Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - 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)

Teaser

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 methodologiesThe prevalence, suffering and financial cost of obesity and its associated inflammatory-related...

Summary

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.

Work performed

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.

Final results

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