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  • Final Report Summary - TIME TO MATURE (Design and validation of a conductive polymer-based system for the functional maturation of human pluripotent stem cell derived cardiomyocytes as a platform for drug testing)

Final Report Summary - TIME TO MATURE (Design and validation of a conductive polymer-based system for the functional maturation of human pluripotent stem cell derived cardiomyocytes as a platform for drug testing)

In spite of the tremendous effort focused on their prevention and treatment, cardiovascular diseases remain the number one health concern worldwide. These diseases impose such an enormous burden on the affected tissue that neither endogenous compensatory mechanisms nor current pharmacological intervention are able to provide long-lasting support, making organ transplantation the only final option for treating the disease. Thus, finding a truly curative therapy for cardiovascular disease is one of the main challenges of modern medicine. However, despite the increasing commitment towards the development of new pharmacological treatments, it has been extremely difficult to find new effective cardiovascular drugs. Currently established in vitro and in vivo tests suffer from a high attrition rate and unexpected side effects. This is mainly because in vitro models lack the adequate complexity of a living tissue, relying on single cell types that inevitably lose functionality, whereas in vivo animal tests raise questions of translatability to humans and bear ethical issues.
TIME TO MATURE aimed to develop a new in vitro model that replicates the features of the native cardiac tissue for drug screening purposes. Only through the implementation of cutting edge interdisciplinary methodology can this question be solved. As a consequence, we aimed to fabricate a system blending the newly developed conductive polymers (CP) and differentiated cardiovascular phenotypes from human pluripotent stem cells (hPSC). With this platform hPSC-derived cardiomyocytes (CM) would be matured, thus making them fully equivalent to their natural adult counterpart. This would provide a new and accurate system for testing new drugs for treating cardiac diseases, increasing the effectiveness of the current methodology, decreasing R&D costs and accelerating discovery of new therapies. This would be achieved by progressing through the following aims:
1. Development and characterization of a CP-based cardiac niche, a worldwide novelty in tissue engineering (TE) and stem cell biology.
2. Generation of a new protocol for the differentiation and isolation of chamber (atrial or ventricular)-specific CM from hPSC.
3. Establishment of a maturation regimen to convert hPSC-CM into phenotypically and functionally adult cells, which is extremely relevant for disease modeling and drug discovery.
4. Validation of the envisioned system for drug screening purposes and proof of concept of its validity as a tool in personalized medicine.
Traditionally, cells selected for such studies are either of rodent origin (HL-1 mouse atrial CM or rat H9c2) or transformed human HEK-293 cells. This is due to the scarcity of human samples from where to obtain primary CM, but also to the rapid dedifferentiation these cells undergo upon culture, reversing to an immature phenotype. The hPSC field has boosted the expectations of patients and scientists alike. hPSC, including embryonic (hESC) and induced pluripotent stem cells (hiPSC), are able to provide the unlimited source of CM as needed by the pharmacological industry. In addition, they are capable of recapitulating patient-specific diseased features, allowing the development of personalized and precision medicine approaches. However, they have not yet been integrated into the drug research pipeline. The main reasons for this are our inability to recreate their natural adult phenotypes as well as the incapability of obtaining pure populations of cells. Indeed, hPSC-CM grown so far display severely immature features. Testing compounds in such heterogeneous cell mixtures is far from adequate and highlights the need for controlled disease-modeling.
TIME TO MATURE was successful overall in achieving its aims. On the biological side, we have managed to set up, optimize and characterize a CM-differentiation protocol from 5 hPSC lines, performing an extensive genetic profile including markers differently regulated during cardiac chamber specification. This provides novel insights on how this set of genes is expressed alongside hPSC differentiation. Additionally, we have designed and produced novel molecular biology tools that will enable the specific direction of hPSC-derived CM towards the atrial or the ventricular fate. Our aim was to provide a far better method of obtaining atrial/ventricular CM as it (1) is more tightly regulated, (2) does not rely on antibiotic selection, (3) produces homogeneous populations and (4) avoids any confounding effects related to the mixed expression of MLC2v in the atria and MCL2a in the ventricle, two traditional markers for ventricular and atrial myocytes and originally selected at the project conception phase.
Work on the material side has opened unexpected avenues of research and has yielded extremely interesting results. We have evaluated the initial candidate, PANi doped with phytic acid. However during this period a fantastic unexpected development has been achieved in the production of an array of conductive protein-based hydrogels that can be doped and turned electrically conductive! We have now conducted an extensive biocompatibility analysis for several candidates of such protein-based hydrogels with extremely positive results. We found that selected substrates support the growth and function of primary cardiac cells (neonatal rat ventricular myocytes) and most importantly of hPSC-derived CM. What is more, we performed an extensive characterization of the influence of the new hydrogel on hPSC-CM at several levels, including a study of functionality. In the final stage, we have interfaced our materials with 2 hiPSC lines: one derived from a patient suffering from a genetic form of hypertrophic cardiomyopathy and a familial healthy control. This has provided critical information on the applicability of these hydrogels for personalized and precision medicine applications. All of this new information will be submitted for publication shortly.
The main results of the present project are the following:
1. Characterization of hPSC-CM differentiation.
2. Generation of molecular biology tools and production of transgenic hPSC lines.
3. Biocompatibility testing of PANi-phytic acid platform.
4. Biocompatibility, gene expression and functional characterization of protein-based hydrogel substrates.
5. Full characterization of the hydrogel-hPSC-CM interaction at gene/protein expression and functional levels.
6. Incorporation of multiple stimuli driving CM maturation: stiffness, adhesion motif, electrical stimulation.
7. Proof of concept of our drug testing platform with healthy and diseased hiPSC-CM.

Therefore, we revealed that protein-based hydrogels offer exciting possibilities as substrates for CM-based studies. Not only we are keeping in line with the use of a conductive substrate, but the new one has surpassed our initial objectives of the project, especially regarding the stiffness of the substrate.
Excitingly, the use of a protein-based hydrogel has paved the way for the development of advanced new strategies where a patient’s proteins and cells are jointly employed to develop a truly personalized tissue engineered drug testbed. It is envisioned that this kind of approaches will increase the efficacy of the drug development pipeline, thus decreasing costs and risks, and at the same time having a huge impact on the large population of cardiovascular patients waiting for new and better treatments. This falls absolutely in line with the aims of TIME TO MATURE and provides a solid base for the development of the next career move.
Over the course of this project, the researcher has gained substantial experimental experience, learning more than a dozen new interdisciplinary techniques. Furthermore, he has mentored 5 PhD and Masters students, and has been invited to give 2 talks at the British Heart Foundation Cardiovascular Regenerative Medicine Centre. These experiences greatly contributed to his career development and opened new connections with international scientists.
Overall the project TIME TO MATURE has been very successful and can act as a platform for future activities in this field.

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