Multimodal imaging to evaluate the distribution and fate of a mesenchymal stromal cell therapy.

Mesenchymal stromal cell (MSC) treatments have shown beneficial outcomes in preclinical models of various diseases, but limited therapeutic effects in clinical trials. This disparity in translation reflects the need to understand the mechanisms involved in the host’s response to therapy. Intravenous injection is the preferred delivery method in the clinics, but it has been observed that using this route leads to MSCs becoming entrapped in the lungs, making this organ an interesting study target. In this thesis, different imaging modalities to study the distribution and fate of administered MSCs in the lung were used. The first aim was to combine the sensitivity of bioluminescence imaging (BLI) with the ability of micro-computed tomography (micro-CT) to image lung tissue to track the cells in vivo. Then, to study the biodistribution of the MSCs in the lung microenvironment at single-cell resolution, an optical tissue clearing protocol was established. Finally, the effect of the MSCs on innate immune cells in the lung and their potential interactions were investigated. Human umbilical cord MSCs (hUC-MSCs) that had been labelled with the genetic reporter Firefly luciferase (FLuc), the fluorescent reporter tandem Tomato (tdTomato), or with gold nanorods were used. The hUC-MSCs were injected into the tail vein of mice, which were then imaged in vivo using BLI and micro-CT. After MSC injection, animals were culled, and the lungs collected and processed for confocal microscopy or flow cytometry. BLI revealed that following intravenous injection, the MSCs localized to the lungs hampering the ability of MSOT to image the MSCs within this organ. Using micro-CT, it was not possible to detect the MSCs, indicating that this method might lack sensitivity to image gold-labelled cells. Next, the CUBIC, a modified stabilized DISCO (s-DISCO) and ethyl cinnamate (ECi) optical tissue clearing protocols were compared to find a suitable method for studying the biodistribution of hUC-MSCs and their interactions with the mouse lung microenvironment. CUBIC was the only method that enabled direct imaging of tdTomato-expressing hUC-MSCs as the other methods quenched the fluorescence of the reporter. Moreover, CUBIC in combination with immunofluorescence allowed the interaction of the hUC-MSCs with cells in the host lung to be investigated. Particularly, it was observed that the hUC-MSCs appeared to be retained in the pulmonary microvasculature as they were not found in large blood vessels. Flow cytometric analysis showed that shortly after hUC-MSC IV injection, neutrophils, monocytes, and macrophages mobilized to the lung and participated in an inflammatory response. Twenty-four hours post cell infusion, the number of innate immune cells in the lungs decreased but a polarization toward an anti-inflammatory phenotype was observed. Moreover, immunofluorescent staining revealed that neutrophils were preferentially distributed in close vicinity to the hUC-MSCs, suggesting that their clearance within 24 h might involve efferocytosis. In summary, using a range of in vivo and ex vivo imaging techniques, it was shown that following intravenous injection into mice, hUC-MSCs appeared to accumulate in the pulmonary vessels and mostly died within 24 h. Within 2 h following administration, the hUC-MSCs caused an inflammatory response in the lung, leading to an increase in neutrophils and pro-inflammatory macrophages. However, by 24 h, neutrophils were at basal levels and there was an increase in anti-inflammatory macrophages. Although there are numerous reports indicating that MSCs polarise macrophage towards an anti-inflammatory phenotype, an interesting finding of this study was that the initial effect of hUC-MSCs on host immune cells was actually pro-inflammatory. This may provide some insight into the potential therapeutic mechanisms of MSCs

Multispectral optoacoustic tomography is more sensitive than micro-computed tomography for tracking gold nanorod labelled mesenchymal stromal cells

Tracking the fate of therapeutic cell types is important for assessing their safety and efficacy. Bioluminescence imaging (BLI) is an effective cell tracking technique, but poor spatial resolution means it has limited ability to precisely map cells in vivo in 3D. This can be overcome by using a bimodal imaging approach that combines BLI with a technique capable of generating high-resolution images. Here we compared the effectiveness of combining either multispectral optoacoustic tomography (MSOT) or micro-computed tomography (micro-CT) with BLI for tracking the fate of luciferase+ human mesenchymal stromal cells (MSCs) labelled with gold nanorods. Following subcutaneous administration in mice, the MSCs could be readily detected with MSOT but not with micro-CT. We conclude that MSOT is more sensitive than micro-CT for tracking gold nanorod-labelled cells in vivo and depending on the route of administration, can be used effectively with BLI to track MSC fate in mice

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications.</jats:p

North Sea Progressive Myoclonus Epilepsy is Exacerbated by Heat, A Phenotype Primarily Associated with Affected Glia

Progressive myoclonic epilepsies (PMEs) comprise a group of rare disorders of different genetic aetiologies, leading to childhood-onset myoclonus, myoclonic seizures and subsequent neurological decline. One of the genetic causes for PME, a mutation in the gene coding for Golgi SNAP receptor 2 (GOSR2), gives rise to a PME-subtype prevalent in Northern Europe and hence referred to as North Sea Progressive Myoclonic Epilepsy (NS-PME). Treatment for NS-PME, as for all PME subtypes, is symptomatic; the pathophysiology of NS-PME is currently unknown, precluding targeted therapy. Here, we investigated the pathophysiology of NS-PME. By means of chart review in combination with interviews with patients (n = 14), we found heat to be an exacerbating factor for a majority of NS-PME patients (86%). To substantiate these findings, we designed a NS-PME Drosophila melanogaster model. Downregulation of the Drosophila GOSR2-orthologue Membrin leads to heat-induced seizure-like behaviour. Specific downregulation of GOSR2/Membrin in glia but not in neuronal cells resulted in a similar phenotype, which was progressive as the flies aged and was partially responsive to treatment with sodium barbital. Our data suggest a role for GOSR2 in glia in the pathophysiology of NS-PME

XXV Curso Monográfico de Psiquiatría Infantil y la Adolescencia: Tópicos de Psicofarmacología Infantil - 2023

El XXV Curso Monográfico de Psiquiatría Infantil y de la Adolescencia, titulado "Tópicos de Psicofarmacología Infantil," fue un evento destacado en el campo de la salud mental infantil y adolescente. Durante tres días en septiembre de 2023, expertos líderes en la materia se reunieron para explorar a fondo la psicofarmacología en este grupo de edad. El evento, dedicado a la memoria del Dr. Francisco Javier Valencia Granados, comenzó con una ceremonia de inauguración en la que participaron autoridades institucionales. Luego, se sucedieron conferencias magistrales que abordaron una amplia variedad de temas cruciales. Estos incluyeron aspectos fundamentales como la neurobioquímica farmacológica y una introducción a la psicofarmacología. El programa se adentró en cuestiones específicas, como el uso de antipsicóticos en paidopsiquiatría, el abordaje de trastornos del aprendizaje, el tratamiento del suicidio desde una perspectiva psicofarmacológica, y la gestión farmacológica del insomnio en niños. Se exploraron temas especializados, como el tratamiento de la esquizofrenia en pacientes infantiles. El segundo día se centró en trastornos emocionales en niños y adolescentes, destacando el tratamiento del trastorno depresivo, los trastornos ansiosos y el espectro autista. Se presentaron enfoques vanguardistas, como el uso de psicodélicos en adolescentes y las novedades en psicofarmacología, como el dextrometorfano y el bupropión. También se discutió el manejo de la epilepsia y la adicción a los videojuegos. El tercer día se enfocó en el tratamiento farmacológico de trastornos pediátricos específicos, como el trastorno bipolar, el déficit de atención e hiperactividad, la enuresis y encopresis, parasomnias, y el abordaje neuropsiquiátrico en pacientes pediátricos con VIH. Se exploraron también trastornos de la conducta alimentaria y la disforia de género. El evento culminó con una reflexión sobre la salud mental en niños y un emotivo tributo al Dr. Francisco Javier

Optical Tissue Clearing to Study the Intra-Pulmonary Biodistribution of Intravenously Delivered Mesenchymal Stromal Cells and Their Interactions with Host Lung Cells

Mesenchymal stromal cells (MSCs) injected intravenously are trapped in the capillaries of the lungs and die within the first 24 h. Studying the biodistribution and fate of labelled therapeutic cells in the 3D pulmonary context is important to understand their function in this organ and gain insights into their mechanisms of action. Optical tissue clearing enables volumetric cell tracking at single-cell resolution. Thus, we compared three optical tissue-clearing protocols (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC), modified stabilised 3D imaging of solvent-cleared organs (s-DISCO) and ethyl cinnamate (ECi)) to evaluate their potential to track the biodistribution of human umbilical cord MSCs expressing the tdTomato fluorescence reporter and investigate how they interact with host cells in the mouse lung. The results showed that although CUBIC clearing is the only method that enables direct imaging of fluorescently labelled MSCs, combining s-DISCO or ECi with immunofluorescence or dye labelling allows the interaction of MSCs with endothelial and immune cells to be studied. Overall, this comparative study offers guidance on selecting an optical tissue-clearing method for cell tracking applications

Intravenous Administration of Human Umbilical Cord Mesenchymal Stromal Cells Leads to an Inflammatory Response in the Lung.

Mesenchymal stromal cells (MSCs) administered intravenously (IV) have shown efficacy in preclinical models of various diseases. This is despite the cells not reaching the site of injury due to entrapment in the lungs. The immunomodulatory properties of MSCs are thought to underlie their therapeutic effects, irrespective of whether they are sourced from bone marrow, adipose tissue, or umbilical cord. To better understand how MSCs affect innate immune cell populations in the lung, we evaluated the distribution and phenotype of neutrophils, monocytes, and macrophages by flow cytometry and histological analyses after delivering human umbilical cord-derived MSCs (hUC-MSCs) IV into immunocompetent mice. After 2 hr, we observed a significant increase in neutrophils, and proinflammatory monocytes and macrophages. Moreover, these immune cells localized in close proximity to the MSCs, suggesting an active role in their clearance. By 24 hr, we detected an increase in anti-inflammatory monocytes and macrophages. These results suggest that the IV injection of hUC-MSCs leads to an initial inflammatory phase in the lung shortly after injection, followed by a resolution phase 24 hr later

Near infrared conjugated polymer nanoparticles (CPN^TM) for tracking cells using fluorescence and optoacoustic imaging

Tracking the biodistribution of cell therapies is crucial for understanding their safety and efficacy. Optical imaging techniques are particularly useful for tracking cells due to their clinical translatability and potential...</jats:p