Periodic Reporting for period 3 - GHOSTS (Genetically enhanced, optically superior tissues (GHOSTS))
Reporting period: 2023-02-01 to 2024-07-31
With our GHOSTs project, we aim to improve the optical properties of living cells and biological tissues to improve optical microscopy of biological structure and dynamic processes that define the living state.
Light microscopy has always been an essential tool to biologists to understand how cells and tissue are organized and how organisms emerge from a fertilized egg during development. Equally, light microscopes are an indispensable in medical research and to understand which and how pharmaceutical compounds may heal certain disease. And while optical microscopes have been engineered to perfection, most medically relevant tissue models are opaque, largely preventing access by microscopy. Knowing about physiological transparency genes will allow unprecedented insights into living tissues. If model tissues in the lab were just 5% as transparent as some glass-like fish found in the deep sea, optical microscopes could unleash their full potential, and enable high resolution views into developmental processes in their native environment. We see further transformative potential especially in the fields of organotypic tissue models, functional brain imaging, as well as pharmaceutical screens in 3D tissue cultures.
We aim to create GHOSTs, meaning Genetically enhanced Optically Superior Tissues.
More specifically, we intend to i) improve the optical properties of biological living cells (proof of concept demonstrated, see figure), and ii) show improved optical microscopy in cleared living mammalian tissues.
Light microscopy has always been an essential tool to biologists to understand how cells and tissue are organized and how organisms emerge from a fertilized egg during development. Equally, light microscopes are an indispensable in medical research and to understand which and how pharmaceutical compounds may heal certain disease. And while optical microscopes have been engineered to perfection, most medically relevant tissue models are opaque, largely preventing access by microscopy. Knowing about physiological transparency genes will allow unprecedented insights into living tissues. If model tissues in the lab were just 5% as transparent as some glass-like fish found in the deep sea, optical microscopes could unleash their full potential, and enable high resolution views into developmental processes in their native environment. We see further transformative potential especially in the fields of organotypic tissue models, functional brain imaging, as well as pharmaceutical screens in 3D tissue cultures.
We aim to create GHOSTs, meaning Genetically enhanced Optically Superior Tissues.
More specifically, we intend to i) improve the optical properties of biological living cells (proof of concept demonstrated, see figure), and ii) show improved optical microscopy in cleared living mammalian tissues.
We followed two conceptionally different approaches in parallel. In these, we aim to improve optical properties of cells (published), and tissue spheroids (ongoing work).
By directed evolution (published in Subramanian et al, J BioPhot 2021), we managed to make living cells considerably more transparent while preserving their physiological state. Specifically, we generated numerous cell lines with improved optical properties (i.e. with reduced side scattering, SSC-A = Integrated side scattering signal per cell) using FACS technology (compare figure 1). Sequencing and computational transcriptome enabled us to find multiple pathways frequently associated with changes in optical properties.
After successful selection of cells with improved optical properties, we performed competitive growth assays to select by fitness such that an heterogenous population of evolved cells was exposed to repeated cell stress, i.e. to freeze–thaw and expansion cycles. This process counter-selected many, but not all, low-scattering cells. A plurality of single-cell origin cell lines from these experiments were sequenced individually. This data revealed that evolved cells fall into transcriptomically distinct groups, that are significantly different not only from the WT cells but also form the mutated, but not optically selected cells. Further, our selection by fitness was indeed successful as also transcriptome reflected a physiological state. That is the mRNA content did not show elevated levels of increased cell stress or apoptosis. Specifically, the GO term ‘repones to stress’ was not found significantly upregulated and there was no strong difference in the expression of important housekeeping genes.
We hypothesized, that improvements in the optical properties of a cell were associated with nuclear architecture or altered chromatin compaction states. Using high resolution microscopy, we indeed observed that nuclear granularity showed a distinct correlation with side scattering. Cell lines showing least light scattering also possessed the lowest granularity.
These results established that a modulation of nuclear architecture of cells in culture, and potentially also in cell spheroids, as a highly promising way to improve their optical properties. Specifically, chromatin compaction states seem a relevant parameter in this regard.
The above results successfully demonstrated a significant optical plasticity of mammalian cells.
This further motivated us to i) get better understanding of the pathways involved, ii) find ways to leverage modern genetics to improve optical properties iii) transition from random mutation to gain of function phenotypes. The finding motivates more systematic screens for genes and pathways affecting these (currently ongoing work).
By directed evolution (published in Subramanian et al, J BioPhot 2021), we managed to make living cells considerably more transparent while preserving their physiological state. Specifically, we generated numerous cell lines with improved optical properties (i.e. with reduced side scattering, SSC-A = Integrated side scattering signal per cell) using FACS technology (compare figure 1). Sequencing and computational transcriptome enabled us to find multiple pathways frequently associated with changes in optical properties.
After successful selection of cells with improved optical properties, we performed competitive growth assays to select by fitness such that an heterogenous population of evolved cells was exposed to repeated cell stress, i.e. to freeze–thaw and expansion cycles. This process counter-selected many, but not all, low-scattering cells. A plurality of single-cell origin cell lines from these experiments were sequenced individually. This data revealed that evolved cells fall into transcriptomically distinct groups, that are significantly different not only from the WT cells but also form the mutated, but not optically selected cells. Further, our selection by fitness was indeed successful as also transcriptome reflected a physiological state. That is the mRNA content did not show elevated levels of increased cell stress or apoptosis. Specifically, the GO term ‘repones to stress’ was not found significantly upregulated and there was no strong difference in the expression of important housekeeping genes.
We hypothesized, that improvements in the optical properties of a cell were associated with nuclear architecture or altered chromatin compaction states. Using high resolution microscopy, we indeed observed that nuclear granularity showed a distinct correlation with side scattering. Cell lines showing least light scattering also possessed the lowest granularity.
These results established that a modulation of nuclear architecture of cells in culture, and potentially also in cell spheroids, as a highly promising way to improve their optical properties. Specifically, chromatin compaction states seem a relevant parameter in this regard.
The above results successfully demonstrated a significant optical plasticity of mammalian cells.
This further motivated us to i) get better understanding of the pathways involved, ii) find ways to leverage modern genetics to improve optical properties iii) transition from random mutation to gain of function phenotypes. The finding motivates more systematic screens for genes and pathways affecting these (currently ongoing work).
We showed for the first time, that living cells can be made considerably more transparent by directed evolution (published). We have newly established:
i) A method for improved cellular transparency without stress and apoptotic responses.
ii) Transparent cell lines cluster into transcriptomically distinct groups with significant impact on molecular pathways.
iii) Evolved transparency frequently goes along with the phenotypic reduction of nuclear granularity.
iv) Nuclear inversion, which is responsible for retina transparency, does not depend on the fluid state of chromatin.
In the next steps, we aim to better understand which pathways are involved in modulating the optical properties of cells and tissues, for which we set up sophisticated tissue assays and screening protocols.
i) A method for improved cellular transparency without stress and apoptotic responses.
ii) Transparent cell lines cluster into transcriptomically distinct groups with significant impact on molecular pathways.
iii) Evolved transparency frequently goes along with the phenotypic reduction of nuclear granularity.
iv) Nuclear inversion, which is responsible for retina transparency, does not depend on the fluid state of chromatin.
In the next steps, we aim to better understand which pathways are involved in modulating the optical properties of cells and tissues, for which we set up sophisticated tissue assays and screening protocols.