Inhibition of GSK3ß Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation.

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3ß inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3ß inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone.

Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.

A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

Near-infrared fluorescence imaging of liver metastases in rats using indocyanine green.

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) is a promising technique to obtain real-time assessment of the extent and number of colorectal liver metastases during surgery. The current study aims to optimize dosage and timing of ICG administration. MATERIALS AND METHODS: Liver tumors were induced in 18 male WAG/Rij rats by subcapsular inoculation of CC531 rat colorectal cancer cells into three distinct liver lobes. Rats were divided in two groups: imaging after 24 and 48 h or 72 and 96 h after intravenous ICG administration. In each time group, rats were allocated to three dose groups: 0.04, 0.08, or 0.16 mg ICG. Intraoperative imaging and ex vivo measurements were performed using the Mini-FLARE imaging system and confirmed by fluorescence microscopy. Fluorescence intensity was quantified using the Mini-FLARE software and the difference between tumor signal and liver signal (tumor-to-liver ratio; TLR) was calculated. RESULTS: In all 18 rats, all colorectal liver metastases (n = 34), some as small as 1.2 mm, were identified using ICG and the Mini-FLARE imaging system. Average tumor-to-liver ratio (TLR) over all groups was 3.0 ± 1.2. TLR was significantly higher in the 72 h time group compared with other time points. ICG dose did not significantly influence TLR, but a trend was found favoring the 0.08 mg dose group. Fluorescence microscopy demonstrated a clear fluorescent rim around the tumor. CONCLUSIONS: This study demonstrates that colorectal cancer liver metastases can be clearly identified during surgery using ICG and the Mini-FLARE imaging system, with optimal timing of 72 h post-injection and an optimal dose of 0.08 mg (0.25 mg/kg) ICG. NIR fluorescence imaging has the potential to improve intraoperative detection of micrometastases and, thus, the completeness of resection.

Optically Coupled PtOEP and DPA Molecules Encapsulated into PLGA-Nanoparticles for Cancer Bioimaging.

Triplet-triplet annihilation upconversion (TTA-UC) nanoparticles (NPs) have emerged as imaging probes and therapeutic probes in recent years due to their excellent optical properties. In contrast to lanthanide ion-doped inorganic materials, highly efficient TTA-UC can be generated by low excitation power density, which makes it suitable for clinical applications. In the present study, we used biodegradable poly(lactic-co-glycolic acid) (PLGA)-NPs as a delivery vehicle for TTA-UC based on the heavy metal porphyrin Platinum(II) octaethylporphyrin (PtOEP) and the polycyclic aromatic hydrocarbon 9,10-diphenylanthracene (DPA) as a photosensitizer/emitter pair. TTA-UC-PLGA-NPs were successfully synthesized according to an oil-in-water emulsion and solvent evaporation method. After physicochemical characterization, UC-efficacy of TTA-UC-PLGA-NPs was assessed in vitro and ex vivo. TTA-UC could be detected in the tumour area 96 h after in vivo administration of TTA-UC-PLGA-NPs, confirming the integrity and suitability of PLGA-NPs as a TTA-UC in vivo delivery system. Thus, this study provides proof-of-concept that the advantageous properties of PLGA can be combined with the unique optical properties of TTA-UC for the development of advanced nanocarriers for simultaneous in vivo molecular imaging and drug delivery.

Image-guided tumor resection using real-time near-infrared fluorescence in a syngeneic rat model of primary breast cancer.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

19F-nanoparticles: Platform for in vivo delivery of fluorinated biomaterials for 19F-MRI.

Fluorine-19 (19F) magnetic resonance imaging (MRI) features one of the most investigated and innovative techniques for quantitative and unambiguous cell tracking, providing information for both localization and number of cells. Because of the relative insensitivity of the MRI technique, a high number of magnetically equivalent fluorine atoms are required to gain detectable signals. However, an increased amount of 19F nuclei induces low solubility in aqueous solutions, making fluorine-based probes not suitable for in vivo imaging applications. In this context, nanoparticle-based platforms play a crucial role, since nanoparticles may carry a high payload of 19F-based contrast agents into the relevant cells or tissues, increase the imaging agents biocompatibility, and provide a highly versatile platform. In this review, we present an overview of the 19F-based nanoprobes for sensitive 19F-MRI, focusing on the main nanotechnologies employed to date, such as fluorine and theranostic nanovectors, including their design and applications.

Novel intraoperative near-infrared fluorescence camera system for optical image-guided cancer surgery.

Current methods of intraoperative tumor margin detection using palpation and visual inspection frequently result in incomplete resections, which is an important problem in surgical oncology. Therefore, real-time visualization of cancer cells is needed to increase the number of patients with a complete tumor resection. For this purpose, near-infrared fluorescence (NIRF) imaging is a promising technique. Here we describe a novel, handheld, intraoperative NIRF camera system equipped with a 690 nm laser; we validated its utility in detecting and guiding resection of cancer tissues in two syngeneic rat models. The camera system was calibrated using an activated cathepsin-sensing probe (ProSense, VisEn Medical, Woburn, MA). Fluorescence intensity was strongly correlated with increased activated-probe concentration (R2= .997). During the intraoperative experiments, a camera exposure time of 10 ms was used, which provided the optimal tumor to background ratio. Primary mammary tumors (n = 20 tumors) were successfully resected under direct fluorescence guidance. The tumor to background ratio was 2.34 using ProSense680 at 10 ms camera exposure time. The background fluorescence of abdominal organs, in particular liver and kidney, was high, thereby limiting the ability to detect peritoneal metastases with cathepsin-sensing probes in these regions. In conclusion, we demonstrated the technical performance of this new camera system and its intraoperative utility in guiding resection of tumors.

An in vitro model that can distinguish between effects on angiogenesis and on established vasculature: actions of TNP-470, marimastat and the tubulin-binding agent Ang-510.

In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature; one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach.

Antigen-adjuvant nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles?

Entrapment of antigens in mucoadhesive nanoparticles prepared from N-trimethyl chitosan (TMC) has been shown to increase their immunogenicity. However, because of their large size compared to soluble antigens, particles poorly diffuse through the nasal epithelium. The aim of this work was to study whether nasal vaccination with a much smaller TMC-antigen nanoconjugate would result in higher antibody responses as compared to TMC nanoparticles. TMC was covalently linked to a model antigen, ovalbumin (OVA), using thiol chemistry. For comparison, TMC/OVA nanoparticles and solutions of OVA and a physical mixture of TMC and OVA were made. As shown previously for TMC/OVA nanoparticles, TMC-OVA conjugate prolonged the nasal residence time of the antigen. TMC-OVA conjugate diffused significantly better through a monolayer of lung carcinoma (Calu-3) cells than TMC/OVA nanoparticles did. Moreover, nasal immunization of mice with the conjugate resulted in significantly more OVA positive DCs in the cervical lymph nodes as compared to TMC/OVA nanoparticles. Mice nasally immunized with TMC-OVA conjugate produced high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG than mice immunized with TMC/OVA nanoparticles after a priming dose. Moreover, as compared to TMC/OVA nanoparticles, TMC-OVA conjugate induced a more balanced IgG1/IgG2a response. In conclusion, the TMC-antigen nanoconjugate improves nasal delivery and immunogenicity of the antigen. This suggests that efficient codelivery of antigen and adjuvant to DCs, rather than a particulate form of the antigen/adjuvant combination, is decisive for the immunogenicity of the antigen.

Design, construction, and biological testing of an implantable porous trilayer scaffold for repairing osteoarthritic cartilage.

Various tissue engineering systems for cartilage repair have been designed and tested over the past two decades, leading to the development of many promising cartilage grafts. However, no one has yet succeeded in devising an optimal system to restore damaged articular cartilage. Here, the design, assembly, and biological testing of a porous, chitosan/collagen-based scaffold as an implant to repair damaged articular cartilage is reported. Its gradient composition and trilayer structure mimic variations in natural cartilage tissue. One of its layers includes hydroxyapatite, a bioactive component that facilitates the integration of growing tissue on local bone in the target area after scaffold implantation. The scaffold was evaluated for surface morphology; rheological performance (storage, loss, complex, and time-relaxation moduli at 1 kHz); physiological stability; in vitro activity and cytotoxicity (on a human chondrocyte C28 cell line); and in vivo performance (tissue growth and biodegradability), in a murine model of osteoarthritis. The scaffold was shown to be mechanically resistant and noncytotoxic, favored tissue growth in vivo, and remained stable for 35 days postimplantation in mice. These encouraging results highlight the potential of this porous chitosan/collagen scaffold for clinical applications in cartilage tissue engineering.

Molecular imaging of tumor angiogenesis using alphavbeta3-integrin targeted multimodal quantum dots.

Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel alphavbeta3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.

Targeting Polymeric Nanobiomaterials as a Platform for Cartilage Tissue Engineering.

Articular cartilage is a connective tissue structure that is found in anatomical areas that are important for the movement of the human body. Osteoarthritis is the ailment that most often affects the articular cartilage. Due to its poor intrinsic healing capacity, damage to the articular cartilage is highly detrimental and at present the reconstructive options for its repair are limited. Tissue engineering and the science of nanobiomaterials are two lines of research that together can contribute to the restoration of damaged tissue. The science of nanobiomaterials focuses on the development of different nanoscale structures that can be used as carriers of drugs / cells to treat and repair damaged tissues such as articular cartilage. This review article is an overview of the composition of articular cartilage, the causes and treatments of osteoarthritis, with a special emphasis on nanomaterials as carriers of drugs and cells, which reduce inflammation, promote the activation of biochemical factors and ultimately contribute to the total restoration of articular cartilage.

Tissue Engineering: An Alternative to Repair Cartilage.

Herein we review the state-of-the-art in tissue engineering for repair of articular cartilage. First, we describe the molecular, cellular, and histologic structure and function of endogenous cartilage, focusing on chondrocytes, collagens, extracellular matrix, and proteoglycans. We then explore in vitro cell culture on scaffolds, discussing the difficulties involved in maintaining or obtaining a chondrocytic phenotype. Next, we discuss the diverse compounds and designs used for these scaffolds, including natural and synthetic biomaterials and porous, fibrous, and multilayer architectures. We then report on the mechanical properties of different cell-loaded scaffolds, and the success of these scaffolds following in vivo implantation in small animals, in terms of generating tissue that structurally and functionally resembles native tissue. Last, we highlight future trends in this field. We conclude that despite major technical advances made over the past 15 years, and continually improving results in cartilage repair experiments in animals, the development of clinically useful implants for regeneration of articular cartilage remains a challenge

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors.

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.

Whole-body optical imaging in animal models to assess cancer development and progression.

Different optical-based imaging models were used to investigate tumor progression and metastasis with particular emphasis on metastasis to bone and bone marrow. We describe how optical imaging can be used to follow important processes in tumor development and treatment response, including angiogenesis, apoptosis, and proteolysis. Finally, we discuss the translation of one optical imaging modality, near-IR fluorescence, from animal validation studies to applications in the clinic related to cancer management.

Prolonged in vivo gene silencing by electroporation-mediated plasmid delivery of small interfering RNA.

For the successful application of RNA interference in vivo, it is desired to achieve (local) delivery of small interfering RNAs (siRNAs) and long-term gene silencing. Nonviral electrodelivery is suitable to obtain local and prolonged expression of transgenes. By intramuscular electrodelivery of a plasmid in which two opposing human polymerase III promoters (H1 and U6) drive the expression of siRNA constructs that form functional double-stranded siRNAs, in combination with in vivo bioluminescence imaging, we were able to knock down exogenous delivered luciferase for at least 100 days in murine calf muscles. This effect was sequence specific, because scrambled siRNA had no effect. Moreover, we were able to demonstrate in vivo reduction of endogenous TLR4 expression for at least 1 week, using a similar vector expressing an siRNA for TLR4 in the muscle. In this study, we demonstrate that in vivo suppression of both endogenous (for at least 1 week) and introduced genes (>100 days) is feasible via plasmid-driven siRNA expression after electroporation-mediated intramuscular gene transfer. With this approach the short-term effect of oligonucleotides and the drawbacks of viral gene delivery, like immunological responses, could be circumvented. Therefore, this application of RNA interference is a useful tool with which to investigate gene function and might be promising as a therapeutic tool for locally acting diseases such as restenosis or tumors.

Advances in optical imaging and novel model systems for cancer metastasis research.

Research into the genetic and physiological interactions of tumours with their host environment requires in vivo assays to address molecular expression patterns and function. In recent years much of this work has been performed using bioluminescent and fluorescent imaging techniques that allow real-time and non-invasive imaging of gene expression and (tumour) tissue development. Luminescence imaging has until now been more or less the only tool that allows the imaging of intra-osseous breast cancer cells and indeed this technique has been pioneered in our laboratory. Here we summarise some recent innovations and developments using cancer cells and some of the first imaging models of multimodal dual luminescence and luminescence combined with fluorescence of intra-osseous tumours. We further engineered our models to incorporate a specific insertion site in the genome and will discuss some of the possible applications. These include the insertion of signalling pathway-specific reporters and studying the fate of multiple injected populations in a single mouse. We conclude that recent improvements in luminescence- and fluorescence-detection platforms now clearly allow multimodal imaging which will greatly enhance our ability to assess gene function and for the first time to visualise multiple gene- and cellular interactions in real time and in vivo.

Optimized Longitudinal Monitoring of Stem Cell Grafts in Mouse Brain Using a Novel Bioluminescent/Near Infrared Fluorescent Fusion Reporter.

Biodistribution and fate of transplanted stem cells via longitudinal monitoring has been successfully achieved in the last decade using optical imaging. However, sensitive longitudinal imaging of transplanted stem cells in deep tissue like the brain remains challenging not only due to low light penetration but because of other factors such as low or inferior expression levels of optical reporters in stem cells and stem cell death after transplantation. Here we describe an optimized imaging protocol for sensitive long-term monitoring of bone marrow-derived human mesenchymal stem cells (hMSCs) expressing a novel bioluminescent/near infrared fluorescent (NIRF) fusion reporter transplanted in mouse brain cortex. Lentivirus expressing the luc2-iRFP720 reporter, a fusion between luc2 codon-optimized firefly luciferase (luc2) and the gene encoding NIRF protein iRFP720, was generated to transduce hMSCs. These cells were analyzed for their fluorescent and bioluminescent emission and checked for their differentiation potential. In vivo experiments were performed by transplanting decreasing amounts of luc2-iRFP720 expressing hMSCs in mouse brain, followed by fluorescence and bioluminescence imaging (BLI) starting 1 wk after cell injection when the blood-brain barrier was restored. Bioluminescent images were acquired when signals peaked and used to compare different luc2 substrate performances, that is, D-luciferin (D-Luc; 25 µM/kg or 943 µM/kg) or CycLuc1 (25 µM/kg). Results showed that luc2-iRFP720 expressing hMSCs maintained a good in vitro differentiation potential toward adipocytes, chondrocytes, and osteocytes, suggesting that lentiviral transduction did not affect cell behavior. Moreover, in vivo experiments allowed us to image as low as 1 × 105 cells using both fluorescence and BLI. The highest bioluminescent signals (∼1 × 107 photons per second) were achieved 15 min after the injection of D-Luc (943 µM/kg). This allowed us to monitor as low as 1 × 105 hMSCs for the subsequent 7 wk without a significant drop in bioluminescent signals, suggesting the sustained viability of hMSCs transplanted into the cortex.