1. From isles of Königsberg to islets of Langerhans: examining the function of the endocrine pancreas through network scienceAndraž Stožer, Marko Šterk, Eva Paradiž, Rene Markovič, Maša Skelin, Cara E. Ellis, Lidija Križančić Bombek, Jurij Dolenšek, Patrick E. MacDonald, Marko Gosak, 2022, pregledni znanstveni članek Opis: Islets of Langerhans are multicellular microorgans located in the pancreas that play a central role in whole-body energy homeostasis. Through secretion of insulin and other hormones they regulate postprandial storage and interprandial usage of energy-rich nutrients. In these clusters of hormone-secreting endocrine cells, intricate cell-cell communication is essential for proper function. Electrical coupling between the insulin-secreting beta cells through gap junctions composed of connexin36 is particularly important, as it provides the required, most important, basis for coordinated responses of the beta cell population. The increasing evidence that gap-junctional communication and its modulation are vital to well-regulated secretion of insulin has stimulated immense interest in how subpopulations of heterogeneous beta cells are functionally arranged throughout the islets and how they mediate intercellular signals. In the last decade, several novel techniques have been proposed to assess cooperation between cells in islets, including the prosperous combination of multicellular imaging and network science. In the present contribution, we review recent advances related to the application of complex network approaches to uncover the functional connectivity patterns among cells within the islets. We first provide an accessible introduction to the basic principles of network theory, enumerating the measures characterizing the intercellular interactions and quantifying the functional integration and segregation of a multicellular system. Then we describe methodological approaches to construct functional beta cell networks, point out possible pitfalls, and specify the functional implications of beta cell network examinations. We continue by highlighting the recent findings obtained through advanced multicellular imaging techniques supported by network-based analyses, giving special emphasis to the current developments in both mouse and human islets, as well as outlining challenges offered by the multilayer network formalism in exploring the collective activity of islet cell populations. Finally, we emphasize that the combination of these imaging techniques and network-based analyses does not only represent an innovative concept that can be used to describe and interpret the physiology of islets, but also provides fertile ground for delineating normal from pathological function and for quantifying the changes in islet communication networks associated with the development of diabetes mellitus. Ključne besede: pancreatic islets, beta cells, calcium imaging, intercellular communication, functional networks, multilayer networks Objavljeno v DKUM: 20.12.2024; Ogledov: 0; Prenosov: 3
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2. Mesenchymal stem cells isolated from paediatric paravertebral adipose tissue show strong osteogenic potentialJan Rožanc, Lidija Gradišnik, Tomaž Velnar, Minja Gregorič, Marko Milojević, Boštjan Vihar, Boris Gole, Uroš Maver, 2022, izvirni znanstveni članek Opis: Mesenchymal stem cells (MSCs) represent the basis of novel clinical concepts in cellular therapy and tissue regeneration. Therefore, the isolation of MSCs from various tissues has become an important endeavour for stem cell biobanking and the development of regenerative therapies. Paravertebral adipose tissue is readily exposed during spinal procedures in children and could be a viable source of stem cells for therapeutic applications. Here, we describe the first case of MSCs isolated from paravertebral adipose tissue (PV-ADMSCs), obtained during a routine spinal surgery on a child. Using quantitative real-time PCR and flow cytometry, we show that PV-ADMSCs have different levels of stem marker expression compared to the MSCs from other sources while having the highest proliferation rate. Furthermore, we evaluate the multipotency of PV-ADMSCs by the three-lineage (adipogenic, osteogenic and chondrogenic) differentiation and compare it to the multipotency of MSCs from other sources. It was found that the PV-ADMSCs have a strong osteogenic potential in particular. Taken together, our data indicate that PV-ADMSCs meet the criteria for successful cell therapy, defined by the International Society for Cellular Therapy (ISCT), and thus, could provide a source of MSCs that is relatively easy to isolate and expand in culture. Due to their strong osteogenic potential, these cells provide a promising basis, especially for orthopaedic applications. Ključne besede: mesenchymal stem cells, paediatric surgery, paravertebral adipose tissue, regenerative medicine, stem marker expression, osteogenic potential, differentiation Objavljeno v DKUM: 16.12.2024; Ogledov: 0; Prenosov: 5
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3. Both electrical and metabolic coupling shape the collective multimodal activity and functional connectivity patterns in beta cell collectives : a computational model perspectiveMarko Šterk, Uroš Barać, Andraž Stožer, Marko Gosak, 2023, izvirni znanstveni članek Opis: Pancreatic beta cells are coupled excitable oscillators that synchronize their activity via different communication pathways. Their oscillatory activity manifests itself on multiple timescales and consists of bursting electrical activity, subsequent oscillations in the intracellular Ca 2 + , as well as oscillations in metabolism and exocytosis. The coordination of the intricate activity on the multicellular level plays a key role in the regulation of physiological pulsatile insulin secretion and is incompletely understood. In this paper, we investigate theoretically the principles that give rise to the synchronized activity of beta cell populations by building up a phenomenological multicellular model that incorporates the basic features of beta cell dynamics. Specifically, the model is composed of coupled slow and fast oscillatory units that reflect metabolic processes and electrical activity, respectively. Using a realistic description of the intercellular interactions, we study how the combination of electrical and metabolic coupling generates collective rhythmicity and shapes functional beta cell networks. It turns out that while electrical coupling solely can synchronize the responses, the addition of metabolic interactions further enhances coordination, the spatial range of interactions increases the number of connections in the functional beta cell networks, and ensures a better consistency with experimental findings. Moreover, our computational results provide additional insights into the relationship between beta cell heterogeneity, their activity profiles, and functional connectivity, supplementing thereby recent experimental results on endocrine networks. Ključne besede: pancreatic, beta cells, oscilators, calcium signaling, cells signaling Objavljeno v DKUM: 10.12.2024; Ogledov: 0; Prenosov: 2
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4. Network representation of multicellular activity in pancreatic islets : Technical considerations for functional connectivity analysisMarko Šterk, Yaowen Zhang, Viljem Pohorec, Eva Paradiž, Jurij Dolenšek, Richard K. P. Benninger, Andraž Stožer, Vira Kravets, Marko Gosak, 2024, izvirni znanstveni članek Opis: Within the islets of Langerhans, beta cells orchestrate synchronized insulin secretion, a pivotal aspect of metabolic homeostasis. Despite the inherent heterogeneity and multimodal activity of individual cells, intercellular coupling acts as a homogenizing force, enabling coordinated responses through the propagation of intercellular waves. Disruptions in this coordination are implicated in irregular insulin secretion, a hallmark of diabetes. Recently, innovative approaches, such as integrating multicellular calcium imaging with network analysis, have emerged for a quantitative assessment of the cellular activity in islets. However, different groups use distinct experimental preparations, microscopic techniques, apply different methods to process the measured signals and use various methods to derive functional connectivity patterns. This makes comparisons between findings and their integration into a bigger picture difficult and has led to disputes in functional connectivity interpretations. To address these issues, we present here a systematic analysis of how different approaches influence the network representation of islet activity. Our findings show that the choice of methods used to construct networks is not crucial, although care is needed when combining data from different islets. Conversely, the conclusions drawn from network analysis can be heavily affected by the pre-processing of the time series, the type of the oscillatory component in the signals, and by the experimental preparation. Our tutorial-like investigation aims to resolve interpretational issues, reconcile conflicting views, advance functional implications, and encourage researchers to adopt connectivity analysis. As we conclude, we outline challenges for future research, emphasizing the broader applicability of our conclusions to other tissues exhibiting complex multicellular dynamics. Ključne besede: islets of Langerhans, beta cells, calcium signaling, intercellular communication, functional networks, myosin model Objavljeno v DKUM: 09.12.2024; Ogledov: 0; Prenosov: 3
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5. NMDA receptor inhibition increases, synchronizes, and stabilizes the collective pancreatic beta cell activity : insights through multilayer network analysisMarko Šterk, Lidija Križančić Bombek, Maša Skelin, Marjan Rupnik, Marko Marhl, Andraž Stožer, Marko Gosak, 2021, izvirni znanstveni članek Opis: NMDA receptors promote repolarization in pancreatic beta cells and thereby reduce glucose-stimulated insulin secretion. Therefore, NMDA receptors are a potential therapeutic target for diabetes. While the mechanism of NMDA receptor inhibition in beta cells is rather well understood at the molecular level, its possible effects on the collective cellular activity have not been addressed to date, even though proper insulin secretion patterns result from well-synchronized beta cell behavior. The latter is enabled by strong intercellular connectivity, which governs propagating calcium waves across the islets and makes the heterogeneous beta cell population work in synchrony. Since a disrupted collective activity is an important and possibly early contributor to impaired insulin secretion and glucose intolerance, it is of utmost importance to understand possible effects of NMDA receptor inhibition on beta cell functional connectivity. To address this issue, we combined confocal functional
multicellular calcium imaging in mouse tissue slices with network science approaches. Our results revealed that NMDA receptor inhibition increases, synchronizes, and stabilizes beta cell activity without affecting the velocity or size of calcium waves. To explore intercellular interactions more precisely, we made use of the multilayer network formalism by regarding each calcium wave as an individual network layer, with weighted directed connections portraying the intercellular propagation. NMDA receptor inhibition stabilized both the role of wave initiators and the course of waves. The findings obtained with the experimental antagonist of NMDA receptors, MK-801, were additionally validated with dextrorphan, the active metabolite of the approved drug dextromethorphan, as well as with experiments on NMDA receptor KO mice. In sum, our results provide additional and new evidence for a possible
role of NMDA receptor inhibition in treatment of type 2 diabetes and introduce the multilayer network paradigm as a general strategy to examine effects of drugs on connectivity in multicellular systems. Ključne besede: pancreas, beta cells, insulin, Islets of Langerhans Objavljeno v DKUM: 29.11.2024; Ogledov: 0; Prenosov: 0
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6. Comparison of canine and feline adipose-derived mesenchymal stem cells/medicinal signaling cells with regard to cell surface marker expression, viability, proliferation, and differentiation potentialMetka Voga, Valerija Kovač, Gregor Majdič, 2021, izvirni znanstveni članek Opis: Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44+CD90+ and CD34−/− cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs. Ključne besede: mesenchymal stem cells, dog, cat, comparison, proliferation, differentiation, cell surface marker, viability Objavljeno v DKUM: 21.10.2024; Ogledov: 0; Prenosov: 6
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7. Isolation of live cells from different mice tissues up to nine days after deathMetka Voga, Ana Pleterski, Gregor Majdič, 2021, izvirni znanstveni članek Opis: Some limited reports suggest that cells can survive in the cadavers for much longer than it was previously thought. In our study we explored how time after death, tissue type (muscle, brain and adipose tissue), storage temperature of cadavers (4 °C or at room temperature) and form of tissue storage (stored as cadavers or tissue pieces in phosphate buffered saline) affect the success of harvesting live cells from mice after death. Cells were isolated from dead tissues and grown in standard conditions. Some cells were used for RNA extraction and RT² Profiler™ PCR Array for cell lineage identification was performed to establish which lineages the cells obtained from post mortem tissues belong to. Results of our study showed that viable cells can be regularly isolated from muscle and brain tissue 3 days post mortem and with difficulty up to 6 days post mortem. Viable cells from brain tissue can be isolated up to 9 days post mortem. No cells were isolated from adipose tissue except immediately after death. In all instances viable cells were isolated only when tissues were stored at 4 °C. Tissue storage did not affect cell isolation. Isolated cells were progenitors from different germ layers. Our results show that live cells could be obtained from mouse cadavers several days after death. Ključne besede: mouse, cadaver, stem cells, brain, muscle, adipose tissue Objavljeno v DKUM: 21.10.2024; Ogledov: 0; Prenosov: 2
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8. The role of cAMP in beta cell stimulus-secretion and intercellular couplingAndraž Stožer, Eva Paradiž, Viljem Pohorec, Jurij Dolenšek, Lidija Križančić Bombek, Marko Gosak, Maša Skelin, 2021, pregledni znanstveni članek Opis: Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus-secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca2+ signal and the most distal exocytosis. In addition to modifying intracellular coupling, the effect of cAMP on insulin secretion could also be at least partly explained by the impact on intercellular coupling. In this review, we systematically describe the possible roles of cAMP at these intra- and inter-cellular signaling nodes, keeping in mind the relevance for the whole organism and translation to humans. Ključne besede: cAMP, beta cells, stimulus-secretion coupling, intercellular coupling, PKA, Epac2A Objavljeno v DKUM: 16.10.2024; Ogledov: 0; Prenosov: 4
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9. Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slicesAndraž Stožer, Maša Skelin, Marko Gosak, Lidija Križančić Bombek, Viljem Pohorec, Marjan Rupnik, Jurij Dolenšek, 2021, izvirni znanstveni članek Opis: Many details of glucose-stimulated intracellular calcium changes in [beta] cells during activation, activity, and deactivation, as well as their concentration-dependence, remain to be analyzed. Classical physiological experiments indicated that in islets, functional differences between individual cells are largely attenuated, but recent findings suggest considerable intercellular heterogeneity, with some cells possibly coordinating the collective responses. To address the above with an emphasis on heterogeneity and describing the relations between classical physiological and functional network properties, we performed functional multicellular calcium imaging in mouse pancreas tissue slices over a wide range of glucose concentrations. During activation, delays to activation of cells and any-cell-to-first-responder delays are shortened, and the sizes of simultaneously responding clusters increased with increasing glucose concentrations. Exactly the opposite characterized deactivation. The frequency of fast calcium oscillations during activity increased with increasing glucose up to 12 mM glucose concentration, beyond which oscillation duration became longer, resulting in a homogenous increase in active time. In terms of functional connectivity, islets progressed from a very segregated network to a single large functional unit with increasing glucose concentration. A comparison between classical physiological and network parameters revealed that the first-responders during activation had longer active times during plateau and the most active cells during the plateau tended to deactivate later. Cells with the most functional connections tended to activate sooner, have longer active times, and deactivate later. Our findings provide a common ground for recent differing views on [beta] cell heterogeneity and an important baseline for future studies of stimulus-secretion and intercellular coupling.
NEW & NOTEWORTHY: We assessed concentration-dependence in coupled [beta] cells, degree of functional heterogeneity, and uncovered possible specialized subpopulations during the different phases of the response to glucose at the level of many individual cells. To this aim, we combined acute mouse pancreas tissue slices with functional multicellular calcium imaging over a wide range from threshold (7 mM) and physiological (8 and 9 mM) to supraphysiological (12 and 16 mM) glucose concentrations, classical physiological, and advanced network analyses. Ključne besede: beta cells, calcium imaging, glucose-dependence, network analysis Objavljeno v DKUM: 15.10.2024; Ogledov: 0; Prenosov: 16
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10. Application of transmission electron microscopy to detect changes in pancreas physiologyMaša Skelin, Jurij Dolenšek, Ismael Valladolid-Acebes, Andraž Stožer, Saška Lipovšek Delakorda, 2022, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Ključne besede: pancreas physiology, exocrine cells, endocrine cells, ultrastructure, metabolic syndrome, type 2 diabetes mellitus, western diet Objavljeno v DKUM: 24.09.2024; Ogledov: 0; Prenosov: 7
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