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ERC

NICHOID Report Summary

Project ID: 646990
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - NICHOID (Mechanobiology of nuclear import of transcription factors modeled within a bioengineered stem cell niche.)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

This project confronts a fundamental question: understanding and controlling stem cell differentiation. This problem is important for society because soon we’ll cure disease with a living cell, not a pill, by innovative therapeutic strategies called “stem cell therapies”, which already have led to the development of new biomedical medicines to repair or recover biological functions of damaged tissues and organs. Examples are the use of bone marrow-derived stem cells to slow down neurodegeneration, or to counteract scar formation after spinal cord injury, a heart attack, or chemotherapy. To accelerate discovery in this field, there is a major ongoing effort in the development of advanced culture substrates to be used as “synthetic niches” for the cells, mimicking the native ones. The goal of this project is to use a synthetic niche cell culture model to test my revolutionary hypothesis that in stem cell differentiation, nuclear import of gene-regulating transcription factors is controlled by the stretch of the nuclear pore complexes. If verified, this idea could lead to a breakthrough in biomimetic approaches to engineering stem cell differentiation.
I investigate this question specifically in mesenchymal stem cells (MSC), because they are adherent and highly mechano-sensitive to architectural cues of the microenvironment. To verify my hypothesis, I will use a combined experimental-computational model of mechanotransduction. I will a) scale-up an existing three-dimensional synthetic niche culture substrate, fabricated by two-photon laser polymerization, b) characterize the effect of tridimensionality on the differentiation fate of MSC cultured in the niches, c) develop a multiphysics/multiscale computational model of nuclear import of transcription factors within differentially-spread cultured cells, and d) integrate the numerical predictions with experimentally-measured import of fluorescently-labelled transcription factors.
This project requires the synergic combination of several advanced bioengineering technologies, including micro/nano fabrication and biomimetics. The use of two-photon laser polymerization for controlling the geometry of the synthetic cell niches is very innovative and will highly impact the fields of bioengineering and biomaterial technology. A successful outcome will lead to a deeper understanding of bioengineering methods to direct stem cell fate and have therefore a significant impact in tissue repair technologies and regenerative medicine.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

At the end of the first reporting period, the implementation of the project is successful and globally on time. The project is subdivided into five main activities: 1) fabrication of synthetic niche culture substrates, 2) study of cell differentiation in the niches, 3) fluorescent labelling of transcription factors, 4) computational modelling of diffusion-deformation, and 5) integration of experiments with computations. Our achievements in the context of these activities, within the first reporting period, are the following:
1) we scaled-up the synthetic niche substrate fabricated, by a nanofabrication technique called “two-photon laser polymerisation”, up to the coating of a culture surface of 0.4 squared cm, a result well compatible with our final goal of coating samples of 1 squared cm;
2) we defined the final protocol for the experiments of mesenchymal stem cells culture on our new “nichoid” substrate. The protocols successfully tested are: cell isolation, cell proliferation, phenotype conditioning towards the osteogenic, adipogenic and myogenic lineages. A new culture medium was also formulated, to replace bovine serum with human-derived platelet lysate. We also selected the specific PCR array to test the cell phenotypic expression;
3) we were able to synthesize two fluorescently-labelled transcription factors: MyoD-GFP and ASCL1-GFP. The proteic products were evaluated with spectrometry to evaluate their quality. This activity is now focused on the characterisation of the capability of these proteins to elicit the correct patterns of gene expression in mesenchymal stem cells;
4) we developed two computational models of diffusion-deformation, one at the cellular microscale, and another one interfacing the cell scale to the nuclear pore nanoscale. The models assign local diffusion coefficients to the cell sub-structures, in function of the cell deformation state. The computational predictions were calibrated on experimentally-measured concentrations of a fluorescent DNA-intercalating dye (Dapi), at different cell deformation states controlled by cell adhesion to the “nichoid” substrate;
5) this final activity is scheduled to start later, at project year three; however, in preparation of this activity, we have already tested different protocols for the measurement of the nuclear fluxes of labelled transcription factors, by lasers canning confocal microscopy. We are experiencing technical limitations related to the confocal microscope available at my lab; we will test other instruments available at major imaging facilities, before taking the final decision on what instrument and which protocol to be used for these measurements.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

"Among cell types, Mesenchymal Stem Cells (MSC) raised a great interest for regenerative medicine for many clinical applications such as orthopaedic, plastic and reconstructive surgery and within preclinical studies in autoimmune or degenerative disease treatments and as immune-suppressors in organ transplantation. In stem cell therapy, cells are injected in the patient after an extensive in vitro manipulation aimed at obtaining a sufficient cell number able to guarantee the therapeutic effect. Currently, stem cell "manufacturing" implies the use of “feeder” cells, such as fibroblasts, and other additives from animal sources, hampering the clinical use of the manipulated cells mainly for safety reasons.
In the context of the ERC Consolidator grant (ERC-CoG) that I currently lead, a novel ground-breaking concept for an easy to use, repeatable, cost-effective, and safe substrate for stem cell expansion, capable of avoiding feeder cells and dangerous additives has been introduced. In particular, we have developed and patented an innovative three-dimensional (3D) nano-engineered substrate that mimics the physical containment to cell migration present in the native 3D “niches”, where MSC reside in the body. Currently, we are able to cover with the nichoid a culture surface up to 0.5 squared cm, in 5 hours of continuous serial laser writing. This fabrication speed is perfectly adequate to generate the samples for basic biological research that I use in the ERC-CoG project that generated the invention, but would be inadequate for a mass production of substrates aimed at their commercialisation for stem cell expansion.
To explore the possibility of an economic exploitation of this invention (already covered by an Italian Patent submitted in September 2015 and already extended as PCT), I have submitted a proposal for an ERC Proof-of-Concept grant (ERC-PoC) to perform a technical and commercial feasibility to move the method developed at the PI’s lab during the ERC-CoG grant to the market. In particular, we plan to speed up the maturity level of the technology further advancing the production process of individual nichoids (fabrication up-scaling by at least a factor of 10, allowing to cover a culture surface of 5 squared cm in five hours), setting of an actionable IPR strategy, assessing the market opportunities in view of identifying the suitable exploitation strategy for valorising the patent/know how (licensing/company creation)."

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