Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - NICHOID (Mechanobiology of nuclear import of transcription factors modeled within a bioengineered stem cell niche.)

Teaser

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...

Summary

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

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 cell diffusion-deformation, and 5) integration of experiments with computations. The implementation of the project is successful and globally on time. Our achievements in the context of these activities are reported below.
1) In the first half of the project, we have been able to pass from samples with a few nichoids, to 0.5 square centimetres of machined surface in half a day, equivalent to around eight thousands nichoids. This surface is adequate for cell culture and allows to fabricate a sample for our research in 12 hours of continuous laser writing, a fabrication time that can be efficiently managed by running the laser fabrication overnight. We managed to achieve this result by subdividing polymer shrinkage on matrices of a limited number of individual niches (e.g. 5x5), instead of fabricating a unique larger matrix covering the whole culture surface. In the field of 2PP, this invention represents a very innovative technological solution, for which we deposited a patent application four months after the project started. We are now able to fabricate all the samples that are necessary for the cell cultures within the project, but also of additional samples that are being employed to expand the application of the nichoid substrate towards some inter and cross-disciplinary developments, as described below. Hereon, I will use the term “nichoid” to refer to our nanofabricated artificial niche substrate. We fabricated all the additional samples that are being employed to expand the application of the nichoid substrate towards some inter and cross-disciplinary developments.
2) Our results so far demonstrate that rat bone marrow mesenchymal stem cells (rBM-MSCs) adhere and grow effectively in the nichoid. The 3D substrate has pronounced effects on cell structure, nuclear dimensions and cytoskeletal organization. Most importantly, the nichoid substrate up-regulates the expression of stemness markers, of cytokines and growth factors by stimulating their autocrine and paracrine secretion by cells, without adding exogenous solutes to the culture medium. In particular, the significant increase in CFS3 gene expression suggests that, in a potential clinical context, MSCs cultured in the nichoid may better contribute to tissue repair, as compared to cells cultured in a traditional 2D substrate.We found that the nichoid substrate has pronounced effects on cell morphology, cytoskeletal organization and gene expression in MSC. We have submitted these results for publication and already resubmitted the paper with modifications.
3) So far, we were successful with MyoD, a transcription factor that promotes myogenic MSC differentiation. To detect the nuclear import of MyoD, we labelled the protein with a fluorescent probe. We designed four variants of MyoD in which we fused the myogenic factor to different forms of the green-fluorescent protein (GFP); Considering the analyses performed using fluorescence confocal microscopy and target genes expression determined by MyoD activity, the variant that best maintains the properties of the native MyoD and is better suited for the intracellular fluorescence detection turned out to be paGFP-MyoD. We are also studying the nuclear import of the protein MyoD fused with a GFP-30 protein and expressed thanks to a transient transfection into MSC cells seeded in glass flat substrate and into the niches, by using the technique of fluorescence recovery after photobleaching (FRAP).We expressed the transcription factor MyoD fused with a GFP-30 with a transient transfection into MSC cells seeded in the nichoid, we characterised the activity of MyoD-GFP on gene expression, we developed a computational model to interpret these results and we measured the nuclear import of MyoD-G

Final results

\"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 12 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 a patent), I have won a ERC Proof-of-Concept grant (ERC-PoC) to perform a technical and commercial feasibility to move to the market the method developed at my lab during the ERC-CoG grant. 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).
In the context of this ERC-CoG grant, I also integrated the nichoid cell culture substrate into an existing miniaturised optically accessible bioreactor (called MOAB). The MOAB allows to culture 3D organoids of few millimetres in size, under continuous perfusion of the culture medium, infusion of the drug to be tested and diagnostics of cell response both in real time and also post-cultivation. Both the original inventions (the nichoid and the MOAB) are covered by Italian patents originated in the frame of the ERC-CoG project and already extended as PCT. On this idea, I won a second ERC-PoC grant to perform a technical and commercial feasibility to move to the market the MOAB device integrating the nichoid-patterned substrate. This second ERC-PoC started in December 2018 and will last 18 months. I will characterize the fluid-dynamics of the bioreactor chamber that has been modified to accommodate the nichoid substrate. Also, I will set a market assessment and an actionable IPR strategy with identification of a suitable exploitation strategy for valorising the patent/know how. I recently founded a start-up company to commercialise the MOAB device, a spin-off of my hosting institution in which the ERC projects are based, Politecnico di Milano.
Last, but not least, I also won a grant by the Italian Ministry of University and Research (MIUR) on a call addressed to ERC grant winners aimed at the development of new research lines linked to the original ERC-funded project. In this new project, I aim at the development of a radically new method and relevant protocols for intravital optical imagi\"

Website & more info

More info: http://www.nichoid.polimi.it.