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Human pluripotent stem cell-based microtechnologies for in vitro modeling of cardiac diseases
Get PDFHuman pluripotent stem cells are quickly emerging as a fundamental tool for in vitro studies. In particular, the advent of “induced pluripotency” opened completely new horizons for in vitro disease modeling and patient-specific disease-on-a-dish therapeutic approach screening. The easy access to cell types of human origin hardly available otherwise, with virtually infinite amounts in a donor-unrestricted manner, unlocked in vitro studies for human tissues such as brain, pancreas and the heart. In the latter case, the need for new models of human cardiac physiology and physiopathology is highlighted by the severe fallouts of heart conditions on worldwide health and economy.
The main focus of this thesis are human cardiomyocytes derived through differentiation of pluripotent stem cells, and their application as an in vitro model of the human cardiac tissue. In particular, the stress point of the work is their early and immature phenotype, that often limits their application and frustrates the potential of a human heart model in a Petri dish.
After introducing the current scenario of study models for heart diseases and describing the main features of human pluripotent stem cells (hPSCs) and their cardiac derivatives (hPSC-CMs), this thesis will separately focus on the two main aspects of the cardiomyocyte physiology: structural and functional features and metabolic profile. From these perspectives, human cardiomyocytes derived from hPSCs display in vitro an early and immature phenotype, closely resembling cardiomyocytes at early stage of the development, such as fetal cardiomyocytes.
Cell ultrastructural organization and functional performance are two strictly related features that find in adult cardiomyocytes perfect synthesis, with a very specialized function performed through a finely orchestrated sequence of events hugely relying on the right spatial distribution of key molecular components. In Chapter 2, biomaterials and microengineered substrates are employed to address the molecular mechanisms triggering cardiac maturation in vitro, in order to provide insight in the process and drive hPSC-CMs towards more adult-like phenotypes, better suiting disease modeling and drug screening.
Cardiac metabolism is likewise a characterizing feature of the tissue supporting in a unique fashion the impressive workload of the heart. In Chapter 3, hPSC-CM metabolism is described and a novel microfluidic technology is developed for metabolic maturation screening of cardiac cultures. With this approach, hPSC-CMs are shown to positively respond to an optimized metabolic maturation protocol, similar to the very rapid fetal-to-adult metabolism switch in hCMs after birth in response to changing metabolite availability.
Finally, in perspective of the maturation approaches previously described and their feasible application to human cardiac cultures, in Chapter 4 are discussed two human genetic diseases affecting the heart muscle. For both Duchenne’s muscular dystrophy and arrhythmogenic right ventricular cardiomyopathy/dysplasia, cardiac cellular models are set up and proven to display in vitro the molecular hallmarks of the disease, thus providing the biological substrate for further studies on human cardiomyocyte cultures
Contraction force of single-cardiomyocyte evaluated through the use of microstructurated elastic substrates combined with the study of calcium dynamics
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Extracellular phosphoprotein regulation is affected by culture system scale-down
Get PDFBackground: Phosphorylated proteins are known to be present in multiple body fluids in normal conditions, and abnormally accumulated under some pathological conditions. The biological significance of their role in the extracellular space has started being elucidated only recently, for example in bone mineralization, neural development, and coagulation. Here, we address some criticalities of conventional culture systems for the study of the extracellular regulation of phosphorylation. Methods: We make use of microfluidics to scale-down the culture volume to a size comparable to the interstitial spaces occurring in vivo. The phosphoprotein content of conditioned media was analyzed by a colorimetric assay that detects global phosphorylation. Results: We found that miniaturization of the culture system increases phosphoprotein accumulation. Moreover, we demonstrated that in conventional culture systems dilution affects the extent of the phosphorylation reactions occurring within the extracellular space. On the other hand, in microfluidics the phosphorylation status was not affected by addition of adenosine triphosphate (ATP) and FAM20C Golgi Associated Secretory Pathway Kinase (FAM20C) ectokinase, as if their concentration was already not limiting for the phosphorylation reaction to occur. Conclusions: The volume of the extracellular environment plays a role in the process of extracellular phosphorylation due to its effect on the concentration of substrates, enzymes and co-factors. General significance: Thus, the biological role of extracellular phosphoregulation may be better appreciated within a microfluidic culture system
Human pluripotent stem cell derived hepatocyte-like cells integrated in microfluidic platform for drug screening applications
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