Facile microwave synthesis of uniform magnetic nanoparticles with minimal sample processing

We present a simple and rapid method for the synthesis of small magnetic nanoparticles (diameters in the order of 5–20 nm) and narrow size distributions (CV's of 20–40%). The magnetite nanoparticles were synthesized in green solvents within minutes and the saturation magnetization of the particles was tunable by changes in the reaction conditions. We show that this particle synthesis method requires minimal processing steps and we present the successful coating of the particles with reactive bisphosphonates after synthesis without washing or centrifugation. We found minimal batch-to-batch variability and show the scalability of the particle synthesis method. We present a full characterization of the particle properties and believe that this synthesis method holds great promise for facile and rapid generation of magnetic nanoparticles with defined surface coatings for magnetic targeting application

Human alveolar progenitors generate dual lineage bronchioalveolar organoids

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3β inhibitor, CHIR99021. By RNA-seq profiling of GSK-3β knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction

Neue biomedizinische Ansätze zur Untersuchung (patho-)physiologischer Bedingungen gesunder und entzündeter Haut in vitro

Atopic dermatitis (AD) is the most common skin disease worldwide and the prevalence continues to increase in industrialized countries. However, due to the complex interplay between barrier disruption and the skin immune system, the pathogenesis of AD is still not fully understood. The use of human-based in vitro skin equivalents grown from patient-derived cells is a promising alternative approach to investigate pathogenic parameters in AD. However, skin biopsies, that are necessary for the collection of patient cells, are often associated with wound healing complications and scar formations. In this thesis, it was shown for the first time that both keratinocytes and fibroblasts can also be isolated from plucked scalp hair follicles and successfully used for generation of skin equivalents. Moreover, the established method laid the foundation for the generation of skin disease models with the help of patient cells. Over the past years, the impact of fibroblasts for disease induction and/or maintenance has been increasingly recognized. However, little is known about their actual contribution in AD. Hence, the impact of AD patient-derived fibroblasts, isolated from plucked hair follicles, on the tissue homeostasis of human-based skin equivalents was investigated. Interestingly, a subset of AD patient-derived fibroblasts induced characteristic features of AD in the skin equivalents such as hyperproliferation, altered expression of tight junction and skin barrier proteins. Notably, the expression of AD-related proteins such as thymic stromal lymphopoietin (TSLP) and protease-activated receptor 2 (PAR2) were significantly increased. A reduced expression of differentiation-associated cytokine leukemia inhibitor factor (LIF) seems to be linked to these effects. Exposure of hyperproliferative skin equivalents to CD4+ T cells resulted in T cell migration into the dermal equivalent, which was not observed in the equivalents grown from normal fibroblasts. Surprisingly, the addition of T cells to the disease equivalents improved the stratum corneum lipid profiles and distinctly attenuated PAR2 expression, probably as a result of increased LIF signaling due to the T cells. In addition to their effects in the skin equivalents, their angiogenic impact was investigated by co-cultivation with dermal microvascular endothelial cells. Interestingly, AD fibroblasts facilitated to the formation of an increased number of endothelial tubes and thus appear to be involved in angiogenesis. Overall, the results of this doctoral thesis demonstrate the pathogenic effects of AD fibroblasts. The major donor differences among the patients support the opinion that AD is a highly heterogeneous disease, which increases the difficulty of unraveling the pathomechanism of AD. In order to a better understanding of this multifactorial disease, the development of a mast cell-competent skin equivalent was introduced. This model might be a promising biomedical approach for the investigation of complex cell-cell interactions in AD lesions, which is currently poorly understood

State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors

Analysis of <i>ACE2</i>, <i>TMPRSS2</i>, and <i>FURIN</i> expression in human alveolar-like organoids.

Expression of ACE2, TMPRSS2, FURIN and GAPDH and β-ACTIN as housekeeping genes measured by qPCR on bulk RNA of human alveolar-like organoids. Shown are Ct values of four different donors (P4-P7) and two technical replicates.</p

Replication kinetics of SARS-CoV-2-infected human alveolar-like organoids.

Human alveolar-like organoids were infected with SARS-CoV-2 (MOI = 1) and viral replication was assessed by plaque assay (A) and via viral E gene-based RT-qPCR (B). Data of seven (plaque assay) and four (RT-qPCR) biological replicates are shown as individual data points. The mean is visualized by a horizontal black line.</p