Benno Naaijkens

Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species

IntroductionAdministration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes.MethodsWe set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates.ResultsOur analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150–200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression.ConclusionsWe demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

Intravital microscopy of localized stem cell delivery using microbubbles and acoustic radiation force

The use of stem cells for the repair of damaged cardiac tissue after a myocardial infarction holds great promise. However, a common finding in experimental studies is the low number of cells delivered at the area at risk. To improve the delivery, we are currently investigating a novel delivery platform in which stem cells are conjugated with targeted microbubbles, creating echogenic complexes dubbed StemBells. These StemBells vibrate in response to incoming ultrasound waves making them susceptible to acoustic radiation force. The acoustic force can then be employed to propel circulating StemBells from the centerline of the vessel to the wall, facilitating localized stem cell delivery. In this study, we investigate the feasibility of manipulating StemBells acoustically in vivo after injection using a chicken embryo model. Bare stem cells or unsaturated stem cells (<5 bubbles/cell) do not respond to ultrasound application (1 MHz, peak negative acoustical pressure P_ = 200 kPa, 10% duty cycle). However, stem cells which are fully saturated with targeted microbubbles (>30 bubbles/cell) can be propelled toward and arrested at the vessel wall. The mean translational velocities measured are 61 and 177 μm/s for P‐ = 200 and 450 kPa, respectively. This technique therefore offers potential for enhanced and well‐controlled stem cell delivery for improved cardiac repair after a myocardial infarction. Biotechnol. Bioeng. 2015;112: 220–227.

Secondary Bjerknes Forces Deform Targeted Microbubbles

Secondary Bjerknes forces can rupture the binding of targeted microbubbles. We have shown before that this effect can be used to quantify the adhesion strength between bubble and target surface [1]. At lower pressures however, microbubbles were observed to snap back to their original position within 100 µs after ultrasound application. In this study the mechanism of this restoring force was investigated in more detail using simultaneous top and side view high speed imaging [2]. Moreover, some results on the process of microbubble detachment (peeling versus uniform rupture) are presented.

Therapeutic Application of Adipose Derived Stem Cells in Acute Myocardial Infarction: Lessons from Animal Models

The majority of patients survive an acute myocardial infarction (AMI). Their outcome is negatively influenced by post-AMI events, such as loss of viable cardiomyocytes due to a post-AMI inflammatory response, eventually resulting in heart failure and/or death. Recent pre-clinical animal studies indicate that mesenchymal stem cells derived from adipose tissue (ASC) are new promising candidates that may facilitate cardiovascular regeneration in the infarcted myocardium. In this review we have compared all animal studies in which ASC were used as a therapy post-AMI and have focused on aspects that might be important for future successful clinical application of ASC.

Wistar rats from different suppliers have a different response in an acute myocardial infarction model.

The Wistar rat is a commonly used strain for experimental animal models. Recently it was shown that results vary between studies using Wistar rats of different suppliers. Therefore we studied whether Wistar rats obtained from Harlan Laboratories (Ha, n=24) and Charles River (CR, n=22) had a different outcome in an acute myocardial infarction (AMI) model. AMI was induced in both Ha and CR Wistar rats by one operator. This resulted in a significantly higher survival rate for Ha (79.2±10.2%) compared with CR rats (54.2±10.2%, p<0.05). Furthermore, CR rats had lost significantly more weight after 7 days (-5.9±3.1%) compared with Ha rats (-0.8±1.7%; p<0.001), indicating a worse health status of the CR rats. Paradoxically, the induced infarct was smaller in CR rats (7.3±3.6% of the heart) compared with Ha rats (12.1±4.7%, p<0.05). This indicates that CR rats were less sensitive for the cardiomyocyte damage subsequent to AMI induction, but remarkably showed more clinical side effects indicating that Wistar rats from two suppliers had a different response within the same AMI model.

Secondary Bjerknes forces deform targeted microbubbles

Secondary Bjerknes forces can rupture the binding of targeted microbubbles. We have shown before that this effect can be used to quantify the adhesion strength between bubble and target surface [1]. At lower pressures however, microbubbles were observed to snap back to their original position within 100 µs after ultrasound application. In this study the mechanism of this restoring force was investigated in more detail using simultaneous top and side view high speed imaging [2]. Moreover, some results on the process of microbubble detachment (peeling versus uniform rupture) are presented.

StemBells: Localized stem cell delivery using targeted microbubbles and acoustic radiation force

The use of stem cells for regenerative tissue repair is hampered by the low number of cells delivered to the site of injury. To increase the delivery, we developed a new technique in which stem cells are coated with functionalized microbubbles, creating echogenic complexes dubbed StemBells. StemBells are highly susceptible to acoustic radiation force; this acoustic force can then be used after injection to deliver the StemBells locally at the treatment site. The dynamics of StemBells during ultrasound insonification was characterized using high-speed optical imaging and is described in an accompanying paper. Here, we investigate the feasibility of manipulating StemBells acoustically after injection employing a chicken embryo model, allowing for the real-time optical observation of the effects of acoustic radiation force in vivo. StemBells were infused by placing a custom-made catheter into one of the vitelline veins. Acoustic radiation force (1 MHz, P = 200–450 kPa, 10% duty cycle) was observed to propel StemBells from the centerline of the microvessels (200–500 μm) to the wall distal from the transducer. Peak translational velocities increased with pressure and varied between 50 μm/s to 300 μm/s. The acoustic radiation force had no effect on the trajectory of bare stem cells.

Microbubble spectroscopy of microbubble-loaded stem cells for targeted cell therapy

Stem cells can be conjugated with targeted microbubbles to form highly echogenic complexes, dubbed StemBells. The complexes can improve stem cell delivery for the local repair of damaged cardiac tissue after a myocardial infarction through propulsion by acoustic radiation forces. While the first in-vivo tests hold great promise, the system would greatly benefit from a mapping of the acoustic parameter space. Here, we develop the theoretical background based on a modified Rayleigh-Plesset type equation to describe the dynamics of the StemBells in response to ultrasound. The complex is shown to resonate as a whole entity and resonance curves are constructed from numerical simulations resembling single bubble responses at a size that relates to the effective complex radius ~10 μm. Ultra high-speed optical imaging of single StemBell complexes at different frequencies using the microbubble spectroscopy method allows for a full characterization with excellent agreement with the developed model. Moreover, from t...

Evaluating the efficacy of subcutaneous C1-esterase inhibitor administration for use in rat models of inflammatory diseases

Abstract Context: C1-esterase inhibitor (C1-inh) therapy is currently administered to patients with C1-inh deficiency through intravenous injections. The possibility of subcutaneous administration is currently being explored since this would alleviate need for hospitalization and increase mobility and well-being of patients. Recently, it was observed in pigs that C1-inh indeed can effectively be applied by subcutaneous injection. For studies on the effectiveness of C1-inh therapy for other indications than acquired and hereditary angioedema, rats are commonly used as model animal. For rats, however, subcutaneous C1-inh administration has never been investigated. Objective: To evaluate the efficacy of subcutaneous C1-inh administration in rats. Materials and methods: Three boli of 100 U/kg human plasma-derived C1-inh were administered to Wistar rats on three consecutive days through subcutaneous injection or intravenous injection. Blood samples were collected from the tail veins 3, 4.5 or 6 h after C1-inh administration for measurement of C1-inh plasma levels. Antigen and activity levels of C1-inh of each plasma sample were determined by means of a specific ELISA. Results: For both C1-inh antigen and C1-inh activity, 21- to 119-fold higher plasma levels were measured after intravenous administration compared with subcutaneous administration. Subcutaneous administration also resulted in C1-inh plasma levels that were less stable and with decreased relative activity. Conclusion: These combined results indicate that in rats, subcutaneous injections in the present formulation are not effective as alternative administration route for C1-inh.