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

Project Information

StemBAT

Grant agreement ID: 669879

DOI

10.3030/669879

Project closed

EC signature date 8 December 2015

Start date 1 January 2016

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 500 000,00

EU contribution

€ 2 500 000,00

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 6 - 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: 2023-07-01 to 2023-12-31

Summary of the context and overall objectives of the project

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.
Obesity results from excess fat accumulation due to an imbalance between intake and energy expenditure, with rising prevalence and associated complications. Current approaches have failed, partly due to limited understanding of the complex mechanisms controlling energy balance.
In StemBAT, we proposed a strategy to combat obesity based on growing and activating brown adipose tissue (BAT) to facilitate negative energy balance and prevent adaptive responses to dietary restriction. BAT specialises in energy dissipation and mediates adaptive thermogenesis but is relatively scarce and poorly characterised in humans. Thus, we proposed to use human stem cells as tools to gain new, unique insights into the biology of human brown adipocytes (BAs).
Our General Objective was to identify therapeutically relevant pathways and factors to promote BAT development and activation. Our specific objectives were to identify pathways that promote BAT development and activation, using human pluripotent stem cell (PSC)-derived BAs, genome engineering, and transplantation into mice for functional validation. We identified candidate genes from GWAS and other studies, generated mutant cell lines, and developed a protocol to convert stem cells into BAs. We are integrating multi-omics to deepen understanding of human BAT biology and have used in-silico drug screening to find repositionable drugs that promote BAT differentiation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Our multi-step differentiation protocol of human PSCs to BAs is fully optimised and published in Stem Cell Report (PMID: 33606988).This protocol is relevant for research groups and industries focusing on human BAs to overcome limited sample access.
We have characterised gene expression patterns across the process and identified specific mature subpopulations in human PSC-BAs that we are currently mapping to human clusters.
Once optimised, we will further enhance the model's characterization using multilayer omics platforms, i.e. transcriptomic, phosphoproteomic and in-depth 3D subcellular imaging. We are developing bioinformatic tools to integrate this data and identify molecular targets link to subcellular changes. These tools are transferable to multiple biological questions beyond the metabolic field.
We have generated more than 40 human PSCs KO cell lines for genes of interest involved in lipid metabolism and remodelling, extracellular matrix remodelling, and signalling. These cells can also be differentiated into other cell types, enabling the expansion/upscale of the program into other organs and the study of intercell communication. We have performed in vitro phenotyping of the most promising cell lines.
We optimised the engraftment of transplanted human PSC-derived BAT cells in immunocompromised mice by supplementing angiogenic factors to improve vascularisation.
The optimisation of the white adipocyte (WA) protocol has proven more challenging than foreseen. We assessed the molecular signature of our 2D and 3D differentiated cells at the gene and protein levels. The cells reach the progenitor state, but at the maturation stage they show lower expression of specific markers than expected, indicating that they have not reached full maturation yet. This problem is common in cell types differentiated from human PSCs.
We have developed collaborative work and conducted an in-silico drug screening using a new bioinformatic methodology to identify compounds that increase the recruitment and activation of our human PSC-derived BAs and promote WAT browning. This method identified known drugs and novel candidates currently used to treat other disorders, and we validated the results in vitro. The manuscript describing this work is being prepared and this methodology is highly transferable to other cell types.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Aim 1: To identify the molecular mechanisms involved in human brown adipose tissue development and activation.
Our multi‐step differentiation protocol was published in Stem Cell Report (PMID: 33606988). We present an in‐depth molecular and functional characterisation of our model, demonstrating its behaviour as bona fide human BAs. We identified critical regulatory molecules and highly specific preadipocyte and adipocyte cell surface markers as well as improving our understanding of the brakes and accelerator mechanisms while fine-tuning the improved protocol. Some of this new information has already fed the system and improved its efficiency. We also generated a list of candidates to enhance brown adipocyte recruitment and distinguished subpopulations via single nuclei RNA sequencing at late differentiation stages.
We established a new collaboration to assess the subcellular architecture of human BAs during differentiation. We developed a pipeline to extract quantitative data from 3D reconstructed images (number of mitochondria, volume, lipid droplets number and size…) and have worked on developing a bioinformatic approach to integrate imaging-derived data with classical omics data. This combination will allow us to extract molecular targets related to the observed changes in the subcellular features.
We implemented innovative algorithms for phosphoproteomic analyses through collaboration with the EBI to amplify the signal, reduce the noise and identify pathways from a limited number of detected phosphosites, as well as coping with the challenge of time-course analyses instead of pair-wise comparisons. We have validated some of the findings in vitro.
We generated a collection of more than 40 human PSCs KO cell lines and characterised some of the most promising ones.
We established an improved transplantation protocol co-injecting angiogenic factors together with the PSC-derived BAs in the lower back region of the mice. We determined that the co-injection of human-derived adipocytes and endothelial cells is necessary to guarantee the proper vascularisation of the fat pads.
Aim 2: To investigate the molecular mechanisms involved in human white adipose tissue browning/brite cell recruitment.
The optimisation of our human PSC to white adipocyte (WA) protocol has proven more challenging than foreseen. We have assessed the molecular signature of 2D and 3D differentiated cells by transcriptomic, immunoblot, and immunofluorescence. Commitment is very efficient. Fully differentiation is not reached yet as observed for other PSCs-derived cell types.
We have identified genes essential for cell membrane homeostasis, the impairment of which is supposed to impact the ability of the adipocytes to differentiate. The characterisation of one of them demonstrates no defects in commitment markers, but defects in some lipid droplets-related markers. Transcriptomic data are being analysed.

Aim 3: To identify therapeutic agents/compounds able to activate human brown adipose tissue/ brite cell recruitment.
Through collaborative work, we performed an in-silico drug screening using novel methodology to identify new compounds increasing the recruitment and activation of PSC-derived BAs and promoting WAT browning. Some of the predicted drugs were validated in vitro.

picture-stembat.png