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

Necroptotic Cell Death in Liver Transplantation and Underlying Diseases: Mechanisms and Clinical Perspective

Cell death is a natural process for the turnover of aged cells, but it can also arise as a result of pathological conditions. Cell death is recognized as a key feature in both acute and chronic hepatobiliary diseases caused by drug, alcohol, and fat uptake; by viral infection; or after surgical intervention. In the case of chronic disease, cell death can lead to (chronic) secondary inflammation, cirrhosis, and the progression to liver cancer. In liver transplantation, graft preservation and ischemia/reperfusion injury are associated with acute cell death. In both cases, so-called programmed cell death modalities are involved. Several distinct types of programmed cell death have been described of which apoptosis and necroptosis are the most well known. Parenchymal liver cells, including hepatocytes and cholangiocytes, are susceptible to both apoptosis and necroptosis, which are triggered by distinct signal transduction pathways. Apoptosis is dependent on a proteolytic cascade of caspase enzymes, whereas necroptosis induction is caspase-independent. Moreover, different from the “silent” apoptotic cell death, necroptosis can cause a secondary inflammatory cascade, so-called necroinflammation, triggered by the release of various damage-associated molecular patterns (DAMPs). These DAMPs activate the innate immune system, leading to both local and systemic inflammatory responses, which can even cause remote organ failure. Therapeutic targeting of necroptosis by pharmacological inhibitors, such as necrostatin-1, shows variable effects in different disease models

Multimodal imaging of hair follicle bulge-derived stem cells in a mouse model of traumatic brain injury

Traumatic brain injury (TBI) is a devastating event for which current therapies are limited. Stem cell transplantation may lead to recovery of function via different mechanisms, such as cell replacement through differentiation, stimulation of angiogenesis and support to the microenvironment. Adult hair follicle bulge-derived stem cells (HFBSCs) possess neuronal differentiation capacity, are easy to harvest and are relatively immune-privileged, which makes them potential candidates for autologous stem cell-based therapy. In this study, we apply in vivo multimodal, optical and magnetic resonance imaging techniques to investigate the behavior of mouse HFBSCs in a mouse model of TBI. HFBSCs expressed Luc2 and copGFP and were examined for their differentiation capacity in vitro. Subsequently, transduced HFBSCs, preloaded with ferumoxytol, were transplanted next to the TBI lesion (cortical region) in nude mice, 2 days after injury. Brains were fixed for immunohistochemistry 58 days after transplantation. Luc2- and copGFP-expressing, ferumoxytol-loaded HFBSCs showed adequate neuronal differentiation potential in vitro. Bioluminescence of the lesioned brain revealed survival of HFBSCs and magnetic resonance imaging identified their localization in the area of transplantation. Immunohistochemistry showed that transplanted cells stained for nestin and neurofilament protein (NF-Pan). Cells also expressed laminin and fibronectin but extracellular matrix masses were not detected. After 58 days, ferumoxytol could be detected in HFBSCs in brain tissue sections. These results show that HFBSCs are able to survive after brain transplantation and suggest that ce

Evaluation of NanoLuc substrates for bioluminescence imaging of transferred cells in mice

NanoLuc luciferase recently gained popularity due to its small size and superior bioluminescence performance. For in vivo imaging applications, NanoLuc has been limited by its substrate furimazine, which has low solubility and bioavailability. Herein, we compared the performances of recently reported NanoLuc luciferase substrates for in vivo imaging in mice. Two substrates with improved aqueous solubility, hydrofurimazine and fluorofurimazine, were evaluated along with three stabilized O-acetylated furimazine analogues, the hikarazines. All 5 analogues, when tested in vitro, displayed greater signal intensity and reaction duration, in comparison to the standard NanoLuc substrate, furimazine. The two best-performing analogues from the in vitro study were selected for further in vivo testing. The NanoLuc/f

Theranostic design of angiopep‐2 conjugated hyaluronic acid nanoparticles (Thera‐ang‐chanps) for dual targeting a

Glioblastoma multiforme (GBM) has a mean survival of only 15 months. Tumour heterogeneity and blood‐brain barrier (BBB) mainly hinder the transport of active agents, leading to late diagnosis, ineffective therapy and inaccurate follow‐up. The use of hydrogel nanoparticles, particularly hyaluronic acid as naturally occurring polymer of the extracellular matrix (ECM), has great potential in improving the transport of drug molecules and, furthermore, in facilitatating the early diagnosis by the effect of hydrodenticity enabling the T1 boosting of Gadolinium chelates for MRI. Here, crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd‐DTPA) and the chemotherapeutic agent irinotecan (Thera-cHANPs) are proposed as theranostic nanovectors, with improved MRI capacities. Irinotecan was selected since currently repurposed as an alternative compound to the poorly effective temozolomide (TMZ), generally approved as the gold standard in GBM clinical care. Also, active crossing and targeting are achieved by theranostic cHANPs decorated with angiopep‐2 (Thera‐ ANG‐cHANPs), a dual‐targeting peptide interacting with low density lipoprotein receptor related protein‐1(LRP‐1) receptors overexpressed by both endothelial cells of the BBB and glioma cells. Results showed preserving the hydrodenticity effect in the advanced formulation and internalization by the active peptide‐mediated uptake of Thera‐cHANPs in U87 and GS‐102 cells. Moreover, Thera‐ANG‐cHANPs proved to reduce ironotecan time response, showing a significant cytotoxic effect in 24 h instead of 48 h

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

Background: Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the colorcoupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings: Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance: We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays

Pre-clinical Evaluation of a Cyanine-Based SPECT Probe for Multimodal Tumor Necrosis Imaging

Purpose: Recently we showed that a number of carboxylated near-infrared fluorescent (NIRF) cyanine dyes possess strong necrosis avid properties in vitro as well as in different mouse models of spontaneous and therapy-induced tumor necrosis, indicating their potential use for cancer diagnostic- and prognostic purposes. In the previous study, the detection of the cyanines was achieved by whole body optical imaging, a technique that, due to the limited penetration of near-infrared light, is not suitable for investigations deeper than 1 cm within the human body. Therefore, in order to facilitate clinical translation, the purpose of the present study was to generate a necrosis avid cyanine-based NIRF probe that could also be used for single photon emission computed tomography (SPECT). For this, the necrosis avid NIRF cyanine HQ4 was radiolabeled with 111indium, via the chelate diethylene triamine pentaacetic acid (DTPA). Procedures: The necrosis avid properties of the radiotracer [111In]DTPA-HQ4 were examined in vitro and in vivo in different breast tumor models in mice using SPECT and optical imaging. Moreover, biodistribution studies were performed to examine the pharmacokinetics of the probe in vivo. Results: Using optical imaging and radioactivity measurements, in vitro, we showed selective accumulation of [111In]DTPA-HQ4 in dead cells. Using SPECT and in biodistribution studies, the necrosis avidity of the radiotracer was confirmed in a 4T1 mouse breast cancer model of spontaneous tumor necrosis and in a MCF-7 human breast cancer model of chemotherapy-induced tumor necrosis. Conclusions: The radiotracer [111In]DTPA-HQ4 possessed strong and selective necrosis avidity in vitro and in various mouse models of tumor necrosis in vivo, indicating its potential to be clinically applied for diagnostic purposes and to monitor anti-cancer treatment efficacy

Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair

Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate.Optical Imaging; Biological Services; Biophysic