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Mechanobiology of nuclear import of transcription factors modeled within a bioengineered stem cell niche.

Periodic Reporting for period 4 - NICHOID (Mechanobiology of nuclear import of transcription factors modeled within a bioengineered stem cell niche.)

Período documentado: 2019-11-01 hasta 2020-07-31

Problem/issue being addressed
Regenerative medicine aims to repair and recover the biological function of damaged tissues and organs often using stem cells. However, this requires the development of culture substrates that mimic the native environment of stem cells and can support their expansion and differentiation. Polymeric scaffolds are emerging as synthetic 3D environments capable of influencing cell growth and proliferation, together with feeder layers or other biological compounds that are employed to maintain the phenotype of stem cells. To overcome safety concerns associated with the use of such additives and feeder layers, the ERC NICHOID project developed a polymeric scaffold with grid architecture using a microfabrication technology called two-photon laser polymerisation. My approach led to the development of the user-friendly and safe Nichoid substrate.

Overall achievements of the project
The Nichoid was tested on mesenchymal stem cells (MSCs) known for their regenerative capacity in orthopaedic, plastic and reconstructive surgery applications, as well as for their immune-suppressive potential in organ transplantation. They are also sensitive to mechanical stimuli from the micro-environment. Mechanical cues transduced within cells are believed to determine cell fate as they alter nuclear shape and hence the import and export of transcription factors that are paramount for regulation of gene expression. With the help of colleagues at the Politecnico di Milano and at the National Research Council of Italy, researchers observed a roundish nuclear configuration in MSCs adhered to the Nichoid microscopic grid, while a spread configuration was seen in cells attached to the flat substrate surrounding the grid. The latter morphology coincided with an increase in nuclear permeability and in the flow of signalling molecules that induce cell differentiation. The Nichoid essentially prevents cell spreading during proliferation by mechanically confining cells in the grid, thus avoiding this master switch towards differentiation. The observation that, in stem cells, nuclear import of transcription factors activating cell differentiation is regulated primarily by nuclear membrane strains was also supported through computational prediction of nuclear import flow as a function of cell morphology.

Importance for society
This geometric control of cell stemness opens an avenue towards reprogramming multipotency or even pluripotency in cultured stem cells, without the need of chemical agents or genetic modifications. The nichoid led to the acquisition of two ERC-PoC grants that led to development of products for biological research integrating the nichoid culture substrate, already into commercialisation. Most importantly, in light of the current COVID-19 pandemic, Raimondi plans to promote the Nichoid substrate for the expansion of MSCs to cure COVID-19, based on a new therapeutic strategy already in clinical trial in China and in the United States. Moreover, she will use all the advanced research tools developed during the NICHOID project to speed up the preclinical testing necessary to bring new antiviral therapies and vaccines to the clinics.
With the help of colleagues at the National Research Council of Italy, we up-scaled the substrate Nichoid quite effectively. We discovered that the Nichoid essentially prevents cell spreading during proliferation by mechanically confining cells in the grid, thus avoiding the master switch towards differentiation. We were able to label cells in order to visualise the nuclear import of the MyoD transcription factor. The observation that in stem cells, nuclear import of transcription factors activating cell differentiation is regulated primarily by nuclear membrane strains was also supported through computational prediction of nuclear import flow as a function of cell morphology. The cell spread morphology coincided with an increase in nuclear permeability and in the flow of signalling molecules that induce cell differentiation, thus providing the validation of the main working hypothesis posed in the project. We plan to link this permeability increase to the nuclear pore complex mechanics in future work. Our achievements in the context of the project activities were reported on several scientific publications and at the major international conferences in the field. I also won two ERC-PoC grants that allowed me to trasfer the nichoid invention developed in this project into a commercial product.
The novel and unconventional methodologies developed within the project are the following.
1) up-scaling of a 3D microstructure, nanofabricated with a spatial resolution down to 100 nm by two-photon polymerization, up to covering a surface in the order of squared cm is a quite unconventional goal and required a strong technological and inventive effort in order to be accomplished.
2) study of cell differentiation in the Nichoid required the development of unconventional protocols to transfer from 2D plastic to a 3D microarchitecture all the necessary cell manipulation and analytical techniques; fluorescence diagnostics, especially, required the development of unconventional techniques for the quantification of the fluorescence intensity in the 3D Nichoids populated with living cells.
3) We developed modified transcription factors that were fluorescently-labelled, rendered able to pass the plasma membrane, diffuse inside the cell nucleus and correctly activate gene transcription; this is an unconventional approach compared to more conventional gene transfection approaches.
4) Computational modelling in this project is based on conventional multiphysics techniques coupling structural mechanics and mass transport models. However, here we defined features of the geometry and mechanical behaviour of the nuclear pore complex based on data measured at the nanoscale, on cells of spread versus roundish morphology, by electron tomography; this approach is very innovative and has never been applied before in the context of nuclear pore complex modelling.
5) In this project, the nuclear import of transcription factors was measured in living cells in the aim to validate a computational model of cell diffusion/deformation. The conventional techniques to quantify intra-cellular transport of fluorescent proteins are 2D in their logic, having been developed for highly spread cells adhered to 2D substrates. We developed innovative methodologies to apply these techniques to live cells adhering inside the 3D lattice of the Nichoid. Estimation of the nuclear import of fluorescent tracers in 3D-cultured cells is a very novel and unconventional field of application of the existing techniques to measure trans-membrane fluxes in live cells.
Image summarising the concept of ERC NICHOID