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Lugo Leija, Hilda Anaid (2020) Stem cell models of adipogenesis: modulating temperature to promote MSC-derived beige adipocytes. PhD thesis, University of Nottingham.

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Abstract

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.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Sottile, Virginie Symonds, Michael Velickovic, Ksenija
Keywords:	adipocyte, MSCs, browning, UCP1, cold temperature, energy expenditure, obesity.
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QS Human anatomy
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	60659
Depositing User:	Lugo Leija, Hilda
Date Deposited:	14 Aug 2020 09:53
Last Modified:	24 Jul 2022 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/60659

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