Characterisation of chamber-specific fibroblasts from adult human heart and investigation of their potential contribution to the function of engineered heart tissue
File(s)
Author(s)
Khalil, Amina
Type
Thesis
Abstract
Research in the past 15 years has shown that the adult heart, including human, contains cardiac fibroblasts exhibiting regenerative capacity. Little is known about the precise characteristics, properties, and differentiation potential of endogenous cardiac fibroblasts (CF) in the four cardiac chambers of the human heart, and how these are affected by the disease process. The fibroblast population in the heart is being redefined from an inert scar-forming cell type into one, which has an active role in terms of paracrine effects and cardiomyocyte coupling. Fibroblast characterisation shows overlap with the variously described mesenchymal stem cell populations, and these cells can be reprogrammed directly to cardiomyocytes. 3D engineered heart tissue (EHT) is made using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSC-CM). The addition of human foetal cardiac fibroblasts to the EHTs provided structural support, enhanced force output, improved calcium handling and induced expression of more mature sarcomeric proteins.
In this thesis, for the first time the human CFs from all four chambers in patients with preserved biventricular function are investigated. A comparison is made between all four chambers and between left and right side of the heart. Fibroblasts identified by a range of markers, are characterised by growth pattern, rate of proliferative, secreted factors and expression of cardiogenic transcription factors. Moreover, the effect of chamber-specific fibroblasts on the function and gene expression of the iPSC-CM in EHT is studied. Finally, for the first time the secretory profile of EHT constructs is elucidated by the proteomic analysis of the medium bathing EHTs. This project aims to investigate the mechanism of this effect regarding cell coupling and paracrine effectors.
The in-vitro gorwth of a truely defined human all four-chamber cardiac fibroblast population depicted that atrial fibroblasts have a better rate of proliferation, growth pattern and lesser predilection for myofriboblastic transformation as compasred to ventricular fibroblasts. The trancription factor expression of atrial fibroblasts is different from ventricular fibroblasts hence pointing towards their different embryological origin. The co-expression of chamber-specific fibroblasts in EHTs with cardiomyocytes of same origin showed that rigth ventricular fibroblasts based EHTs had the best kinetics. Finally the proteomic expression of EHTs is mainly dependent on presence of cardiomyocytes. The right ventricular fibroblasts based EHTs showed least amount of proteins expression in conjuction with best kinetics eluding to the fact that in-vivo many proteins expressed by cardiomyocytes and non-cardiomyocytes might have an inhibitory effect.
This thesis provides an insight into phenotypic variablity, transcription factor expression, impact on kinetics and protein co-epxression in EHTs of chamber-specific fibroblasts with cardiomyocytes. Further investigations into the role of chamber-specific fibroblasts in EHTs in patients with impaired ventricular function can be explored subsequently.
In this thesis, for the first time the human CFs from all four chambers in patients with preserved biventricular function are investigated. A comparison is made between all four chambers and between left and right side of the heart. Fibroblasts identified by a range of markers, are characterised by growth pattern, rate of proliferative, secreted factors and expression of cardiogenic transcription factors. Moreover, the effect of chamber-specific fibroblasts on the function and gene expression of the iPSC-CM in EHT is studied. Finally, for the first time the secretory profile of EHT constructs is elucidated by the proteomic analysis of the medium bathing EHTs. This project aims to investigate the mechanism of this effect regarding cell coupling and paracrine effectors.
The in-vitro gorwth of a truely defined human all four-chamber cardiac fibroblast population depicted that atrial fibroblasts have a better rate of proliferation, growth pattern and lesser predilection for myofriboblastic transformation as compasred to ventricular fibroblasts. The trancription factor expression of atrial fibroblasts is different from ventricular fibroblasts hence pointing towards their different embryological origin. The co-expression of chamber-specific fibroblasts in EHTs with cardiomyocytes of same origin showed that rigth ventricular fibroblasts based EHTs had the best kinetics. Finally the proteomic expression of EHTs is mainly dependent on presence of cardiomyocytes. The right ventricular fibroblasts based EHTs showed least amount of proteins expression in conjuction with best kinetics eluding to the fact that in-vivo many proteins expressed by cardiomyocytes and non-cardiomyocytes might have an inhibitory effect.
This thesis provides an insight into phenotypic variablity, transcription factor expression, impact on kinetics and protein co-epxression in EHTs of chamber-specific fibroblasts with cardiomyocytes. Further investigations into the role of chamber-specific fibroblasts in EHTs in patients with impaired ventricular function can be explored subsequently.
Version
Open Access
Date Issued
2019-04
Date Awarded
2020-07
Copyright Statement
Creative Common Attribution Non-commercial No Derivative license
Advisor
Punjabi, Prakash
Harding, Sian
Sponsor
British Heart Foundation
Grant Number
BHF Clinical Research Training Fellowship no. FS/15/79/31736 - Amina Khalil
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Medicine (Research) MD (Res)