Cardiomyocytes generation by programming human pluripotent stem cell fate in microfluidics: from Wnt pathway modulators to synthetic modified mRNA

Cardiovascular disease (CVD) is still one of the major cause of morbidity and mortality in the world, with ischemic heart disease representing the majority of deaths over the past 10 years. The high burden of the disease, both immediate and chronic, associated with the high costs for the healthcare systems, claim for the development of novel therapeutic strategies. The main issue of current pharmacological and interventional therapeutic approaches is their inability to compensate the great and irreversible loss of functional cardiomycytes (CMs). Because of the limited regenerative capacity of post-natal CMs and the difficulty to obtain and isolate heart bioptic tissue, very limited supplies of these cells are available at present for dedicated studies. Moreover, even if animal models are surely the best tool to study and understand in vivo the mechanisms of specific human pathologies in a complex organism, they are not fully predictive and representative of the human condition; from an economic point of view, animal maintenance and the related experimentations are time consuming and very expensive. In this scenario, human pluripotent stem cells (hPSCs), including human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs), play an important role in the cardiovascular research field, because they can be indefinitely expanded in culture without loosing their stemness, and differentiated into cells of the three germ layers, such as CMs. A great breakthrough in science has occurred in 2007 with the discovery of hiPSCs by the Nobel Prize Shinya Yamanaka. This has been the starting point for deriving patient-specific hiPSCs from the reprogramming of somatic cells obtained with less- or non-invasive procedures (skin biopsies, blood, urine…), useful for the generation of tissues for autologous-repair, bypassing the ethical and political debates surrounding the hESCs derivation. The researchers have made several efforts to develop strategies to efficiently direct hPSCs cardiac differentiation and the existing methods for deriving CMs involve stage-specific perturbations of different signaling pathways using growth factors (GFs) or small molecules that recapitulate key steps of the cardiac development observed in vivo. However, these strategies are accompanied by some limitations including: high intra- and inter-experimental variability, low efficiencies, presence of xeno-contaminants, undefined medium components and differences in the expression of cytokines of endogenous signaling pathways. Other strategies are based on the direct lineage conversion of somatic cells, especially fibroblasts, via the overexpression of cardiac transcription factors (TFs) combinations through integrating and non-integrating vectors. However, also these approaches are characterized by low efficiencies, combined with the risk of genomic integration and insertional mutagenesis when using integrating vectors or the need for stringent steps of purification when using non-integrating techniques. Because of the difficulty to specifically direct hPSCs cardiac fate in a robust way, combined with the scarce ability of conventional culture systems to reproduce in vitro, the environment in which cells reside in vivo, the CMs produced to date are immature and more similar to fetal cardiac cells. In 2010, Warren L. and co-workers pioneered a novel, non-integrating strategy based on repeated transfection with cathionic vehicles of synthetic modified messenger RNA (mmRNA), specifically designed to avoid innate immune response from the cell, demonstrating the possibility to both reprogram somatic cells to pluripotency and to programm hPSCs fate into terminally differentiated myogenic cells. Hence, the aim of this PhD thesis is the development of an efficient and robust method for cardiac differentiation of hPSCs by combining the mmRNA with the microfluidic technology. Repeated transfections with mmRNA encoding 6 cardiac TFs are employed to force the endogenous protein expression in the cells and to drive the differentiation toward functional maturation of CMs. The integration of cardiac differentiation within an ad hoc microfluidic platform, facbricated in BioERA laboratory, allows a more precise control of culture conditions, enabling a high mmRNA transfection efficiency, thanks to the high volume/surface ratio, and the in vitro reproduction of physiological niches. In fact, the small scale offered by microfluidics, best mimics the cellular dynamics, which occur in the soluble microenvironment in vivo. Moreover, the microfluidic technology offers the possibility to perform combinatorial, multiparametric, parallelized and highthroughput experiments at one time in a cost-effective manner, not achievable and not economically sustainable in macroscopic conventional culture systems. Chapter 1 starts with the definition of regenerative medicine and introduces the complexity of cardiac development, with the network of TFs that cooperate in this process. The state of the art regarding the derivation of CMs from hPSCs and from the transdifferentiation of somatic cells is described, together with the current limitations and challenges. Finally, the general aim of this PhD thesis is presented. Chapter 2 will focus on hPSCs (hES and hiPS) employed during this project, describing their most important characteristics. It will be also presented a monolayer-based cardiac differentiation protocol of hPSCs that, to date is considered the gold standard for the fast generation of a high yield of beating CMs in conventional culture systems. This protocol relies on the temporal modulation of Wnt pathway via the administration of small molecules. In addition, a hES line, dual reporter for 2 cardiac TFs will be described and always adopted as a tool to monitor the progression of cardiac differentiation. The results obtained in standard cultures will be showed. Chapter 3 will review the state of the art of microfluidic technology for cell culture in regenerative medicine applications. Then, the microfluidic platform fabrication will be described and employed, followed by the optimization of culture, expansion and cardiac differentiation of hPSCs with the gold standard protocol deriving form the translation from macro- to micro-scale. Chapter 4 will introduce the novel mmRNA strategy for reprogramming and programming cell fate: also in this case the state of the art will be discussed. Then, the experimental strategies developed to program cardiac differentiation of hPSCs toward a more mature CM phenotype will be presented, together with the results obtained and the related structural, functional and molecular characterizations. In this work, for the first time, it has been possible to derive CMs from hPSCs with repeated transfections of mmRNA encoding 6 cardiac TFs in microfluidics, with efficiencies higher to current methods described in literature, performed in standard systems. Finally, Chapter 5 will present the general discussion and conclusions, with the future perspectives regarding the use of mmRNA combined with microfluidic technology for deriving different CMs phenotypes, just varying the combination of TFs delivered. To conclude, the experiments developed during this project provide proof-of-principle that it is possible to program hPSCs fate toward cardiac lineage and cardiac maturation in microfluidics; moreover, thanks to the non-integrating characteristic of mmRNA, the CMs obtained are clinical-grade and could potentially be employed in the next future for clinical applications of autologous tissue self-repair and for personalized drug screening.