Periodic Reporting for period 1 - NEVADA (Novel microengineered environments for mouse embryonic stem cell (mESC) differentiation towards cardiomyocytes)
Reporting period: 2018-09-01 to 2020-08-31
Many forms of heart disease are associated with a decrease in the number of functional cardiomyocytes (CMs). Since the proliferative capacity of CMs decreases significantly in postnatal life, elucidation of the molecular mechanisms that govern cell pluripotent state and lineage commitment in CM renewal bring an additional avenue for therapeutic interventions and disease modeling. Mouse embryonic stem cells (mESCs) constitute an excellent model system for studying these mechanisms due to the availability of various protocols for differentiation and the relative simplicity for genetic manipulation. However, heterogeneity during mESC differentiation has posed a particular problem in the formation of lineage-specific cell populations.
Most of the currently employed approaches in cell- and tissue-based engineering studies still involve two-dimensional (2D) surfaces, or monolayer cell cultures, that offer unnatural growth kinetics and cell attachments. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic three-dimensional (3D) microenvironments. 3D synthetic microscaffolds, fabricated by two-photon polymerization (2-PP) photolithography, offer favorable cell responses due to tunable chemical, physical and mechanical properties. 2-PP technology allows the fabrication of volumetric structures of arbitrary shape by directly writing the intended geometry within a photosensitive material. Due to the unprecedented flexibility of 2-PP, matrix architecture and pore size can be controlled with a nanometer resolution. Indeed, none of the currently available microfabrication approaches has been able to investigate cell mechanical properties in 3D with the accuracy of 2-PP photolithography. Chemically defined media combined with the 3D architectures, which more accurately resemble the extracellular environment, offer a powerful tool to mimic specific tissues in vitro.
The scientific objective for the project was to establish an advanced cardiomyocytes model to study mESC cardiac differentiation in a biomimetic three-dimensional (3D) environment. Two goals were proposed for this project:
• Design of a 3D tailored conductive microscaffold to facilitate spatial cell spreading and to achieve precise stimulation patterns to individual cells in vitro.
• Precise quantification of the molecular mechanisms underlying mESC pluripotent state and cardiac differentiation under mechanical and/or electrical stimulation.
Most of the currently employed approaches in cell- and tissue-based engineering studies still involve two-dimensional (2D) surfaces, or monolayer cell cultures, that offer unnatural growth kinetics and cell attachments. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic three-dimensional (3D) microenvironments. 3D synthetic microscaffolds, fabricated by two-photon polymerization (2-PP) photolithography, offer favorable cell responses due to tunable chemical, physical and mechanical properties. 2-PP technology allows the fabrication of volumetric structures of arbitrary shape by directly writing the intended geometry within a photosensitive material. Due to the unprecedented flexibility of 2-PP, matrix architecture and pore size can be controlled with a nanometer resolution. Indeed, none of the currently available microfabrication approaches has been able to investigate cell mechanical properties in 3D with the accuracy of 2-PP photolithography. Chemically defined media combined with the 3D architectures, which more accurately resemble the extracellular environment, offer a powerful tool to mimic specific tissues in vitro.
The scientific objective for the project was to establish an advanced cardiomyocytes model to study mESC cardiac differentiation in a biomimetic three-dimensional (3D) environment. Two goals were proposed for this project:
• Design of a 3D tailored conductive microscaffold to facilitate spatial cell spreading and to achieve precise stimulation patterns to individual cells in vitro.
• Precise quantification of the molecular mechanisms underlying mESC pluripotent state and cardiac differentiation under mechanical and/or electrical stimulation.
Towards the achievement of NEVADA’s goals, we presented several designs of 3D microscaffold arrays, fabricated by 2-PP photolithography, to mimic tissue-specific architecture, enabling cell-to-matrix interaction and cell-to-cell communication in vitro. To prove that cell culture in 3D microscaffold arrays, better mimics the in vivo states and thus maintain a more homogenous cell population, we used a model of pre-implantation embryos, in which pluripotent mESC populations were maintained under controlled culture conditions. We performed several experiments to demonstrate that 3D microscaffolds reinforce the pluripotency gene expression program. First, we used light microscopy to assess the morphology of the mESC colonies growing on 2D and 3D scaffolds. Second, we used alkaline phosphatase (AP) staining to prove that cell colonies are expressing stem cell marker. Finally, to support our preliminary observations, we performed quantitative immunofluorescence analysis of three key pluripotency markers - NANOG, OCT4 and ESRRB. Our data demonstrated that in comparison to 2D surface, mESCs in 3D microscaffold arrays exhibit enhanced expression levels of these three pluripotency markers and a homogenous expression of two pluripotency markers - NANOG and ESRRB. We thus hypothesized that mESCs in 3D microscaffold arrays might have a stronger self-renewal ability resembling more closely the pre-implantation embryos.
NEVADA project has achieved some of its objectives and milestones. It has faced disruptions and delays because of COVID containment measures. Due to the coronavirus epidemic, unprecedented actions have been undertaken against COVID-19 at ETH Zurich. This has greatly impact experimental lab-based research activity and have brought a massive disruption to our project.
NEVADA project has achieved some of its objectives and milestones. It has faced disruptions and delays because of COVID containment measures. Due to the coronavirus epidemic, unprecedented actions have been undertaken against COVID-19 at ETH Zurich. This has greatly impact experimental lab-based research activity and have brought a massive disruption to our project.
The primary outcome of the project is a prototype - 2PP-MICRO_SLIDE. The slide took the advantages of 3D microscaffold array to mimic architectural aspects of tissue and ensure the uniform distribution of nutrients, chemical cues and oxygen in vitro. It has the necessary infrastructure for long-term cell culture and is accessible for in situ high-resolution, real-time microscopic observation by immunofluorescence imaging due to geometry of 3D microscaffolds, transparent materials and size of a standard microscopy slide. As a second generation of the slide, we developed a modular and low cost microfluidic chip-based platform - 2PP-MICRO_CHIP - for parallel molecular and cellular analysis of several cell populations. This significantly increases the operational advantages for the end-user (e.g. subjecting several cell population to a complex experimental protocol with a decreased reagent consumption) while retaining cost-effectiveness and ease of fabrication.
The architectural concepts, that have been developed within NEVADA, are the basis for creating more complex structural designs. The design of 3D tailored conductive scaffolds and the array dimensions can be easily adapted to any cell type. A customized design of 3D scaffolds will only involve a 3D computer model development using a computer-aided design program (Solidworks Corp., USA). Due to unprecedented flexibility of the 3D patterning approach and superior advantages in terms of material characteristics, the entire manufacturing process can be performed in a very time-effective manner. Thus, the technology is widely applicable to study other biological systems for which 3D environment is of crucial importance for functioning. It will be possible to use them in:
• Basic & Applied Research that aims to increase the understanding of early mammalian development. Potential applications: induced pluripotent stem cells, human embryonic stem cells and cancer modeling.
• Translational Research focused on the development of treatment protocols and boost rapid transition of new scientific findings into clinical application. Potential applications: pharmaceutical compound libraries studies, drug discovery and screening with decrease reagent consumption.
• Pharma & Biotech companies for improving drug selection process for personalized medicine. Potential applications: screening of drug agents for patient-derived tumor cells, biomarker-based testing and personalized pharmacological therapies for degenerative diseases.
Therefore, NEVADA project affords a unique and irreplaceable tool to model human diseases and study mammalian development in a systematic and automated manner. The new concept explored in this project results in advanced cell culture, live imaging and manipulation tool on the Regenerative Medicine and Microfluidics markets.
The architectural concepts, that have been developed within NEVADA, are the basis for creating more complex structural designs. The design of 3D tailored conductive scaffolds and the array dimensions can be easily adapted to any cell type. A customized design of 3D scaffolds will only involve a 3D computer model development using a computer-aided design program (Solidworks Corp., USA). Due to unprecedented flexibility of the 3D patterning approach and superior advantages in terms of material characteristics, the entire manufacturing process can be performed in a very time-effective manner. Thus, the technology is widely applicable to study other biological systems for which 3D environment is of crucial importance for functioning. It will be possible to use them in:
• Basic & Applied Research that aims to increase the understanding of early mammalian development. Potential applications: induced pluripotent stem cells, human embryonic stem cells and cancer modeling.
• Translational Research focused on the development of treatment protocols and boost rapid transition of new scientific findings into clinical application. Potential applications: pharmaceutical compound libraries studies, drug discovery and screening with decrease reagent consumption.
• Pharma & Biotech companies for improving drug selection process for personalized medicine. Potential applications: screening of drug agents for patient-derived tumor cells, biomarker-based testing and personalized pharmacological therapies for degenerative diseases.
Therefore, NEVADA project affords a unique and irreplaceable tool to model human diseases and study mammalian development in a systematic and automated manner. The new concept explored in this project results in advanced cell culture, live imaging and manipulation tool on the Regenerative Medicine and Microfluidics markets.