Periodic Reporting for period 5 - 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)
Período documentado: 2021-07-01 hasta 2023-06-30
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 complications are large and ntinue 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 how much we eatand how much we expend through processes such as physical activity and increased brown fat activation. 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 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 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.
The prevalence, suffering and financial cost of obesity and its associated complications are large and ntinue 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 how much we eatand how much we expend through processes such as physical activity and increased brown fat activation. 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 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 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.
Our multi-step protocol of differentiation of human PSCs to human brown adipocytes has been published in Stem Cell Report (PMID: 33606988). A in-depth bioinformatic analysis of the bulk and Single nuclei trascriptome of the hPSC-BA model is in progress. Other omics analysis i.e phosphoproteomic has also been performed, and it is being analysed at the moment.
We have generated and collected more than 40 human PSCs KO cell lines for genes of interest identified from epidemiology studies, the analysis of the bulk RNAseq from our cells, and previous background work performed in our lab. We have completed the in vitro phenotyping of some of the lines and we are progressing analysis others.
We have performed some optimisation studies for the transplantation of our human PSC-derived BAT cells in immunocompromised mice attempting to improve the vascularisation of the newly formed fat pad. Further analysis of the morphology of the pad are in progress.
We progressed quite substantially in optimising the chemically defined protocol for the differentiation of human PSCs into WAT both in 2D and 3D. We also assessed the molecular signature of our differentiated cells for the expression of key markers of immature and mature white adipose tissue. The transcriptome analysis of the human PSC derived WAT cell model has been performed, confirming the expression of human WAT specific markers. The next step will be the collection of samples from human PSC-derived WAT cells undergoing browning for transcriptome analysis. The molecular and functional characterisation of the KO cell lines for genes of interest for human white and beige adipocyte differentiation and function is in progress.
We are performing an in silico drug screening to identify new compounds to increase the recruitment and activation of our human PSC derived BAs and promote WAT browning.
We have generated and collected more than 40 human PSCs KO cell lines for genes of interest identified from epidemiology studies, the analysis of the bulk RNAseq from our cells, and previous background work performed in our lab. We have completed the in vitro phenotyping of some of the lines and we are progressing analysis others.
We have performed some optimisation studies for the transplantation of our human PSC-derived BAT cells in immunocompromised mice attempting to improve the vascularisation of the newly formed fat pad. Further analysis of the morphology of the pad are in progress.
We progressed quite substantially in optimising the chemically defined protocol for the differentiation of human PSCs into WAT both in 2D and 3D. We also assessed the molecular signature of our differentiated cells for the expression of key markers of immature and mature white adipose tissue. The transcriptome analysis of the human PSC derived WAT cell model has been performed, confirming the expression of human WAT specific markers. The next step will be the collection of samples from human PSC-derived WAT cells undergoing browning for transcriptome analysis. The molecular and functional characterisation of the KO cell lines for genes of interest for human white and beige adipocyte differentiation and function is in progress.
We are performing an in silico drug screening to identify new compounds to increase the recruitment and activation of our human PSC derived BAs and promote WAT browning.
Aim 1: To identify the molecular mechanisms involved in human brown adipose tissue development and activation.
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.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. 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.
We will engineer bi-allelic Loss of Function null mutations in human PSC using Cas9/CRISPR methodology. We will investigate the molecular and the metabolic phenotype of these mutated cells to gain insight about the genes pathways and other characteristics/parameters 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 at systemic level.
Aim 2: To investigate the molecular mechanisms involved in human white adipose tissue browning/brite cells recruitment
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. 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. 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 similarly to aim 1.
Aim 3: To identify therapeutic agents/compounds able to activate human brown adipose tissue/ brite cells recruitment
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.Compounds will be made available through a collaboration with industry.
We will perform in vitro validation and in vivo validation of activation of BAT and brite cells recruitment by the candidate compounds.
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.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. 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.
We will engineer bi-allelic Loss of Function null mutations in human PSC using Cas9/CRISPR methodology. We will investigate the molecular and the metabolic phenotype of these mutated cells to gain insight about the genes pathways and other characteristics/parameters 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 at systemic level.
Aim 2: To investigate the molecular mechanisms involved in human white adipose tissue browning/brite cells recruitment
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. 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. 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 similarly to aim 1.
Aim 3: To identify therapeutic agents/compounds able to activate human brown adipose tissue/ brite cells recruitment
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.Compounds will be made available through a collaboration with industry.
We will perform in vitro validation and in vivo validation of activation of BAT and brite cells recruitment by the candidate compounds.