Andrea Dalmao Fernansez

• Martinez, Mikel Amirola; Combriat, Thomas Michel Daniel; Ferencevic, Katharina; Wilhelmsen, Ingrid; Dalmao-Fernandez, Andrea & Olsen, Petter Angell [Show all 9 contributors for this article] (2025). Aspects of zone-like identity and holotomographic tracking of human stem cell-derived liver sinusoidal endothelial cells. Frontiers in Cell and Developmental Biology. 13. doi: 10.3389/fcell.2025.1528991. Full text in Research Archive Show summary

  Introduction: Liver sinusoidal endothelial cells (LSEC) are specialized endothelial cells with unique metabolic and barrier functions adapted to the needs of the liver sinusoid. LSECs are highly sensitive to their environment, and this fragile nature causes challenges in analyzing their phenotype under in vitro conditions. Methods: In this work, we first differentiated LSEC-like cells (scLSECs) from two human pluripotent stem cell lines and characterized them by a panel of qPCR markers, immunohistochemistry, substrate oxidation for energy metabolism, scavenger function, and nitric oxide secretion. We then introduced holotomography, a technique that allows to recover quantitative and three-dimensional information about the refractive indexes of cell components, as a tool to image and track scLSEC in vitro in a minimally intrusive, label-free manner. Results and discussion: Holotomography and developed machine learning-based algorithm for image processing allowed us to describe and monitor changes in intracellular pore-like structures over time. Finally, we tested the possibility of inducing aspects of zone-specific LSEC phenotype and metabolism using culture-based treatments, which resulted in modest shifts in marker expression and metabolic activity. The presented strategy provides an advanced tool kit for investigating liver endothelial cells.

• Dalmao-Fernandez, Andrea; Katare, Parmeshwar Bajirao; Bakke, Hege Gilbø; Hamarsland, Håvard; Ellefsen, Stian & Singh, Sachin [Show all 10 contributors for this article] (2025). The use of three-dimensional primary human myospheres to explore skeletal muscle effects of in vivo krill oil supplementation. In vitro models. ISSN 2731-3433. 4(2), p. 145–155. doi: 10.1007/s44164-025-00087-6. Full text in Research Archive Show summary

  Supplementation with krill oil has shown effects on whole-body lipid and glucose metabolism, as well as on skeletal muscle strength and function. We previously showed that krill oil intervention in vivo promoted fatty acid metabolism and protein synthesis in cultured human myotubes in a two-dimensional (2D) model. The aim of this study was to explore the effects of krill oil supplementation in vivo in a 3D myosphere model, and to compare a the human skeletal muscle 3D cell model to a 2D model. Myospheres were formed from myoblasts obtained before and after 7 weeks of in vivo krill oil intervention. Glucose and oleic acid metabolism were assessed, and transcriptomic and proteomic analyses were performed. In vivo intervention with krill oil increased glucose metabolism in myospheres, while no effect was observed on fatty acid metabolism. Transcriptomic analyses of myospheres after krill oil intervention showed increased expression of genes involved in pathways like motor proteins and hypertrophy, as well as in calcium signaling, of which motor proteins and hypertrophy pathways have not been described in 2D myotube cultures. Proteomic analyses after krill oil intervention showed increased expression of proteins in glycolysis/gluconeogenesis and fatty acid degradation. Comparison of proteins expressed in the 3D myosphere model and the 2D myotube model at the basal level showed that in myospheres, mitochondrial gene expression and translation dominated, while in 2D cultures, mitochondrial organization and response to oxidative stress were more important. These findings suggest that in vivo krill oil intervention induces different metabolic effects when comparing 3D and 2D cultures. In contrast to the 2D model, data obtained with the 3D model showed gene expression changes that are more compatible with previously observed results in vivo concerning skeletal muscle motoric function. Hence, the 3D cell model might better reflect krill oil-induced modifications in skeletal muscle performance in vivo than the 2D model.

• Wilhelmsen, Ingrid; Combriat, Thomas Michel Daniel; Dalmao-Fernandez, Andrea; Stokowiec, Justyna; Wang, Chencheng & Olsen, Petter Angell [Show all 10 contributors for this article] (2024). The effects of TGF-β-induced activation and starvation of vitamin A and palmitic acid on human stem cell-derived hepatic stellate cells. Stem Cell Research & Therapy. 15(223). doi: 10.1186/s13287-024-03852-8. Full text in Research Archive Show summary

  Background Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. Methods In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-β. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. Results The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-β-induced activation-related transcriptome. However, TGF-β-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-β. Conclusions scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-β-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.

• Dalmao-Fernandez, Andrea; Hermida-Gómez, Tamara; Nogueira-Recalde, Uxia; Rego-Pérez, Ignacio; Blanco-Garcia, Francisco J. & Fernández-Moreno, Mercedes (2024). Mitochondrial Role on Cellular Apoptosis, Autophagy, and Senescence during Osteoarthritis Pathogenesis. Cells. 13(11). doi: 10.3390/cells13110976. Full text in Research Archive
• Aizenshtadt, Aleksandra; Wang, Chencheng; Abadpour, Shadab; Menezes, Pedro Duarte; Wilhelmsen, Ingrid & Dalmao-Fernandez, Andrea [Show all 15 contributors for this article] (2024). Pump-Less, Recirculating Organ-on-Chip (rOoC) Platform to Model the Metabolic Crosstalk between Islets and Liver. Advanced Healthcare Materials. ISSN 2192-2640. 13(13). doi: 10.1002/adhm.202303785. Full text in Research Archive Show summary

  Type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD) are epidemiologically correlated disorders with a worldwide growing prevalence. While the mechanisms leading to the onset and development of these conditions are not fully understood, predictive tissue representations for studying the coordinated interactions between central organs that regulate energy metabolism, particularly the liver and pancreatic islets, are needed. Here, a dual pump-less recirculating organ-on-chip platform that combines human pluripotent stem cell (sc)-derived sc-liver and sc-islet organoids is presented. The platform reproduces key aspects of the metabolic cross-talk between both organs, including glucose levels and selected hormones, and supports the viability and functionality of both sc-islet and sc-liver organoids while preserving a reduced release of pro-inflammatory cytokines. In a model of metabolic disruption in response to treatment with high lipids and fructose, sc-liver organoids exhibit hallmarks of steatosis and insulin resistance, while sc-islets produce pro-inflammatory cytokines on-chip. Finally, the platform reproduces known effects of anti-diabetic drugs on-chip. Taken together, the platform provides a basis for functional studies of obesity, T2DM, and MASLD on-chip, as well as for testing potential therapeutic interventions.

• Wang, Chencheng; Abadpour, Shadab; Aizenshtadt, Aleksandra; Dalmao-Fernandez, Andrea; Høyem, Merete & Wilhelmsen, Ingrid [Show all 13 contributors for this article] (2024). Cell identity dynamics and insight into insulin secretagogues when employing stem cell-derived islets for disease modeling. Frontiers in Bioengineering and Biotechnology. 12. doi: 10.3389/fbioe.2024.1392575. Full text in Research Archive Show summary

  Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

• Katare, Parmeshwar Bajirao; Dalmao-Fernandez, Andrea; Mengeste, Abel Mulu; Navabakbar, Farnaz; Hamarsland, Håvard & Ellefsen, Stian [Show all 12 contributors for this article] (2024). Krill oil supplementation in vivo promotes increased fuel metabolism and protein synthesis in cultured human skeletal muscle cells. Frontiers in Nutrition. 11:1452768, p. 1–14. doi: 10.3389/fnut.2024.1452768. Full text in Research Archive Show summary

  Introduction: Krill oil is a dietary supplement derived from Antarctic krill; a small crustacean found in the ocean. Krill oil is a rich source of omega-3 fatty acids, specifically eicosapentaenoic acid and docosahexaenoic acid, as well as the antioxidant astaxanthin. The aim of this study was to investigate the effects of krill oil supplementation, compared to placebo oil (high oleic sunflower oil added astaxanthin), in vivo on energy metabolism and substrate turnover in human skeletal muscle cells. Methods: Skeletal muscle cells (myotubes) were obtained before and after a 7-week krill oil or placebo oil intervention, and glucose and oleic acid metabolism and leucine accumulation, as well as effects of different stimuli in vitro, were studied in the myotubes. The functional data were combined with proteomic and transcriptomic analyses. Results: In vivo intervention with krill oil increased oleic acid oxidation and leucine accumulation in skeletal muscle cells, however no effects were observed on glucose metabolism. The krill oil-intervention-induced increase in oleic acid oxidation correlated negatively with changes in serum low-density lipoprotein (LDL) concentration. In addition, myotubes were also exposed to krill oil in vitro. The in vitro study revealed that 24 h of krill oil treatment increased both glucose and oleic acid metabolism in myotubes, enhancing energy substrate utilization. Transcriptomic analysis comparing myotubes obtained before and after krill oil supplementation identified differentially expressed genes associated with e.g., glycolysis/gluconeogenesis, metabolic pathways and calcium signaling pathway, while proteomic analysis demonstrated upregulation of e.g., LDL-receptor in myotubes obtained after the krill oil intervention. Conclusion: These findings suggest that krill oil intervention promotes increased fuel metabolism and protein synthesis in human skeletal muscle cells, with potential implications for metabolic health.

• Stevanovic, Stanislava; Dalmao-Fernandez, Andrea; Mohamed, Derya; Nyman, Tuula Anneli; Kostovski, Emil & Iversen, Per Ole [Show all 11 contributors for this article] (2024). Time-dependent reduction in oxidative capacity among cultured myotubes from spinal cord injured individuals. Acta Physiologica. ISSN 1748-1708. 240:e14156(7), p. 1–15. doi: 10.1111/apha.14156. Full text in Research Archive Show summary

  Background Skeletal muscle adapts in reaction to contractile activity to efficiently utilize energy substrates, primarily glucose and free fatty acids (FA). Inactivity leads to atrophy and a change in energy utilization in individuals with spinal cord injury (SCI). The present study aimed to characterize possible inactivity-related differences in the energy metabolism between skeletal muscle cells cultured from satellite cells isolated 1- and 12-months post-SCI. Methods To characterize inactivity-related disturbances in spinal cord injury, we studied skeletal muscle cells isolated from SCI subjects. Cell cultures were established from biopsy samples from musculus vastus lateralis from subjects with SCI 1 and 12 months after the injury. The myoblasts were proliferated and differentiated into myotubes before fatty acid and glucose metabolism were assessed and gene and protein expressions were measured. Results The results showed that glucose uptake was increased, while oleic acid oxidation was reduced at 12 months compared to 1 month. mRNA expressions of PPARGC1α, the master regulator of mitochondrial biogenesis, and MYH2, a determinant of muscle fiber type, were significantly reduced at 12 months. Proteomic analysis showed reduced expression of several mitochondrial proteins. Conclusion In conclusion, skeletal muscle cells isolated from immobilized subjects 12 months compared to 1 month after SCI showed reduced fatty acid metabolism and reduced expression of mitochondrial proteins, indicating an increased loss of oxidative capacity with time after injury.

• Lunde, Ngoc Nguyen; Osoble, Nimo Mukhtar Mohamud; Dalmao-Fernandez, Andrea; Lukacs, Alfreda Serwah; Jafari, Abbas & Singh, Sachin [Show all 10 contributors for this article] (2023). Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism. Biomedicines. 11:2908(11), p. 1–20. doi: 10.3390/biomedicines11112908. Full text in Research Archive Show summary

  The interplay between skeletal muscle and bone is primarily mechanical; however, biochemical crosstalk by secreted mediators has recently gained increased attention. The aim of this study was to investigate metabolic effects of conditioned medium from osteoblasts (OB-CM) on myotubes and vice versa. Human skeletal muscle cells incubated with OB-CM showed increased glucose uptake and oxidation, and mRNA expression of the glucose transporter (GLUT) 1, while fatty acid uptake and oxidation, and mRNA expression of the fatty acid transporter CD36 were decreased. This was supported by proteomic analysis, where expression of proteins involved in glucose uptake, glycolytic pathways, and the TCA cycle were enhanced, and expression of several proteins involved in fatty acid metabolism were reduced. Similar effects on energy metabolism were observed in human bone marrow stromal cells differentiated to osteoblastic cells incubated with conditioned medium from myotubes (SKM-CM), with increased glucose uptake and reduced oleic acid uptake. Proteomic analyses of the two conditioned media revealed many common proteins. Thus, our data may indicate a shift in fuel preference from fatty acid to glucose metabolism in both cell types, induced by conditioned media from the opposite cell type, possibly indicating a more general pattern in communication between these tissues.

• Dalmao-Fernandez, Andrea; Aizenshtadt, Aleksandra; Bakke, Hege Gilbø; Krauss, Stefan Johannes Karl; Rustan, Arild Christian & Thoresen, G. Hege [Show all 7 contributors for this article] (2023). Development of three-dimensional primary human myospheres as culture model of skeletal muscle cells for metabolic studies. Frontiers in Bioengineering and Biotechnology. 11, p. 1–13. doi: 10.3389/fbioe.2023.1130693. Full text in Research Archive Show summary

  Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism. Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid. Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

• Mengeste, Abel Mulu; Nikolic, Natasa; Fernandez, Andrea Dalmao; Feng, Yuan Zeng; Nyman, Tuula Anneli & Kersten, Sander [Show all 11 contributors for this article] (2022). Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome. Frontiers in Physiology. 13. doi: 10.3389/fphys.2022.928195. Full text in Research Archive Show summary

  Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

• Katare, Parmeshwar Bajirao; Fernandez, Andrea Dalmao; Mengeste, Abel Mulu; Hamarsland, Håvard; Ellefsen, Stian & Bakke, Hege Gilbø [Show all 9 contributors for this article] (2022). Energy metabolism in skeletal muscle cells from donors with different body mass index. Frontiers in Physiology. 13. doi: 10.3389/fphys.2022.982842. Full text in Research Archive
• Mengeste, Abel Mulu; Katare, Parmeshwar Bajirao; Fernandez, Andrea Dalmao; Lund, Jenny; Bakke, Hege Gilbø & Baker, David [Show all 11 contributors for this article] (2022). Knockdown of sarcolipin (SLN) impairs substrate utilization in human skeletal muscle cells. Molecular Biology Reports. ISSN 0301-4851. p. 6005–6017. doi: 10.1007/s11033-022-07387-0. Full text in Research Archive Show summary

  Abstract Background Recent studies have highlighted that uncoupling of sarco-/endoplasmic reticulum Ca 2+ -ATPase (SERCA) by sarcolipin (SLN) increases ATP consumption and contributes to heat liberation. Exploiting this thermogenic mechanism in skeletal muscle may provide an attractive strategy to counteract obesity and associated metabolic disorders. In the present study, we have investigated the role of SLN on substrate metabolism in human skeletal muscle cells. Methods and results After generation of skeletal muscle cells with stable SLN knockdown (SLN-KD), cell viability, glucose and oleic acid (OA) metabolism, mitochondrial function, as well as gene expressions were determined. Depletion of SLN did not influence cell viability. However, glucose and OA oxidation were diminished in SLN-KD cells compared to control myotubes. Basal respiration measured by respirometry was also observed to be reduced in cells with SLN-KD. The metabolic perturbation in SLN-KD cells was reflected by reduced gene expression levels of peroxisome proliferator-activated receptor γ coactivator 1α ( PGC1α ) and forkhead box O1 ( FOXO1 ). Furthermore, accumulation of OA was increased in cells with SLN-KD compared to control cells. These effects were accompanied by increased lipid formation and incorporation of OA into complex lipids. Additionally, formation of complex lipids and free fatty acid from de novo lipogenesis with acetate as substrate was enhanced in SLN-KD cells. Detection of lipid droplets using Oil red O staining also showed increased lipid accumulation in SLN-KD cells. Conclusions Overall, our study sheds light on the importance of SLN in maintaining metabolic homeostasis in human skeletal muscle. Findings from the current study suggest that therapeutic strategies involving SLN-mediated futile cycling of SERCA might have significant implications in the treatment of obesity and associated metabolic disorders. Graphical abstract

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