Guidelines and best practices in successfully using Zebrabow for lineage tracing multiple cells within tissues.

Labelling cells and following their progeny, also known as lineage tracing, has provided important insights into the cellular origins of tissues. Traditional lineage tracing experiments have been limited to following single or small groups of cells with classic techniques such as dye injections and Cre/LoxP labelling of cells of interest. Brainbow is a fluorescent dependent, lineage tracing technique that allows a broader visualization and analysis of multiple cells within a tissue, initially deployed to examine lineages within neural tissues. This technique has now been adapted to zebrafish (Zebrabow) and takes advantages of the imaging capabilities that this system provides over other animal models. In this paper we shall describe how Zebrabow is performed as well as some guides on some of the common pitfalls encountered when using this labelling strategy.

Cell tracking, survival, and differentiation capacity of adipose-derived stem cells after engraftment in rat tissue.

Adipose tissue is an important source of adipose derived stem cells (ADSCs). These cells have the potential of being used for certain therapies, in which the main objective is to recover the function of a tissue/organ affected by a disease. In order to contribute to repair of the tissue, these cells should be able to survive and carry out their functions in unfavorable conditions after being transplanted. This process requires a better understanding of the biology involved: such as the time cells remain in the implant site, how long they stay there, and whether or not they differentiate into host tissue cells. This report focuses on these questions. ADSC were injected into three different tissues (substantia nigra, ventricle, liver) and they were tracked in vivo with a dual GFP-Luc reporter system. The results show that ADSCs were able to survive up to 4 months after the engraftment and some of them started showing resident cell tissue phenotype. These results demonstrate their long-term capacity of survival and differentiation when injected in vivo.

A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice.

Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: (1) real-time PCR-based detection of a novel male-specific, multiple-copy gene, Rbmy, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and (2) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 10(5) male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75 % of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6 ± 2.1 % of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8 ± 0.5 % (i.e., 1,224 ± 230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10 % of the cells injected; at 35 days, ~1 %). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking.

Magnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells. Cells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. Using the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 × 1016 19F atoms) were detected with 19F MRI, with SNR > 5. MPI has the potential to be more sensitive than 19F MRI for cell tracking. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

Cellular imaging using temporally flickering nanoparticles.

Utilizing the surface plasmon resonance effect in gold nanoparticles enables their use as contrast agents in a variety of applications for compound cellular imaging. However, most techniques suffer from poor signal to noise ratio (SNR) statistics due to high shot noise that is associated with low photon count in addition to high background noise. We demonstrate an effective way to improve the SNR, in particular when the inspected signal is indistinguishable in the given noisy environment. We excite the temporal flickering of the scattered light from gold nanoparticle that labels a biological sample. By preforming temporal spectral analysis of the received spatial image and by inspecting the proper spectral component corresponding to the modulation frequency, we separate the signal from the wide spread spectral noise (lock-in amplification).

Background migration of USPIO/MLs is a major drawback for in situ labeling of endogenous neural progenitor cells.

MR-labeling of endogenous neural progenitor cells (NPCs) to follow up cellular migration with in vivo magnetic resonance imaging (MRI) is a very promising tool in the rapidly growing field of cellular imaging. To date, most of the in situ labeling work has been performed using micron-sized iron oxide particles. In this work magnetoliposomes (MLs), i.e. ultrasmall superparamagnetic iron oxide cores (USPIOs), each individually coated by a phospholipid bilayer, were used as the MR contrast agent. One of the main advantages of MLs is that the phospholipid bilayer allows easy modification of the surface, which creates the opportunity to construct a wide range of MLs optimized for specific biomedical applications. We have investigated the ability of MLs to label endogenous NPCs after direct injection into the adult mouse brain. Whereas MRI revealed contrast relocation towards the olfactory bulb, our data strongly imply that this relocation is independent of the migration of endogenous NPCs but represents background migration of MLs along a white matter tract. Our findings suggest that the small size of USPIOs/MLs intrinsically limits their potential for in situ labeling of NPCs.

Stem cell tracking in human trials: a meta-regression.

The potential effectiveness of cell therapies is dependent upon homing of transplanted cells to relevant target organs. In this study we firstly characterise the range of methods employed in all human therapeutic-cell studies published to date investigated with cell-tracking. Secondly, we determine factors that predict target-organ cell uptake efficiency by meta-regression. Following a comprehensive literature search, we identified 19 relevant trials, representing 145 patients over the following 7 diseases: myocardial infarction; Chagasic cardiomyopathy; ischemic stroke; traumatic injury of brain or spinal cord; diabetes and cirrhosis. Cell-labelling strategies employed were: 18-fluorodeoxyglucose-PET, 111-indium-SPECT; 99-technetium-SPECT, and iron oxide-MRI. The following methodological parameters were extracted: label type; label dose; labelling efficiency; stability; cell dose; percentage labelled cells; disease type and chronicity; cell purity; cell type; and cell uptake efficiency. Meta-regression techniques were used to identify predictors of cell-labelling efficiency; viability and cell uptake efficiency. These analyses found that labelling efficiency is proportionate to cell dose, while cell viability is lowest with indium and long label incubation times. Uptake efficiency of cells is predicted by stem cell purity (positive association) and cell infusion number (negative association), although these two variables are themselves strongly negatively correlated between studies. In summary the methodological factors associated with enhanced therapeutic-cell homing from both our own analysis, and within-trial comparisons, are: acute (versus chronic) disease, selective stem cells (versus unselected cells), and intra-arterial (versus intravenous) delivery. However, future trials need to keep cell doses and imaging times constant so as to enable meaningful comparisons in uptake efficiency.