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New players in human BAT differentiation and activation: a human PSC-derived BAT approach combined with state of the art genome engineering and –omics based methodologies

Periodic Reporting for period 3 - StemBAT (New players in human BAT differentiation and activation: a human PSC-derived BAT approach combined with state of the art genome engineering and –omics based methodologies)

Reporting period: 2019-01-01 to 2020-06-30

StemBAT-New players in human BAT differentiation and activation: a human PSC‐derived BAT approach combined with state of the art genome engineering and -omics based methodologies

The prevalence, suffering and financial cost of obesity and its associated inflammatory-related and metabolic complications are large and worryingly continue to grow. No effective long-term treatments exist for obesity other than bariatric surgery. Obesity, defined as the accumulation of excess fat causing disease, occurs as a result of a mismatch between energy intake (how much we eat) and energy expenditure (how much we expend through processes such as physical activity and increased brown fat activation). Despite years of advice on healthy eating and recognised benefits of increasing physical activity, obesity rates continue to increase worldwide. Current approaches to tackle the epidemic of obesity and complications have not been successful, in part because the incomplete knowledge about the mechanisms controlling energy balance, and particularly because of a complexity and functional redundancy.
In StemBAT we propose a strategy to control body weight and prevent/reverse obesity based on growing and activating brown adipose tissue (BAT) to facilitate negative energy balance and prevent adaptive responses to dietary restriction. Brown adipose tissue is a form of adipose tissue specialised for energy dissipation. This contrasts with the predominantly anabolic energy storing function of white adipose tissue (WAT). BAT is the main site of non-shivering thermogenesis in mammals and is activated by cold and hypercaloric intake, mediating a process referred as adaptive thermogenesis. BAT in humans is relatively inaccessible, relatively scarce and poorly characterised. Thus, we propose to use human stem cells as tools to gain new unique insights into the biology of human brown adipocytes.

Our General Objective is to identify pathways and factors of therapeutic relevance that can be used to promote BAT development and/or activation/recruitment. For this we will be using a stem cell based BAT differentiation approach involving genome engineering of human stem cell derived adipocytes and in vivo by transplanting these cells into mice to functionally validate the role of these factors in vitro and in vivo.

Specific Aims are: 1. To identify molecular mechanisms involved in human brown adipose tissue development and activation. 2. To investigate the molecular mechanisms involved in human white adipose tissue browning/brite cells recruitment 3. To identify new agents/compounds of therapeutic value, able to activate or recruit human brown adipose tissue/brite cells.

Experimental strategy: We will use human pluripotent stem cell (PSC) lines differentiated into brown and white adipocytes to identify genetic factors that may contribute to brown adipocyte differentiation/activation and white adipocyte browning. Following the identification of candidate genes, we will initially knock out, constitutively and/or inducible, both alleles of these genes in human PSC cells, producing a total loss of function. Following the in vitro phenotyping of the cells we will proceed to the in vivo validation of the functional properties/phenotype of human PSC derived brown/brite adipocytes (wild-type and loss of function) by transplanting these cells into mice. Using these reporter tools we will also perform in vitro pharmacological screening and in vivo validation of new compounds that stimulate BAT activation and WAT browning.
We have proceeded to the final optimisation of the chemically define protocol for the differentiation of human PSC into brown adipocyte both in a 2D and a 3D system aiming to reach greater differentiation and optimise options for upscaling. The differentiation of the adipocytes is influenced by mechanical cues and stiffness of the matrix on which they are cultured, so given the biological advantages provided by a more physiological 3D setting we decided to develop a 3D system that mimics more closely the physiological environment of the adipose tissue in vivo and sustain a better differentiation.
We also validated the molecular signature of our differentiated cells i.e. we have checked that they expressed specific markers of BAT similarly and comparably to human brown adipocyte immortalised cell lines and online transcriptome databases available from human BAT primary cells as well as their functionality i.e. oxygen consumption. We are currently collecting samples from our human PSC-derived brown adipocytes at different stage of differentiation, to perform an in-depth transcriptome analysis i.e. RNAseq to identify new key factors (genes/paths) involved in early BAT differentiation and development. Lipidomic and proteomic analysis will complement and be integrated with transcriptome profile. We have also progressed in the optimisation of the chemically define protocol for the differentiation of human PSCs into WAT. We are using for this the same 2D and 3D approaches that for the BAT. As a backup approach, we are also generated genetically modified human PSC cell lines carrying a tet-on system controlling the expression of key transcription factors for the generation of BAT and WAT adipocyte.

In parallel we have initiated the generation of KO human PSC cell lines for candidate genes identified in epidemiology studies,that we anticipate to play an important role in BAT adipogenesis and development through the High-throughput Gene Editing pipeline at Sanger. We anticipate more candidate genes will be identified following our RNAseq analysis. Thanks to our collaboration with other research groups at Sanger we will also have access to already generated human PSC cell lines for candidate genes to play an important role in lipid metabolism and that we can test in our cellular model.

We are evaluating the possibility to screen a library of natural compounds to which we will have access through a collaboration and that are considered good candidate for BAT recruitment/activation on our human PSC-derived BAT/WAT cellular models.
Aim 1: To identify the molecular mechanisms involved in human brown adipose tissue development and activation.
Rationale/State of the art. There is limited knowledge about the development of human BAT and its potential recruitment in adults, therefore we rely on insights from knockout and Cre-recombinase-based lineage tracing mouse models. Murine studies suggest that canonical brown adipose tissue might share a common somitic developmental origin with skeletal myoblasts and dermal precursors It is crucial to validate these insights in a human developmental model system since understanding the origins of the BAT lineage and mapping the transcriptomes and signalling pathways of BAT precursors is key to understanding BAT function in the adult and in metabolic disease. Almost nothing is known about the activated state of human BAT and gaining more insights into this bottleneck is key to the development of effective therapeutic interventions based on BAT. Limitations of current human BAT cellular models involve deficient proliferative capacity, heterogeneous and progressively impaired differentiation and lack of reliability to produce large numbers of pure brown adipocytes [To overcome these issues a human pluripotent stem cell based system is ideal. Using PSCs would allow the generation of progenitor populations with a large proliferative capacity that are able to differentiate.

Taking advantage of our PSC cellular model of human BAT differentiation, we will identify factors involved in early BAT differentiation through transcriptome analysis applied to cell populations corresponding to the specific stages of development “defined” in the differentiation protocol (i.e. pluripotent, late primitive streak, presomitic mesoderm (PSM), somitic mesoderm (SM) and differentiated adipocyte.
Identification of factors involved in BAT activation. Our human BAT-PSC derived cellular system will be terminally differentiated and treated/untreated with β3-adrenergic agonists, and analysed to identify new players involved in the induction of the thermogenic program using a systems biology approach.
Systems biology approach: Transcriptome analysis of our human BAT-PSC derived cells at different stage of differentiation will be carried out and complemented with metabolome/lipidome and/or proteome analysis following the results and the key pathway identified in the transcriptome analysis.
Bioinformatics analysis of the omics analysis and data integration will be performed and the information obtained from the integration of omics data will be used as a selection rationale of genes/pathways for the generation of mutated cell lines.
Engineered Mutations: In collaboration with gene targeting pipelines at the Sanger Institute we will engineer bi-allelic Loss of Function (LOF) null mutations in human pluripotent stem cells (PSC) using powerful Cas9/CRISPR methodology. This powerful approach will allow clean genetic evaluation of the role of various candidate genes in either differentiation of human PSCs to BAT or BAT function.
Mutant cell line phenotyping will assess the ability of the genetically modified cell line to a) proliferate b) to differentiate into BAT and c) to be activated by thermogenic stimuli. We will investigate the molecular and the metabolic phenotype of these mutated cells to gain insight about the genes pathways and other characteristics/parameters of the cells affected by the depletion of the genes of interest.
Moreover, using a transplantation system of human derived brown adipocyte precursors into immunocompromised mice we will confirm the functionality and the phenotype of these cells in vivo and examine their interactions with neural or vascular endothelial cells, as both neuronal growth and vasculature are essential for the physiological function of BAT as well as the impact of the mutation at systemic level.

In summary, the data produced from this screen will be invaluable for understanding the development of human BAT and will allow the design of new therapeutic strategies to treat and prevent obesity. Understanding the biology behind BAT formation will enable specific target interventions to increase BAT mass and activity, to improve the metabolic profile of an individual.

Aim 2: To investigate the molecular mechanisms involved in human white adipose tissue browning/brite cells recruitment
Rationale/State of the art: The two main hypotheses regarding how brite adipocyte recruitment occurs in murine WAT depots following cold exposure or adrenergic stimulation are: a) through trans-differentiation from white to brown-like adipocytes and b) via bi-potential common early progenitors. There is evidence that humans also develop browning in their WAT. However the knowledge about brite cell recruitment is limited, though some insights indicate that compared to brown adipocytes, human brite cells may have a different genetic signature and exhibit selective responsiveness to specific differentiating factors. Because of interspecies differences, it is essential to be able to investigate the molecular mechanisms and genetic factors implicated in brite cell recruitment in a human cellular model.

Taking advantage of our PSC cellular model of human WAT/beige cells differentiation, we will identify of genetic factors involved in human WAT browning by applying our chemically defined protocol for WAT differentiation to our PSC cell lines and investigating the molecular mechanisms involved in human recruitment of brite adipocytes. We will validate the WAT differentiation protocol by analysing the expression of white adipocyte specific markers vs brown/ brite cells markers respectively. The browning will be assessed by determining the levels of UCP1 expression. We will perform a transcriptome analysis of the brown-like cells, what will be followed by bioinformatics and pathway analysis to identify new candidate genes involved in WAT browning.
Genetic engineering: The role of the newly identified WAT browning candidate genes will be evaluated by bi-allelic loss of function using the same CRISPRS/Cas9 approach as planned for the human BAT study.
Mutant cell in vitro and in vivo phenotyping: Browning of control and homozygous knockout differentiated adipocytes treated with thermogenic activators will be assessed by transcriptome and protein expression profiling of thermogenic genes and also molecular markers of brite cells. The functionality and metabolic phenotyping of the cells will also be assessed. In the most promising cases we will validate the effect of browning in vivo in a transplant system using the approach described above for brown adipocytes.
All in all, our investigation into brite cell recruitment in WAT using human stem cells will allow a better elucidation of the key players involved in these processes in humans. Understanding the biology behind human WAT browning will allow the development of specific target interventions that help to increase the number of brite cells in white fat depots and improve the metabolic profile of an individual. Our work will also facilitate the identification of new specific brite cell markers which are so far very few in numbers.

Aim 3: To identify therapeutic agents/compounds able to activate human brown adipose tissue/ brite cells recruitment
Rationale/State of the art. The need for and importance of a human mature brown adipocyte and WAT browning screening system derives from the differences between rodent and human BAT regarding their thermogenic capacity and pharmacological responsiveness. So far, the access to suitable human tissue has been difficult due to its limited availability. A human PSC based reporter cell line represents a simple and straightforward system for drug screening and also gives access to an unlimited amount of material required for –omics based approaches.

In vitro pharmacological screening will be performed to test new compounds stimulating BAT formation/activation and brite cells recruitment in WAT. For the screening we plan to use of a human PSC based UCP1 reporter cell line to obtain a homogeneous population of brown adipocyte. This cellular system will be used to identify potential BAT pharmacological activators and the read out for BAT activation will be an increase in the fluorescence of the reporter system. The same reporter cell line will be differentiated in white adipocytes and brite cells recruitment will be monitored by looking at an increased fluorescence of the reporter gene under the control of the UCP1 promoter. Compounds will be made available through a collaboration with industry.
In vitro validation of activation of BAT and brite cells recruitment by the candidate compounds will be assessed by gene/protein expression analysis of markers of the thermogenic program activation and by functional assays.
In vivo validation will require injecting subcutaneously or administrating via drinking water the agent to mouse models of diet-induced obesity and/or of genetic-induced obesity and subsequent evaluation of their metabolic phenotype. This will be complemented by histological and molecular characterisation of BAT activation/WAT browning.
In summary, our human stem cell based differentiation model (of BAT and browned WAT) combined with genetic engineering technology (UCP1 reporter system) represents a straightforward approach for pharmaceutical/chemical screening purposes, facilitating the discovery of compounds of therapeutic use. Moreover, this strategy could be applied to other metabolically relevant cell types.