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Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.

Although several investigations have shown a relationship between increased epicardial adipose tissue (EAT) and atrial fibrillation (AF), the association between EAT and ventricular tachycardia (VT) has not been evaluated. We investigated the association between EAT and post-ablation VT recurrence. In this study, sixty-one consecutive patients (mean age=62.0±13.9) undergoing VT ablation with pre-procedural cardiac magnetic resonance imaging (MRI) were recruited. EAT thickness was measured using cardiac MRI in the right and left atrioventricular grooves (AVGs), RV free wall, anterior, inferior, and superior interventricular grooves (IVGs). During a mean follow-up period of 392.9±180.2 days, post-ablation VT recurrence occurred in 15 (24.6%) patients. EAT thickness was significantly higher in the VT recurrence group than that in the non-recurrent VT at the right (18.7±5.7 vs. 14.1±4.4 mm; p=0.012) and left (13.3±3.9 vs. 10.4±4.1; p=0.020) AVGs. The best cut-off points for predicting VT recurrence were calculated as 15.5 mm for the right AVG (area under ROC curve=0.74) and 11.5 mm for the left AVG (area under ROC curve=0.72). Multivariate Cox regression analysis showed that pre procedural right AVG-EAT (HR: 1.2; 95% CI: [1.06-1.39], p=0.004) was the only independent predictor of VT recurrence after adjustment for covariates. Kaplan–Meier analysis showed a difference for post-ablation VT recurrence between the two groups with right AVG-EAT thickness cut-off value of <15.5 mm versus ≥15.5 mm (log-rank, p=0.003). Based on the finding of this study, we suggested a new possible imaging marker for risk stratification of post-ablation VT recurrence. A higher EAT may be associated with VT recurrence after catheter ablation of VTs.:Epicardial Adipose Tissue Anatomy Embryology Physiology and Pathophysiology Measurement of EAT EAT and heart disorders Future direction VT Catheter Ablation History of VT ablation Catheter ablation for VT in structural and non-structural heart Outcome of VT catheter ablation Predictors of VT recurrence after catheter ablation Objectives of the thesis Publication Summary References

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<p>Obesity is a major global health concern and is associated with the development of numerous non-communicable diseases. A thorough understanding of the onset of obesity is critical to the development of effective therapeutic strategies against this disease state. Recently, obesity has been described as a complex disease characterized by chronic low grade inflammation. Abnormal adipose tissue expansion is accompanied by an increased presence of proinflammatory immune cells, dysregulated adipokine expression, oxidative stress, and is associated with significant changes in the bacterial composition of the gut. While interleukin-15 (IL-15) has been studied extensively for its immunological effects, this cytokine has recently been shown to influence body weight and fat mass. The focus of this thesis was to elucidate the role of and mechanism by which IL-15 modulates adipose tissue. Our first study demonstrated that low levels of IL-15 expression are associated with adiposity and promotes an obese state in IL-15-/- mice and human subjects, while IL-15 overexpression was associated with a lean phenotype in IL-15tg mice when compared to appropriate controls. To uncover the underlining mechanisms by which IL- 15 mediates differences in body weight, we subsequently determined that IL-15 mediated weight loss occurred independently of lymphocytes. In another study, we showed that IL-15tg mice had increased mitochondrial activity and mass specific to adipose tissue compared to IL-15-/- and B6 mice, while acute IL-15 administration induced the expression of FAO markers in adipose tissue. Lastly, IL-15 treatment increased mitochondrial membrane potential and decreased lipid deposition in cultured adipocytes, suggesting that IL-15 may mediate its effects directly on adipose tissue. The experimental results presented in this thesis demonstrate that IL-15 is an important regulator of adipose tissue and body weight. Future studies examining the effects of IL- 15 on adipose tissue will further our knowledge on IL-15 biology, and may contribute to novel therapeutic strategies for the treatment and prevention of obesity.</p> / Doctor of Philosophy (PhD)

Brown adipose tissue (BAT) serves as a crucial thermogenic organ, extracting glucose and lipids from circulation to generate heat. Enhancing BAT activity holds potential as a therapy for treating metabolic diseases, such as obesity and diabetes. The sympathetic nervous system (SNS) is the main regulator of BAT activity by increasing extraction and oxidation of substrates. However, the SNS role in metabolic disorders is complex. In obesity, there is increased sympathetic tone, yet reduced BAT responsiveness. Furthermore, increasing systemic sympathetic tone in individuals already at heightened cardiovascular risk leads to adverse complications, as demonstrated by recent clinical trials. As a result, BAT’s impact on overall health in humans has been challenged in recent years, largely due to the lack of methods to selectively activate BAT without affecting other organs. Here, I used chemogenetics and retrograde viral injections in the interscapular BAT (iBAT) of mice to selectively activate only the neurons projecting to this tissue. Targeted activation of BAT did increase thermogenesis and improved glucose homeostasis. Leveraging on the single-cell RNA sequencing from our laboratory, we identified two sympathetic populations innervating iBAT: one primarily targets the small arterioles, while the other innervates the parenchyma. These populations mediate non-overlapping sympathetic-functions in iBAT: activating only the vascular projecting neurons lowers blood glucose without affecting thermogenesis, while activating the other population results in increased energy expenditure, local thermogenesis, and blood flow, with no effect on glycemia. The findings from this work could pave the way to the development of targeted strategies against metabolic disorders characterized by hyperglycemia, highlighting the potential of selectively activating specific SNS components to normalize blood glucose levels.

G protein-coupled receptors (GPCRs) play a major role in physiological functions by transducing extracellular information into intracellular responses, thereby, allowing auto-, para , and endocrine communication. In combination with their ubiquitous expression, GPCRs are of special interest for developing therapeutic approaches. Due to their high targetability, GPCRs are also interesting for obesity research. Even though the prevalence for obesity and its comorbidities like type 2 diabetes mellitus is rapidly rising worldwide, the physiological function of many GPCRs is not well resolved in adipose tissue (AT). Here, the high percentage of orphan GPCRs (65 %), meaning GPCRs characterized by lacking information about an endogenous ligand, signal transduction and/or physiological functions, is an enormous restricting factor to understand AT (patho)physiology. In this study the overall goal was the identification and characterization of GPCRs hitherto unrecognized in AT and adipocyte functionality. Therefore, the first part of this study focused on incorporating a web application for the analysis of publicly available RNA-seq data of human and mouse AT and adipocytes, called FATTLAS. With this application, the GPCRome was analysed to identify GPCRs connected to AT function. Furthermore, those GPCRs were characterized in 3T3-L1, a murine preadipocyte cell line with adipogenic potential. Following, the main results are described: 1. Analysing the GPCR expression patterns in AT of lean individuals and individuals with obesity lead to the identification of a multitude of highly and differentially expressed genes, many of them being already characterized in AT or adipocyte function. Nevertheless, four GPCRs were identified being either highly expressed in both conditions (Gpr146) or differentially expressed in individuals with obesity (Fzd5, Mrgprf, and Ptger2) having an unknown function in AT. For all four receptors an agonist has been previously described. 2. Besides expression in AT, all four receptors are also present in primary and 3T3-L1 (pre)adipocytes. Thus, GPR146, FZD5, MRGPRF, and PTGER2 are suitable for characterizing their role in adipocyte function. 3. Investigating receptor overexpression in the heterologous expression system HEK293T, FZD5, MRGPRF and PTGER2 were found to be expressed at the cell surface, whereas GPR146 was mainly located in endosomes. 4. While analysing receptor signalling in HEK293T cells high basal receptor activity was detected for PTGER2 (Gɑs protein) and GPR146 (Gɑi protein). As the published agonists could not induce receptor activation in 3T3-L1 (pre)adipocytes, siRNA-mediated knockdown was the method of choice for the characterization of endogenously expressed receptors in 3T3-L1 preadipocytes. 5. Receptor knockdown analysis for these four receptors revealed a reduced adipogenesis acting via the PPARγ axis. Here, MRGPRF and PTGER2 act via a cAMP-dependent mechanism. Furthermore, the receptors are involved in preadipocyte viability, which also contribute to the regulation of adipogenesis. As the receptor knockdown in preadipocytes leads to a reduced viability and adipogenesis, adipocyte function was analysed after knockdown in adipocytes. 6. Before the investigation of adipocyte function, the effect of receptor knockdown in mature adipocytes on adipocyte viability was analysed with only Mrgprf knockdown showing an effect. 7. In adipocyte function, MRGPRF was involved in adiponectin secretion most likely by changing intracellular cAMP accumulation. Moreover, GPR146 regulates lipolysis via basal Gɑi-protein signalling. PTGER2 and FZD5 did not show an involvement in the analysed adipocyte functionalities. Taken together, in this study an interactive public database (FATTLAS) was implemented, incorporating publicly available RNA-seq data for AT and adipocytes for improved access and analysis of these complex datasets. Using this database, four hitherto unrecognized GPCRs were identified and their involvement in adipogenesis and AT function was proved. In the second part of this thesis, the subgroup adhesion GPCRs (aGPCRs) of the GPCRome, were analysed. Genome-wide association studies have linked this orphan GPCR class to AT function and metabolic dysfunction (e.g. obesity). However, they have not been thoroughly investigated yet. By performing a comprehensive study, the following main findings about aGPCR expression in AT and adipocytes and about their role in adipogenesis and adipocyte function were revealed: 1. In mice 30 aGPCRs are genomically encoded. 25 receptors were identified to be expressed in subcutaneous AT (scAT), 28 in visceral AT (vAT). Only Emr4, Gpr133 and Gpr116 showed a differential expression between scAT and vAT. 2. Under high-fat diet seven receptors (Lphn1-3, Eltd1, Emr1, Gpr124, Gpr116, and Gpr56) were significantly higher expressed in scAT, whereas in vAT three receptors were upregulated (Emr4, Gpr124, and Celsr3) and four downregulated (Gpr113, Gpr116, Gpr64, and Gpr97). 3. As AT consists of diverse cell types, cell-specific expression of aGPCRs in adipocytes and the stroma vascular fraction (SVF) was determined. Here, four receptors were significantly higher expressed in adipocytes (Lphn2, Gpr125, Gpr111, and Gpr64), six receptors were lower expressed (Emr1, Emr4, Gpr133, Gpr113, and Gpr97, Gpr126). However, most aGPCRs are expressed in adipocytes making them interesting for investigating their role in adipocyte function. 4. In human scAT CELSR1, CELSR2, EMR2, and GPR126 were upregulated comparing lean and obese conditions, GPR64 and GPR97 were downregulated. The expression of aGPCRs in human scAT was comparable to mouse scAT indicating a transferability of aGPCR function in mouse and human samples. 5. Analysing aGPCR expression during adipogenesis in 3T3-L1, three distinct expression patterns were found: no changes (Lphn1-3, Cd97, Gpr124, Gpr125, Gpr116, Gpr56, Gpr64, Gpr97, and Gpr126), steady upregulation (Gpr124 and Gpr126), and downregulation (Cd97, Gpr116, Gpr56, and Gpr64). 6. Knockdown of Lphn2, Gpr124, Gpr125, Gpr116, Gpr64, and Gpr126 in 3T3-L1 lead to a reduced lipid accumulation and droplet size indicating an impaired differentiation into mature adipocytes. 7. Exemplarily, GPR64 was selected to study the role of aGPCRs in adipocyte function due to its significantly reduced expression in obesity and its role in adipogenesis. Knockdown and stimulation by a tethered agonist-derived peptide uncovered Gαs protein-mediated signalling. Furthermore, peptide stimulation resulted in a reduced adiponectin secretion and glucose uptake in 3T3-L1 adipocytes. Lipolysis was induced in 3T3-L1 and primary adipocytes after peptide stimulation indicating a transferability of cell culture experiments to ex vivo analysis. In summary, this comprehensive study describes aGPCR expression in human and mouse AT, adipocytes and SVF in depth. Furthermore, their involvement in adipogenesis under physiological and knockdown conditions was studied in the model cell line 3T3-L1. The functional role of aGPCRs was exemplarily analysed for GPR64. As the model cell line 3T3-L1 is difficult to transfect and knockdown only reduces gene expression, the suitability of CRISPR/Cas technology for generating a receptor knockout (KO) in 3T3-L1 cell line was tested. For validation, the aGPCRs, Gpr64 and Gpr126, were chosen. The main results of the third part of this study are the following: 1. Gpr64- and Gpr126 KO cells were generated using a commercially avaible 3T3-L1 cell line constitutively overexpressing Cas9 (3T3-L1 Cas9). 2. An impaired adipogenesis and adipocyte function was found already in 3T3-L1 Cas9 compared to 3T3-L1 wildtype. Creating a self-made Cas9-overexpressing 3T3-L1 was not feasible. Thus, Cas9-overexpressing 3T3-L1 cells are not suitable for analysing adipocyte function after KO. 3. Evaluating other approaches for CRISPR/Cas9-mediated KO strategies, transient overexpression of Cas9 using plasmid-based methods were not feasible, too. However, introducing Cas9 protein was successful and did not interfere with adipogenesis making this approach the method of choice for CRISPR/Cas9-mediated KO in 3T3-L1. In brief, KO studies in 3T3-L1 cell line using CRISPR/Cas technology should not be carried out using plasmid-based approaches. However, transfecting the Cas9 protein or its ribonucleoprotein complex is feasible to create 3T3-L1 KO cell lines without interfering with adipogenesis and adipocyte function. In conclusion, this thesis provides a comprehensive study about GPCR expression in AT of lean individuals and individuals with obesity. The identification and characterization of GPCRs hitherto unrecognized in AT or even orphan GPCRs supports the understanding of AT (patho)physiology. In particular, the expression of all GPCRs in AT of lean individuals and individuals with obesity was uncovered for human scAT as well as mouse scAT and vAT. Furthermore, three rhodopsin-like GPCRs (GPR146, MRGPRF, and PTGER2), one Fzd-like receptor (FZD5) and the adhesion GPCR class were basally characterized regarding adipogenesis and (pre)adipocyte functionality using the model cell line 3T3-L1. These data are fundamental for understanding the importance of the whole repertoire of GPCR in AT (patho)physiology and can be used as a starting point for their characterization in AT and adipocyte function in depth, possibly even leading to novel therapeutic approaches in the future.

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.