubMed Amir Madany-Mamlouk Tim Becker Daniel Rapoport Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Stem cell therapies are increasingly recognized as the future direction of regenerative medicine, but the biological fate of the administrated stem cells remains a major concern for clinical translation, which calls for an approach to efficiently monitoring the stem cell behaviors in vivo. Magnetic particle imaging (MPI) is an emerging technology for cell tracking; however, its utility has been largely restricted due to the lack of optimal magnetic nanoparticle tracers. Herein, by controlled engineering of the size and shape of magnetic nanoparticles tailored to MPI physics theory, a specialized MPI tracer, based on cubic iron oxide nanoparticles with an edge length of 22 nm (CIONs-22), is developed. Due to the inherent lower proportion of disordered surface spins, CIONs-22 exhibit significantly larger saturation magnetization than that of spherical ones, while they possess similar saturation magnetization but smaller coercivity compared to larger-sized CIONs. These magnetic properties of CIONs-22 warrant high sensitivity and resolution of MPI. With their efficient cellular uptake, CIONs-22 exhibit superior MPI performance for stem cell labeling and tracking compared to the commercialized tracer Vivotrax. By virtue of these advantages, CIONs-22 enable real-time and prolonged monitoring of the spatiotemporal trajectory of stem cells transplanted to hindlimb ischemia mice, which demonstrates the great potential of CIONs-22 as MPI tracers to advance stem cell therapies.

Stem cell therapy is rapidly moving toward translation to clinical application. To elucidate the therapeutic effect, a robust method that allows tracking of the stem cells over an extended period of time is required. Herein, semiconducting polymer dots (Pdots) are demonstrated for their use in bright labeling and tracking of human mesenchymal stem cells (MSCs) in vitro and in vivo. The Pdots coated with a cell-penetrating peptide (R8) showed remarkable endocytic uptake efficiency that was 15 times higher than that of carboxyl Pdots and more than 200 times than that of bare Pdots. The Pdot-labeled MSCs can be traced for 15 generations in vitro and tracked over 2 weeks in vivo after subcutaneous transplantation. The labeled MSCs administered through the tail vein were preferentially accumulated in the lung; this was distinctive from the distribution of free Pdots, which were primarily distributed in the liver. Based on the properties of bright labeling, excellent tracking capability, and great biocompatibility, the Pdots will be valuable in the applications of stem cell biology and regenerative medicine.

Bone marrow (BM)-derived stem and immune cells play critical roles in maintaining the health, regeneration, and repair of many tissues. Given their important functions in tissue regeneration and therapy, tracking the dynamic behaviors of BM-derived cells has been a long-standing research goal of both biologists and engineers. Because of the complex cellular-level processes involved, real-time imaging technologies that have sufficient spatial and temporal resolution to visualize them are needed. In addition, in order to track cellular dynamics, special attention is needed to account for changes in the microenvironment where the cells reside, for example, tissue contraction, stretching, development, etc. In this chapter, we introduce methods for real-time imaging and longitudinal tracking of BM-derived immune and stem cells in in vivo three-dimensional (3-D) tissue environments with an integrated optical microscope. The integrated microscope combines multiple imaging functions derived from optical coherence tomography (OCT) and multiphoton microscopy (MPM), including optical coherence microscopy (OCM), microvasculature imaging, two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) microscopy. Short- and long-term tracking of the dynamic behavior of BM-derived cells involved in cutaneous wound healing and skin grafting in green fluorescent protein (GFP) BM-transplanted mice is demonstrated. Methods and algorithms for nonrigid registration of time-lapse images are introduced, which allows for long-term tracking of cell dynamics over several months.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 × 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 × 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

Automatic tracking of neural stem cells in sequential digital images

High MR sensitive fluorescent magnetite nanocluster for stem cell tracking in ischemic mouse brain

Photoacoustic (PA) imaging and tracking of stem cells plays an important role in the real-time assessment of cell-based therapies. Nevertheless, the limitations of conventional inorganic PA contrast agents and the narrow range of the excitation wavelength in the first near-infrared (NIR-I) window hamper the applications of PA imaging in living subjects. Herein, we report the design and synthesis of a second near-infrared (NIR-II) absorptive organic semiconducting polymer (OSP)-based nanoprobe (OSPN+) for PA imaging and tracking of stem cells. Comparison studies in biological tissue show that NIR-II light excited PA imaging of the OSPN+ has significantly higher signal-to-noise ratio than NIR-I light excited PA imaging, thereby demonstrating the superiority of the OSPN+ for deep tissue imaging. With good biocompatibility, appropriate size, and optimized surface property, the OSPN+ shows enhanced cellular uptake for highly efficient PA labeling of stem cells. In vivo investigations reveal significant NIR-II PA contrast enhancement of the transplanted OSPN+-labeled human mesenchymal stem cells by 40.6- and 21.7-fold in subcutaneous and brain imaging, respectively, compared with unlabeled cases. Our work demonstrates a class of OSP-based nanomaterials for NIR-II PA stem cell imaging to facilitate a better understanding and evaluation of stem cell-based therapies.

Stem cells hold great promise for the treatment of multiple human diseases and disorders. Tracking and monitoring of stem cells in vivo after transplantation can supply important information for determining the efficacy of stem cell therapy. Magnetic resonance imaging (MRI) combined with contrast agents is believed to be the most effective and safest non-invasive technique for stem cell tracking in living bodies. Commercial superparamagnetic iron oxide nanoparticles (SPIONs) in the aid of transfection agents (TAs) have been applied to labeling stem cells. However, owing to the potential toxicity of TAs, more attentions have been paid to develop novel SPIONs with specific surface coating or functional moieties which facilitate effective cell internalization in the absence of TAs. This review aims to summarize the recent progress in the design and preparation of SPIONs as cellular MRI probes, to discuss their applications and current problems facing in stem cell labeling and tracking, and to offer perspectives and solutions for the future development of SPIONs in this field.

Reliable long-term in vivo tracking of stem cells is of great importance in stem cell-based therapy and research. Fluorescence imaging with in situ excitation has significant autofluorescence background, which results in poor signal-to-noise ratio (SNR). Here we report TAT penetrating peptide-bioconjugated long persistent luminescence nanoparticles (LPLNP-TAT) for long-term tracking of adipose-derived stem cells (ASC) without constant external excitation. LPLNP-TAT exhibits near-infrared emitting, red light renewable capability, and superior in vivo imaging depth and SNR compared with conventional organic dye and quantum dots. Our findings show that LPLNP-TAT can successfully label ASC without impairing their proliferation and differentiation and can effectively track ASC in skin-regeneration and tumor-homing models. We believe that LPLNP-TAT represents a new generation of cell tracking probes and will have broad application in diagnosis and therapy.

SOD-Functionalized gold nanoparticles as ROS scavenger and CT contrast agent for protection and imaging tracking of mesenchymal stem cells in Idiopathic pulmonary fibrosis treatment

Human stem cells from various sources have potential therapeutic applications. The clinical implementation of these therapies introduces the need for methods of noninvasive tracking of cells. The purpose of this study was to evaluate a high resolution magnetic resonance imaging (MRI) technique for in vivo detection and tracking of superparamagnetic micron sized iron oxide particle (MPIO)-labeled human amniotic fluid stem (hAFS) cells injected in the mouse heart. Because of the small subject size, MR signal and resolution of the in vivo MRI were increased using strong gradients, a 7.0 Tesla magnet, and an ECG and respiratory gated gradient echo sequence. MRI images of mouse heart were acquired during a 4 week course of this longitudinal study. At the end of the study, histological analysis was used to correlate cell localization with the MRI results. Introduction of MPIOs into hAFS had no significant effect upon cell proliferation and differentiation. Results of flow cytometry analysis indicated that hAFS cells remained labeled for up to 4 weeks. MRI of MPIO-labeled hAFS cells injected in agarose gels resulted in significant hypointense regions. Labeled hAFS cells injected into mouse hearts produced hypointense regions in the MR images that could be detected 24 hours and 7, 14, 21 and 28 days post injection. The co-localization of labeled cells within the hypointense regions was confirmed by histological analysis. These results indicate that high resolution MRI can be used successfully for noninvasive longitudinal tracking of hAFS cells injected in the mouse heart. The potential utility of this finding is that injected stem cells can be tracked in vivo and might serve to monitor cell survival, proliferation and integration into myocardial tissue.