Periodic Reporting for period 3 - SYNBIO.ECM (SYNBIO.ECM: Designer extracellular matrices to program healthy and diseased cardiac morphogenesis)
Periodo di rendicontazione: 2023-12-01 al 2025-05-31
In this project, we seek to define how extracellular matrix (ECM) components control the behavior of fetal cardiac cells in-vitro. With this knowledge, we seek to program custom-designed ECM mixtures that can induce programmable tissue-level behavior. Specifically, we want to replicate a key aspect of healthy heart development, myocardial trabeculation, and the defects seen in congenital heart disease (CHD).
Why did we decide to focus on CHD? Engineered tissue models are becoming increasingly important for society as they offer more predictive platforms for drug development. In fact, more predictive results than those obtained on traditional in-vitro and animal models were obtained when testing compounds on human cells grown on engineered substrates that mimic native microenvironments. Moreover, this approach is 3R complaint and promises to replace animal models entirely. At present, tho, no engineered platform focuses on CHD, a family of structural disorders that affect 1 in 100 European newborns.
Based on the PI previous work and existing literature in development biology, biophysics, and bioengineering, we hypothesized that three ECM components (agrin, hyaluronic acid, and laminin) and two cell types (fetal cardiomyocytes and endothelial cells) are responsible for myocardial trabeculation.
To test this hypothesis, we will follow a classical engineering bottom-up approach. First, we will develop a high-throughput platform to study ECM-cell interactions using live microscopy. We call it our TestBench and expect it to yield a library of well-characterized parts. Second, we will develop a computer-aided design tool to model ECM-cell interactions based on particle dynamics since we are specifically interested in modeling tissues that change during embryonic development and CHD. And third, we will leverage these well-characterized and computationally-modeled ECM-cell interactions to create designer-ECM that can program healthy and diseased myocardial trabeculation.
Importantly, we believe that the knowledge of the parts and their predicted interactions will help us precisely and mechanistically design engineered CHD in-vitro models, thus introducing new products (CHD in-vitro models) and a new process (programmable tissue models) in cardiac tissue engineering.
Why did we decide to focus on CHD? Engineered tissue models are becoming increasingly important for society as they offer more predictive platforms for drug development. In fact, more predictive results than those obtained on traditional in-vitro and animal models were obtained when testing compounds on human cells grown on engineered substrates that mimic native microenvironments. Moreover, this approach is 3R complaint and promises to replace animal models entirely. At present, tho, no engineered platform focuses on CHD, a family of structural disorders that affect 1 in 100 European newborns.
Based on the PI previous work and existing literature in development biology, biophysics, and bioengineering, we hypothesized that three ECM components (agrin, hyaluronic acid, and laminin) and two cell types (fetal cardiomyocytes and endothelial cells) are responsible for myocardial trabeculation.
To test this hypothesis, we will follow a classical engineering bottom-up approach. First, we will develop a high-throughput platform to study ECM-cell interactions using live microscopy. We call it our TestBench and expect it to yield a library of well-characterized parts. Second, we will develop a computer-aided design tool to model ECM-cell interactions based on particle dynamics since we are specifically interested in modeling tissues that change during embryonic development and CHD. And third, we will leverage these well-characterized and computationally-modeled ECM-cell interactions to create designer-ECM that can program healthy and diseased myocardial trabeculation.
Importantly, we believe that the knowledge of the parts and their predicted interactions will help us precisely and mechanistically design engineered CHD in-vitro models, thus introducing new products (CHD in-vitro models) and a new process (programmable tissue models) in cardiac tissue engineering.
Despite massive COVID delays, the team has progressed well and accomplished a number of intermediate outcomes:
a) We developed a new manufacturing approach to scale the throughput of our TestBench using liquid handling robots instead of 3D bioprinting. We validate the system with various hydrogels and are currently optimizing this setup for the three ECM components needed for this project.
b) We developed a new genetically encoded fluorescent cell cycle indicator (FUCCIplex) and combined it with existing structural (LifeAct) and functional (GCaMP6f and fluo-4) indicators. We genetically engineered these sensors in a human cell line (HaCat) and on the reference hiPSC line from the Gladstone Institute in the USA. These 2- and 4-color reporter hiPSCs are new global reference lines, for which we reached a re-distribution agreement with Gladstone.
c) We also developed dedicated imaging protocols to track all four fluorescent signals during long-term (48 hr) live imaging confocal experiments. Critical steps include imaging at low laser intensities, AI-based image denoising, buffer formulations, etc.
d) We developed a new version of the particle dynamics software SEM++ (Milde, 2014) and recompiled/built it around updated version of the supporting packages, including LAMMPS. We installed it on architectures ranging from personal laptops to high-performance computing workstations and clusters. We then improved upon this version developing new approaches to model cell spreading, migration, and proliferation on adherent substrates. Also, we combined this new version of SEM++ with a novel framework for discrete multiscale mechanics in cells and tissues and called it subcellular element modeling and mechanics, or SEM2.
e) We explored novel ways to solve tissue-mechanics problems in the heart using more classical approaches with partial differential equations (PDE). Here, we leveraged isogeometric analysis (IGA) instead of the classical finite element methods to treat the cardiac electro-mechanical problems in novel and more efficient ways.
We also started creating models of heart development using cardiac organoids and have assembled a light-sheet microscope to track myocardial morphogenesis using the genetically-engineered cells we developed. We will then use the computational methods we introduced to model ECM-cell interactions in complex organoids based on the relationships inferred in the simpler ECM-matched hydrogels.
a) We developed a new manufacturing approach to scale the throughput of our TestBench using liquid handling robots instead of 3D bioprinting. We validate the system with various hydrogels and are currently optimizing this setup for the three ECM components needed for this project.
b) We developed a new genetically encoded fluorescent cell cycle indicator (FUCCIplex) and combined it with existing structural (LifeAct) and functional (GCaMP6f and fluo-4) indicators. We genetically engineered these sensors in a human cell line (HaCat) and on the reference hiPSC line from the Gladstone Institute in the USA. These 2- and 4-color reporter hiPSCs are new global reference lines, for which we reached a re-distribution agreement with Gladstone.
c) We also developed dedicated imaging protocols to track all four fluorescent signals during long-term (48 hr) live imaging confocal experiments. Critical steps include imaging at low laser intensities, AI-based image denoising, buffer formulations, etc.
d) We developed a new version of the particle dynamics software SEM++ (Milde, 2014) and recompiled/built it around updated version of the supporting packages, including LAMMPS. We installed it on architectures ranging from personal laptops to high-performance computing workstations and clusters. We then improved upon this version developing new approaches to model cell spreading, migration, and proliferation on adherent substrates. Also, we combined this new version of SEM++ with a novel framework for discrete multiscale mechanics in cells and tissues and called it subcellular element modeling and mechanics, or SEM2.
e) We explored novel ways to solve tissue-mechanics problems in the heart using more classical approaches with partial differential equations (PDE). Here, we leveraged isogeometric analysis (IGA) instead of the classical finite element methods to treat the cardiac electro-mechanical problems in novel and more efficient ways.
We also started creating models of heart development using cardiac organoids and have assembled a light-sheet microscope to track myocardial morphogenesis using the genetically-engineered cells we developed. We will then use the computational methods we introduced to model ECM-cell interactions in complex organoids based on the relationships inferred in the simpler ECM-matched hydrogels.
Our progress beyond the state of the art (SOTA) currently includes the following items.
1. We used liquid handling automation to create engineered substrates directly in multiwell plate formats. Currently, we are working on downstream processing of these platforms using photofabrication to create the 2D and 3D patterns to potentiate the predictivity of the migration, proliferation, and maturation experiments. We expect these experiments to provide the most comprehensive characterization of the proliferation, migration, and maturation of hiPSC-derived cell types.
2. We engineered a line of hiPSCs that features functional and structural fluorescent reporters in the form of a calcium-sensitive protein (GCaMP) and a fluorescent tag to a cytoskeletal protein (actin). Moreover, we added a cell cycle sensor, effectively creating a new reference line for the field. We have also secured a re-distribution agreement with Gladstone so that other groups can access the line.
3. We introduced SEM2 as the new standard particle dynamics software to study cell and tissue morphogenesis and mechanics. We are now introducing a novel treatment of the cell nucleus to be able to model its changes in shape as well as their effect on cellular mechanics. We expect to use the current software version to model cells and tissues in 2D and 3D photo-patterns and to introduce particles whose dynamics model changes in the cytoskeletal organization.
4. Together with colleagues in the department, we developed IGA-based approaches for the cardiac electromechanical problem that haven’t been presented before.
5. We created cardiac organoids following recently published protocols but using the 2- and 4-color reporter lines to study migration, proliferation, and maturation directly in an in-vitro platform that recapitulates heart development.
1. We used liquid handling automation to create engineered substrates directly in multiwell plate formats. Currently, we are working on downstream processing of these platforms using photofabrication to create the 2D and 3D patterns to potentiate the predictivity of the migration, proliferation, and maturation experiments. We expect these experiments to provide the most comprehensive characterization of the proliferation, migration, and maturation of hiPSC-derived cell types.
2. We engineered a line of hiPSCs that features functional and structural fluorescent reporters in the form of a calcium-sensitive protein (GCaMP) and a fluorescent tag to a cytoskeletal protein (actin). Moreover, we added a cell cycle sensor, effectively creating a new reference line for the field. We have also secured a re-distribution agreement with Gladstone so that other groups can access the line.
3. We introduced SEM2 as the new standard particle dynamics software to study cell and tissue morphogenesis and mechanics. We are now introducing a novel treatment of the cell nucleus to be able to model its changes in shape as well as their effect on cellular mechanics. We expect to use the current software version to model cells and tissues in 2D and 3D photo-patterns and to introduce particles whose dynamics model changes in the cytoskeletal organization.
4. Together with colleagues in the department, we developed IGA-based approaches for the cardiac electromechanical problem that haven’t been presented before.
5. We created cardiac organoids following recently published protocols but using the 2- and 4-color reporter lines to study migration, proliferation, and maturation directly in an in-vitro platform that recapitulates heart development.