Periodic Reporting for period 1 - SiGNATURE (Selection of human iPSC-derived cardiomyocytes by sinGle cell geNe expression and pAtch clamp for a maTUre caRdiac modEl)
Período documentado: 2020-02-01 hasta 2022-01-31
Heart disease is a burden for the European population, as cardiovascular disease is the most common cause of death Europe and the number of people suffering from heart rhythm abnormalities (cardiac arrhythmia) is increasing. The mechanisms of arrhythmias are in some cases poorly understood, because of the lack of proper models able to accurately reproduce the human heart in a more simplified environment. Animal models have been and still are widely used to study genetic mutations leading to arrhythmia, but their differences with the human species impeded a precise reproduction of patients’ diseases. Moreover, reducing the use of animal testing for ethical reasons is one of the objectives of EU.
In the last years efforts to find an alternative model for heart disease have directed the attention to human induced pluripotent stem cells (hiPSCs). These cells can be derived by reverting to the stem cell state a patient’s cells, therefore carry the same genes and mutations. They can then generate virtually unlimited amounts of human cardiomyocytes, the heart contracting cells. The use of hiPSC-derived cardiomyocytes (hiPSC-CMs) has indeed provided many insights on genetic mutations associated with heart disease. However, current technology for cardiac differentiation from stem cells is not yet producing cardiomyocytes mature enough to resemble adult heart cells for functionality and gene expression. This is particularly relevant when using the hiPSC-CMs to test drugs for cardiopathic patients who are most commonly adults: their responses may not correspond. Moreover, genetic heart diseases due to mutations in genes not expressed in immature hiPSC-CMs cannot currently be studied.
In this context, SiGNATURE project aimed to improve the human cardiac model, by producing more mature and thus reliable hiPSC-CMs. The approach was to promote maturation by better mimicking the heart, which has a three-dimensional (3D) structure and is composed by different cell types, the main being CMs, fibroblasts and vascular cells. The goal was to produce a 3D multicellular culture system as simple as possible and with contained production costs, to be usable by many researchers in both academic and industrial laboratories. Moreover, the system should allow analysis at the single cell level, to investigate the precise mechanisms underlying genetic heart diseases. Combining analysis of specific gene expression and single cardiomyocyte electrical activity, the project aims at demonstrating the utility of this 3D maturation system by revealing the effects of mutations masked in immature hiPSC-CMs, providing a more consistent model to test drugs.
In the last years efforts to find an alternative model for heart disease have directed the attention to human induced pluripotent stem cells (hiPSCs). These cells can be derived by reverting to the stem cell state a patient’s cells, therefore carry the same genes and mutations. They can then generate virtually unlimited amounts of human cardiomyocytes, the heart contracting cells. The use of hiPSC-derived cardiomyocytes (hiPSC-CMs) has indeed provided many insights on genetic mutations associated with heart disease. However, current technology for cardiac differentiation from stem cells is not yet producing cardiomyocytes mature enough to resemble adult heart cells for functionality and gene expression. This is particularly relevant when using the hiPSC-CMs to test drugs for cardiopathic patients who are most commonly adults: their responses may not correspond. Moreover, genetic heart diseases due to mutations in genes not expressed in immature hiPSC-CMs cannot currently be studied.
In this context, SiGNATURE project aimed to improve the human cardiac model, by producing more mature and thus reliable hiPSC-CMs. The approach was to promote maturation by better mimicking the heart, which has a three-dimensional (3D) structure and is composed by different cell types, the main being CMs, fibroblasts and vascular cells. The goal was to produce a 3D multicellular culture system as simple as possible and with contained production costs, to be usable by many researchers in both academic and industrial laboratories. Moreover, the system should allow analysis at the single cell level, to investigate the precise mechanisms underlying genetic heart diseases. Combining analysis of specific gene expression and single cardiomyocyte electrical activity, the project aims at demonstrating the utility of this 3D maturation system by revealing the effects of mutations masked in immature hiPSC-CMs, providing a more consistent model to test drugs.
SiGNATURE project started with developing a maturation culture system for hiPSC-CMs, consisting in 3D microtissues composed by hiPSC- CMs, -fibroblasts and -vascular cells. The interaction between the different cell types was shown to promote maturation in different important aspects: structure, function, gene expression and metabolism. Strikingly, maturation of hiPSC-CMs was maintained even after they were dissociated from the tissue. The microtissues are composed by only 5000 cells which are able to auto-aggregate into spheric spontaneously beating structures, meaning the model is simple to produce and very cost-effective. The model also demonstrated to be very flexible: the three composing cell types can be derived by either healthy individuals or cardiac patients, with different possible combinations. These results were disseminated with two publications in high-impact scientific journals (Giacomelli, Meraviglia, Campostrini al. Cell Stem Cell, 2020; Campostrini et al. Nature Protocols, 2021) and advertised via press release and social media (Facebook, Linkedin).
The microtissue model was then used to demonstrate its utility to study heart disease. We used hiPSC-CMs from two patients, carrying a mutation in SCN5A and KCNQ1 gene, respectively. These genes encode for two important ion channels determining the electrical activity of cardiomyocytes that are subjected to complex regulation of their expression during development. SCN5A mutation was in a form only expressed postnatally, whereas the mutation of KCNQ1 inherited from the father is initially silenced in embryonic development. For these reasons, both mutations were not detectable in immature hiPSC-CMs. When we included patient hiPSC-CMs in microtissues, the expression of both mutations was significantly increased and the functional impact on the electrical activity could be evaluated. Thanks to the flexibility of the microtissue system, we further demonstrated that a specific factor MBNL1 was necessary for the expression of a postnatal form of SCN5A, since maturation effect on SCN5A was absent in hiPSC-CMs lacking MBNL1. These results are now in press in a specialized scientific journal (Campostrini et al. Cardiovascular Research, 2022).
The microtissue model was then used to demonstrate its utility to study heart disease. We used hiPSC-CMs from two patients, carrying a mutation in SCN5A and KCNQ1 gene, respectively. These genes encode for two important ion channels determining the electrical activity of cardiomyocytes that are subjected to complex regulation of their expression during development. SCN5A mutation was in a form only expressed postnatally, whereas the mutation of KCNQ1 inherited from the father is initially silenced in embryonic development. For these reasons, both mutations were not detectable in immature hiPSC-CMs. When we included patient hiPSC-CMs in microtissues, the expression of both mutations was significantly increased and the functional impact on the electrical activity could be evaluated. Thanks to the flexibility of the microtissue system, we further demonstrated that a specific factor MBNL1 was necessary for the expression of a postnatal form of SCN5A, since maturation effect on SCN5A was absent in hiPSC-CMs lacking MBNL1. These results are now in press in a specialized scientific journal (Campostrini et al. Cardiovascular Research, 2022).
SiGNATURE project represented a step forward in the state of the art for modelling the human heart. The 3D cardiac microtissue developed during the project demonstrated several advantages compared to previously available culture systems, the main being:
1) Production of more mature hiPSC-CMs for a broad range of characteristics, particularly mature functionality and postnatal gene expression regulation
2) Accessibility in terms of culture techniques and costs
3) Possibility to use different combinations of patient-derived cells (healthy/diseased) of the three composing cardiac cell types
4) Possibility to be dissociated into single cells which maintain the mature features
The results from the project will positively impact not only heart disease modelling, extending the range of diseases that can be studied, but also drug testing. Large scale production of 3D cardiac microtissues can be implemented by industry for screening of thousands of drugs, whose response will be more reliable because of the closer resemblance with the human heart compared to standard hiPSC-CM cultures. This is expected to contribute establishing hiPSC-derived cells as a preclinical model, which would reduce the time and costs of preclinical trials and the use of animal testing.
1) Production of more mature hiPSC-CMs for a broad range of characteristics, particularly mature functionality and postnatal gene expression regulation
2) Accessibility in terms of culture techniques and costs
3) Possibility to use different combinations of patient-derived cells (healthy/diseased) of the three composing cardiac cell types
4) Possibility to be dissociated into single cells which maintain the mature features
The results from the project will positively impact not only heart disease modelling, extending the range of diseases that can be studied, but also drug testing. Large scale production of 3D cardiac microtissues can be implemented by industry for screening of thousands of drugs, whose response will be more reliable because of the closer resemblance with the human heart compared to standard hiPSC-CM cultures. This is expected to contribute establishing hiPSC-derived cells as a preclinical model, which would reduce the time and costs of preclinical trials and the use of animal testing.