Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Transparent Adult Zebrafish as a Tool for In Vivo Transplantation Analysis

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/epdf/10.1634/stemcells.2007-0594

Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

Raman spectroscopy is a newly developed, noninvasive preclinical imaging technique that offers picomolar sensitivity and multiplexing capabilities to the field of molecular imaging. In this study, we demonstrate the ability of Raman spectroscopy to separate the spectral fingerprints of up to 10 different types of surface enhanced Raman scattering (SERS) nanoparticles in a living mouse after s.c. injection. Based on these spectral results, we simultaneously injected the five most intense and spectrally unique SERS nanoparticles i.v. to image their natural accumulation in the liver. All five types of SERS nanoparticles were successfully identified and spectrally separated using our optimized noninvasive Raman imaging system. In addition, we were able to linearly correlate Raman signal with SERS concentration after injecting four spectrally unique SERS nanoparticles either s.c. (R2 = 0.998) or i.v. (R2 = 0.992). These results show great potential for multiplexed imaging in living subjects in cases in which several targeted SERS probes could offer better detection of multiple biomarkers associated with a specific disease.

https://www.pnas.org/content/pnas/106/32/13511.full.pdf

Multiplexed imaging of surface enhanced Raman scattering nanotags in living mice using noninvasive Raman spectroscopy

Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells to Cardiomyocytes: A Methods Overview

Background— Recent studies have shown that stem cell therapy can promote tissue regeneration; however, monitoring stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. Methods and Results— Murine embryonic stem (ES) cells were stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). ES cell viability, proliferation, and differentiation ability were not adversely affected by either reporter genes or reporter probes compared with nontransduced control cells (P=NS). Afterward, 1×107 of ES cells carrying the TF reporter gene (ES-TF) were injected into the myocardium of adult nude rats (n=20). Control animals received nontransduced ES cells (n=6). At day 4, the bioluminescence and positron emission tomography signals in study animals were 3.7×107±5.8×106 photons · s−1 · cm−2 per steradian (s...

/pdf/in-vivo-visualization-of-embryonic-stem-cell-survival-22hf4nvavv.pdf

In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery

Background: Semiconductor quantum dots (QDs) hold increasing potential for cellular imaging both in vitro and in vivo. In this report, we aimed to evaluate in vivo multiplex imaging of mouse embryonic stem (ES) cells labeled with Qtracker delivered quantum dots (QDs). Results: Murine embryonic stem (ES) cells were labeled with six different QDs using Qtracker. ES cell viability, proliferation, and differentiation were not adversely affected by QDs compared with non-labeled control cells (P = NS). Afterward, labeled ES cells were injected subcutaneously onto the backs of athymic nude mice. These labeled ES cells could be imaged with good contrast with one single excitation wavelength. With the same excitation wavelength, the signal intensity, defined as (total signal-background)/exposure time in millisecond was 11 ± 2 for cells labeled with QD 525, 12 ± 9 for QD 565, 176 ± 81 for QD 605, 176 ± 136 for QD 655, 167 ± 104 for QD 705, and 1,713 ± 482 for QD 800. Finally, we have shown that QD 800 offers greater fluorescent intensity than the other QDs tested. Conclusion: In summary, this is the first demonstration of in vivo multiplex imaging of mouse ES cells labeled QDs. Upon further improvements, QDs will have a greater potential for tracking stem cells within deep tissues. These results provide a promising tool for imaging stem cell therapy noninvasively in vivo.

/pdf/quantum-dot-imaging-for-embryonic-stem-cells-19x26mhe2k.pdf

Quantum dot imaging for embryonic stem cells.

Numerous studies have demonstrated the potential use of stem cells for the repair and regeneration of injured tissues. However, tracking transplanted stem cell fate and function in vivo remains problematic. To address these issues, murine embryonic stem (ES) cells were stably transduced with self-inactivating lentiviral vectors carrying either a triple fusion (TF) or double fusion (DF) reporter gene construct. The TF consisted of monomeric red fluorescence protein (mrfp), firefly luciferase (Fluc), and herpes simplex virus truncated thymidine kinase (HSV-ttk) reporter genes. The DF consisted of enhanced green fluorescence protein (egfp) and Fluc reporter genes but lacked HSV-ttk. Stably transduced ES-TF or ES-DF cells were selected by fluorescence activated cell sorting based on either mrfp (TF) or egfp (DF) expression. Afterwards, cells were injected subcutaneously into the right (ES-TF cells) and left (ES-DF cells) shoulders of adult female nude mice. Cell survival was tracked noninvasively by bioluminescence and positron emission tomography imaging of Fluc and HSV-ttk reporter genes, respectively. Imaging signals progressively increased from day 2 to day 14, consistent with ES cell survival and proliferation in vivo. However, teratoma formation occurred in all nude mice after 5 weeks. Administration of ganciclovir (GCV), targeting the HSV-ttk gene, resulted in selective ablation of teratomas arising from the ES-TF cells but not ES-DF cells. These data demonstrate the novel use of multimodality imaging techniques to (1) monitor transplanted ES cell survival and proliferation in vivo and (2) assess the efficacy of suicide gene therapy as a backup safety measure against teratoma formation.

Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation.

Stem cell therapy offers exciting promise for treatment of ischemic heart disease Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations Microarray studies comparing gene expression in nontransduced control ES cells vs stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells

Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation.

Purpose
The therapeutic benefits of cell transplantation may depend on the survival of sufficient numbers of grafted cells. We evaluate four potent immunosuppressive medications aimed at preventing acute donor cell death.

Shuan Lin

Papers

In Vivo Visualization of Embryonic Stem Cell Survival, Proliferation, and Migration After Cardiac Delivery

Quantum dot imaging for embryonic stem cells.

Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation.

Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation.

Noninvasive evaluation of immunosuppressive drug efficacy on acute donor cell survival.