Modelling the non-coding genome using RNA and CRISPRi/a approaches to define vascular heterogeneity in health and disease
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Molecular. Genetics & Pop Health
Abstract
Despite initially being regarded as a simple monolayer barrier, the role of the vascular endothelium in vascular homeostasis has become increasingly apparent in recent years. Its function in maintaining the structure of the vessel wall coupled with its ability to respond to physical and chemical stimuli by regulation of vascular tone, coagulation, and inflammation highlights its importance in vascular biology. Vascular endothelium dysfunction has been demonstrated to occur following tissue ischemia. In addition to the necrosis of endothelial cells in response to the initial ischaemic injury, reperfusion results in cellular abnormalities evoking apoptosis. Consequently, a potential therapeutic approach for cardiovascular disease would be to utilise endothelial-regenerating cells to improve the perfusion of ischaemic tissue. Prior attempts to initiate angiogenesis using various stem cells have been disappointing. Improved understanding of vascular endothelial cell specification is required to fully utilise the therapeutic potential of endothelial-regenerating cells.
In response to the limitations in existing regenerative approaches, this project aims to characterise the influence of the non-coding genome in endothelial cell maturation and specification. The Professor Andrew Baker group have established a clinically-compliant protocol to generate mature CD31+/CD144+ endothelial cells from human embryonic stem cells, thus providing an ideal model to investigate endothelial specification. A comprehensive understanding of endothelial cell specification is particularly important when considering the highly heterogeneous nature of the vascular endothelium. Such heterogeneity exists within the various vessel types as well as in an organ specific context, thus facilitating varying physiological functions.
Owing to the vascular endothelial heterogeneity, single cell RNA sequencing will be conducted in various endothelial cells types to identify coding and non-coding RNA molecules implicated in cell maturation and specification. Prospective lncRNAs will then be targeted in CRISPR-Cas9 activation and interference screening, thus demonstrating their causative effect on endothelial cell maturation. lncRNA are involved in numerous biological functions, with cell specific expression patterns being attributed to coordinating cell state and differentiation. By conducting both CRISPR-Cas9 interference and activation screens, identification of complementary data will verify the causal role of candidate lncRNAs. CRISPR-Cas9 activation will be achieved by the use of the dCas9-VPR system.
The expression, regulation, and function of candidate lncRNAs and protein coding genes will then be investigated in the model system, in which human embryonic stem cells give rise to mature endothelial cells. This will involve using techniques such as RT-qPCR, RNA-FISH and possibly genome editing utilising CRISPR-Cas9 methods. Findings from in vitro experiments will then be tested in murine models of peripheral and myocardial ischaemia.
Finally, ethical approval has been obtained to use RNA samples from individuals with and without vascular disease to asses the relevance of lncRNAs in human vascular pathology. Since dysfunction of the vascular endothelium is known to precede atherosclerosis, then determining the role of lncRNAs in this process may contribute valuable information to vascular pathogenesis thus informing future precision medicine approaches.
In response to the limitations in existing regenerative approaches, this project aims to characterise the influence of the non-coding genome in endothelial cell maturation and specification. The Professor Andrew Baker group have established a clinically-compliant protocol to generate mature CD31+/CD144+ endothelial cells from human embryonic stem cells, thus providing an ideal model to investigate endothelial specification. A comprehensive understanding of endothelial cell specification is particularly important when considering the highly heterogeneous nature of the vascular endothelium. Such heterogeneity exists within the various vessel types as well as in an organ specific context, thus facilitating varying physiological functions.
Owing to the vascular endothelial heterogeneity, single cell RNA sequencing will be conducted in various endothelial cells types to identify coding and non-coding RNA molecules implicated in cell maturation and specification. Prospective lncRNAs will then be targeted in CRISPR-Cas9 activation and interference screening, thus demonstrating their causative effect on endothelial cell maturation. lncRNA are involved in numerous biological functions, with cell specific expression patterns being attributed to coordinating cell state and differentiation. By conducting both CRISPR-Cas9 interference and activation screens, identification of complementary data will verify the causal role of candidate lncRNAs. CRISPR-Cas9 activation will be achieved by the use of the dCas9-VPR system.
The expression, regulation, and function of candidate lncRNAs and protein coding genes will then be investigated in the model system, in which human embryonic stem cells give rise to mature endothelial cells. This will involve using techniques such as RT-qPCR, RNA-FISH and possibly genome editing utilising CRISPR-Cas9 methods. Findings from in vitro experiments will then be tested in murine models of peripheral and myocardial ischaemia.
Finally, ethical approval has been obtained to use RNA samples from individuals with and without vascular disease to asses the relevance of lncRNAs in human vascular pathology. Since dysfunction of the vascular endothelium is known to precede atherosclerosis, then determining the role of lncRNAs in this process may contribute valuable information to vascular pathogenesis thus informing future precision medicine approaches.
Organisations
People |
ORCID iD |
Andrew Baker (Primary Supervisor) | |
Ian McCracken (Student) |
Publications
McCracken IR
(2020)
Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.
in European heart journal
McCracken IR
(2018)
Unravelling atherosclerotic heterogeneity by single cell RNA sequencing.
in Current opinion in lipidology
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
MR/N013166/1 | 01/10/2016 | 30/09/2025 | |||
1939057 | Studentship | MR/N013166/1 | 01/09/2017 | 31/08/2021 | Ian McCracken |
Title | scRNA-seq dataset of hESC-EC differentiation |
Description | Carried out 10X scRNA-seq on cells taken at various point along differentiation from human ES cells to endothelial cells. |
Type Of Material | Database/Collection of data |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | Important contribution to field of regenerative biology and regenerative medicine. Data formed basis of a publication in the European Heart Journal ('Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing'). The importance of this publication was highlighted in an editorial from Pinto et al (Mapping human pluripotent stem cell-endothelial cell differentiation using scRNA-seq: a step towards therapeutic angiogenesis). |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131736 |
Description | CafeSci talk in Glasgow |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Gave a talk at Cafe Scientifique event in Glasgow about the uses of stem cells to treat heart disease. |
Year(s) Of Engagement Activity | 2019 |
URL | http://cafescientifique.org/uk/glasgow/previous-events-glasgow |
Description | Edinburgh International Science Festival |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Demonstrated at the Edinburgh International Science Festival explaining the process of atherosclerotic plaque formation and benefits of healthy eating via a workshop aimed for children aged 10-15. |
Year(s) Of Engagement Activity | 2018 |
Description | InterSCi presentation |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | Gave a talk at InterSci event in Edinburgh about ageing and immortality. |
Year(s) Of Engagement Activity | 2018 |
URL | https://intersci.org/conversations/meet-the-scientists/series-6-meet-the-scientists/ |
Description | Tour of facilities at the Centre for Cardiovascular Sciences |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Participated in giving BHF volunteers a tour of facilities at the Centre for Cardiovascular Sciences. |
Year(s) Of Engagement Activity | 2018 |