Cold-induced beigeing of stem cell-derived adipocytes is not fully reversible after return to normothermia

Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid-laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (i.e. 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige-like morphology and exhibited an intermediate gene expression profile, with both beige-like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post-transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (e.g. carbonyl cyanide-4- phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown-like features after 3 weeks of acclimatisation at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible

Targeting glutamine synthesis inhibits stem cell adipogenesis in vitro

Background/Aims: Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on differentiation of adipocytes and lipid droplet accumulation. Methods: Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine – MSO) during differentiation and their response compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). Results: Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. Conclusion: These results suggest that the glutamine pathway may have a previously overlooked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity

Caffeine exposure induces browning features in adipose tissue in vitro and in vivo

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans

Cell imaging by phonon microscopy: sub-optical wavelength ultrasound for non-invasive imaging

The mechanical properties of cells play an important role in cell function and behavior. This paper presents recent developments that have enabled the use of laser-generated phonons (ultrasound) with sub-optical wavelengths to look inside living cells. The phonons reveal contrast from changes in the elasticity of the cell and can provide high resolution three dimensional images

Stem cell models of adipogenesis: modulating temperature to promote MSC-derived beige adipocytes

Since the discovery of brown adipose tissue (BAT), emerging strategies to promote its activation and recruitment have been examined due to the thermogenic capacity of BAT, which makes it an encouraging approach to regulate obesity and related diseases. Uncoupling protein 1 (UCP1) is a specific protein located in the inner mitochondria of brown adipocytes, when activated, uncouples substrate oxidation from ATP production by dissipating energy in the form of heat. Beige adipocytes have been recently discovered and are cells which possess the morphological and biochemical characteristics of brown adipocytes, including UCP1. It has been previously reported that human BAT expresses beige cell-selective genes instead of classical brown markers which underline the importance of understanding beige adipogenic differentiation, induction and maintenance. All adipocytes share a mesodermal origin. Mesenchymal stem cells (MSCs) are able to differentiate into a variety of lineages, including adipocytes. To date, the most potent stimulus to activate UCP1 in BAT is cold exposure. However, whether cells sense temperature and differentiate into brown and beige lineages is not fully understood. The aim of this thesis is to obtain a better understanding on how low temperature affects the differentiation of MSCs into white, beige and brown adipocytes and if these traits acquired are maintained after being switched to normal culture conditions (37ºC). Additionally, cold exposure evaluation during adipogenic differentiation of human MSCs derived from cord tissue taken from diabetic and healthy pregnancies at term were examined. Cold temperature (32ºC) was investigated for its potential to activate browning in adipocytes. Here, MSCs differentiated under normal culture conditions showed a dual leptin and UCP1 protein expression in the majority of differentiated cells. Hypothermic conditions applied while the MSCs were under adipogenic differentiation promoted a brown/beige morphological and molecular signature. These changes were accompanied with enhanced UCP1 protein abundance, uncoupled respiration and metabolic adaptation. Hypothermic differentiated cells showed a marked translocation of leptin into adipocyte nuclei, which indicates a potential role for leptin in the browning process. These results indicated that MSCs could be driven to form beige like adipocytes by differentiating them in a cold environment. To further investigate whether these MSC-derived adipocytes could adapt overtime and retain their brown/beige traits, brown-like cells produced under hypothermic conditions were transferred to normal culture conditions. MSCs differentiated at 32ºC and then rewarmed maintained an intermediate brown/beige phenotype and gene profile with UCP1 protein expression found after 21 days of warm acclimation (37ºC). Additionally, these cells showed an intermediate mitochondrial respiration level, lipolysis, fatty acid oxidation, glycolysis rate and intracellular calcium expression, suggesting a shared mechanism of calcium mediated UCP1-independent thermogenesis. Moreover, these cells stored lipids and produce heat when stimulated. Altogether these results highlight the plasticity of MSCs to generate and preserve de novo beige adipocytes. Several isolation and culture methods were then tested in order to establish optimal conditions for human umbilical cord tissue MSCs (hUCT-MSCs) isolation, growth and differentiation. For hUCT-MSCs isolation, an enzymatic method using collagenase B (0.4 U/ml per cm) was used. After testing different culture mediums, it was found that DMEM low glucose,1% L-glutamine, 1% penicillin/streptomycin and 10% human platelet lysate was the best growth medium for these cells, while for adipogenic differentiation the best suitable medium was DMEM F12, 1% penicillin/streptomycin and 10% foetal bovine serum supplemented with 1 M dexamethasone, 500 M isobutyl-methylxanthine, 1 M rosiglitazone and 10 g/ml insulin. Finally, the hypothermic differentiation model of adipogenesis was tested in hUCT-MSCs from gestational diabetic and healthy backgrounds. A decreased metabolic activity was found in diabetic cells at lower temperature. Healthy cells showed less adipogenic potential at hypothermic conditions, however no difference in lipid content was found between temperatures and different metabolic backgrounds. Using these hUCT-MSCs models, upregulation of AdipoQ, PPAR, Glut4 mRNA and UCP1 protein expression in MSCs-adipocytes derived from healthy cells was detected while diabetic adipocytes displayed increased 3-AR and PPAR and decreased PRDM16 mRNA at normal culture conditions. 3-AR was identified as the only upregulated gene in diabetic cells differentiated under hypothermic conditions. These results indicate 3-AR as a potential signalling pathway for functional evaluation in recruiting stored fat and energy expenditure in human adipocyte differentiation. In conclusion, results presented here suggest that MSCs differentiated under hypothermic conditions could provide a source of thermogenic beige adipocytes which can preserve induced molecular and metabolic features. Maintenance of these properties suggest a mechanism of differentiation memory particularly when combined with browning stimulus in order to activate energy expenditure in obese populations. Cold temperature (32ºC) as an adipogenic differentiation strategy in MSCs represents a potential approach to understand beige and brown adipogenesis. Overall these results highlight MSCs as an available cell source to model the differentiation and maintenance of functional beige adipocytes

Stem cell models of adipogenesis: modulating temperature to promote MSC-derived beige adipocytes

Since the discovery of brown adipose tissue (BAT), emerging strategies to promote its activation and recruitment have been examined due to the thermogenic capacity of BAT, which makes it an encouraging approach to regulate obesity and related diseases. Uncoupling protein 1 (UCP1) is a specific protein located in the inner mitochondria of brown adipocytes, when activated, uncouples substrate oxidation from ATP production by dissipating energy in the form of heat. Beige adipocytes have been recently discovered and are cells which possess the morphological and biochemical characteristics of brown adipocytes, including UCP1. It has been previously reported that human BAT expresses beige cell-selective genes instead of classical brown markers which underline the importance of understanding beige adipogenic differentiation, induction and maintenance. All adipocytes share a mesodermal origin. Mesenchymal stem cells (MSCs) are able to differentiate into a variety of lineages, including adipocytes. To date, the most potent stimulus to activate UCP1 in BAT is cold exposure. However, whether cells sense temperature and differentiate into brown and beige lineages is not fully understood. The aim of this thesis is to obtain a better understanding on how low temperature affects the differentiation of MSCs into white, beige and brown adipocytes and if these traits acquired are maintained after being switched to normal culture conditions (37ºC). Additionally, cold exposure evaluation during adipogenic differentiation of human MSCs derived from cord tissue taken from diabetic and healthy pregnancies at term were examined. Cold temperature (32ºC) was investigated for its potential to activate browning in adipocytes. Here, MSCs differentiated under normal culture conditions showed a dual leptin and UCP1 protein expression in the majority of differentiated cells. Hypothermic conditions applied while the MSCs were under adipogenic differentiation promoted a brown/beige morphological and molecular signature. These changes were accompanied with enhanced UCP1 protein abundance, uncoupled respiration and metabolic adaptation. Hypothermic differentiated cells showed a marked translocation of leptin into adipocyte nuclei, which indicates a potential role for leptin in the browning process. These results indicated that MSCs could be driven to form beige like adipocytes by differentiating them in a cold environment. To further investigate whether these MSC-derived adipocytes could adapt overtime and retain their brown/beige traits, brown-like cells produced under hypothermic conditions were transferred to normal culture conditions. MSCs differentiated at 32ºC and then rewarmed maintained an intermediate brown/beige phenotype and gene profile with UCP1 protein expression found after 21 days of warm acclimation (37ºC). Additionally, these cells showed an intermediate mitochondrial respiration level, lipolysis, fatty acid oxidation, glycolysis rate and intracellular calcium expression, suggesting a shared mechanism of calcium mediated UCP1-independent thermogenesis. Moreover, these cells stored lipids and produce heat when stimulated. Altogether these results highlight the plasticity of MSCs to generate and preserve de novo beige adipocytes. Several isolation and culture methods were then tested in order to establish optimal conditions for human umbilical cord tissue MSCs (hUCT-MSCs) isolation, growth and differentiation. For hUCT-MSCs isolation, an enzymatic method using collagenase B (0.4 U/ml per cm) was used. After testing different culture mediums, it was found that DMEM low glucose,1% L-glutamine, 1% penicillin/streptomycin and 10% human platelet lysate was the best growth medium for these cells, while for adipogenic differentiation the best suitable medium was DMEM F12, 1% penicillin/streptomycin and 10% foetal bovine serum supplemented with 1 M dexamethasone, 500 M isobutyl-methylxanthine, 1 M rosiglitazone and 10 g/ml insulin. Finally, the hypothermic differentiation model of adipogenesis was tested in hUCT-MSCs from gestational diabetic and healthy backgrounds. A decreased metabolic activity was found in diabetic cells at lower temperature. Healthy cells showed less adipogenic potential at hypothermic conditions, however no difference in lipid content was found between temperatures and different metabolic backgrounds. Using these hUCT-MSCs models, upregulation of AdipoQ, PPAR, Glut4 mRNA and UCP1 protein expression in MSCs-adipocytes derived from healthy cells was detected while diabetic adipocytes displayed increased 3-AR and PPAR and decreased PRDM16 mRNA at normal culture conditions. 3-AR was identified as the only upregulated gene in diabetic cells differentiated under hypothermic conditions. These results indicate 3-AR as a potential signalling pathway for functional evaluation in recruiting stored fat and energy expenditure in human adipocyte differentiation. In conclusion, results presented here suggest that MSCs differentiated under hypothermic conditions could provide a source of thermogenic beige adipocytes which can preserve induced molecular and metabolic features. Maintenance of these properties suggest a mechanism of differentiation memory particularly when combined with browning stimulus in order to activate energy expenditure in obese populations. Cold temperature (32ºC) as an adipogenic differentiation strategy in MSCs represents a potential approach to understand beige and brown adipogenesis. Overall these results highlight MSCs as an available cell source to model the differentiation and maintenance of functional beige adipocytes

Leptin deficiency impairs adipogenesis and browning response in mouse mesenchymal progenitors

Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning

Magnetically Assisted Control of Stem Cells Applied in 2D, 3D and In Situ Models of Cell Migration

The success of cell therapy approaches is greatly dependent on the ability to precisely deliver and monitor transplanted stem cell grafts at treated sites. Iron oxide particles, traditionally used in vivo for magnetic resonance imaging (MRI), have been shown to also represent a safe and efficient in vitro labelling agent for mesenchymal stem cells (MSCs). Here, stem cells were labelled with magnetic particles, and their resulting response to magnetic forces was studied using 2D and 3D models. Labelled cells exhibited magnetic responsiveness, which promoted localised retention and patterned cell seeding when exposed to magnet arrangements in vitro. Directed migration was observed in 2D culture when adherent cells were exposed to a magnetic field, and also when cells were seeded into a 3D gel. Finally, a model of cell injection into the rodent leg was used to test the enhanced localised retention of labelled stem cells when applying magnetic forces, using whole body imaging to confirm the potential use of magnetic particles in strategies seeking to better control cell distribution for in vivo cell delivery