The IIT@Sapienza laboratory, born after a Research Project CLNS submitted in 2010, revolves around two topics of biomedical interest where technological innovation is key to reach the goals.
The first regards neurodegenerative disorders, hereditary and sporadic conditions characterized by progressive nervous system dysfunction. The focus of the second will be brain tumours, the most life-threatening diseases of adulthood and childhood.
These challenging projects benefit from the establishment and convergence of common technological platforms. They avail themselves of state-of-the-art commercial equipment and, at the same time, foster and support the development of new techniques and instrumentation.
Amiotrophic Lateral Sclerosis (ALS) is one of the most devastating degenerative neuromuscular disorders, characterized by motoneuron (MN) progressive degeneration, muscle atrophy and fatal paralysis.
Even though biomedical research in the field witnessed major efforts profused in the last years, many facets of ALS etiopathology remains elusive, concerning in particular the mechanism through which physiological functions are corrupted in diseased conditions.
The A1 project aims at applying state-of-the-art nanotechnologies as tools to elucidate the molecular, cellular and tissutal processes underlying nervous system differentiation and homeostasis, as a prerequisite to understand their pathological mis-regulation in the occurring neuromuscular degeneration.
Mouse motoneuron unit
Among the numerous genes involved in ALS onset and progression, the unit is focusing on the pleiotropic RNA-binding protein FUS/TLS (FUS), whose mutations have been found in 4% of familial cases of ALS. We are taking advantage of mouse Embryonic Stem Cells (mESCs) as a powerful model where investigating the role of FUS in ALS molecular etiopathogenesis.
Human iPSCs Unit
Induced Pluripotent Stem Cells (iPSCs) provide an opportunity to study human diseases in those cases where appropriate models are not available. We have derived human ALS-iPSCs that can generate motoneurons or muscle cells in vitro. This cell system recapitulates key hallmarks of the pathology, such as aberrant localization of mutated proteins in response to cellular stress, and can be exploited to investigate the correlation between mutations and ALS ethiopathogenesis.
Mouse skeletal muscle unit
The muscle unit is focused on the histological, molecular, functional and biomechanical analysis on both skeletal muscle and nervous tissue of the ALS animal models.
The unit develops electrphysiological and time-lapse imaging recording of neuronal and microglial cells activity to characterize their interactions in physiological and pathological conditions (ALS and brain tumours).
The unit is specialized in protein engineering by genetic and chemical modification methods. In particular, transport protein variants are designed, cloned, expressed and purified. Protein carriers are thus exploited for delivery of pharma products, diagnostic probes and nucleic acids to cells and tissues.
Brain tumours are the most life-threatening diseases of adulthood and childhood. Despite the recent advances in diagnosis and treatment, the prognosis remains poor. Thus, improving surgical and imaging technologies as well as a better genetic/epigenetic characterization of selected tumour cell populations are needed.
An important issue deals with the presence of brain tumour cancer stem cells (CSC), which represent the reservoir of tumour initiating cells sustaining tumour maintenance and progression.
The project is mainly focused: 1) to develop in vivo molecular imaging technologies to improve tumour detection during intraoperative brain tumour delineation; 2) to understand the biology and the dynamics of CSC population and the pathogenic signalling pathways able to sustain CSC (such as Hedgehog and Notch signalling); the final aim is to identify theranostic (diagnostic & therapeutic) agents to be used in diagnostic and follow-up procedures, with specific emphasis to multifunctional theranostic nanoparticle-based delivery systems targeting CSC-specific signalling pathways and neoangiogenesis.
Laboratories and Supporting experimental platforms
This challenging project is being conducted in the CLNS@Sapienza laboratories: an open space of fully equipped workstations for molecular and cellular biology, including sterile tissue/cell culture room, biohazard cell culture laboratory BSL3 safety culture, microbiology room, dark room and instrument room. Among the available instruments there are: Real Time Viia7, thermal cyclers, Luminometer, Chemidoc, Nanodrop, cryostat.
Supporting experimental platforms
The Genomics Platform welcomes sequencing and expression analysis projects from investigators within and outside the Centre. This integrated platform provides comprehensive services to the scientific community and is dedicated to pursue operational excellence, advanced process design, data analysis and visualization, and technology development. The Genomics platform is equipped with Illumina Hiseq 1500, Miseq, NanoString nCounter and conventional instrumentations and technologies to support the full workflow of samples preparation and the generation of validated libraries.
The bioinformatics facility is a research, support and service core to assist IIT researchers with Next Generation Sequencing (NGS) data analysis. The facility is supported by a dedicated High Performance Computing platform whose architecture is specifically suited to genomics data analysis:
- Gateway to Illumina Miseq and Hiseq sequencers
- HP Enclosure with 11 blades
- HPC cluster with 9 nodes for parallel computing
- 130+ cores up to 512GB RAM
- 60+ Terabytes of SAN storage
- Lustre parallel file system supporting HPC simulation environment
- Tape library.
The facility participates in multiple projects and collaborations, providing the most used NGS data analysis software, designing specific pipelines for diverse genomics data types and developing customized bioinformatics tools. Standard and advanced level of NGS analysis are provided, including: RNA-seq, miR-seq, ChIP-seq, whole exome and genome sequencing (WES, WGS).
Bioprinting is a technology that creates living cellular constructs through the layer-by-layer, 3D deposition of biological materials, consisting in cells and extracellular matrix components (the bio-ink). It finds application in the development of biomimetic tissue models for in vitro studies or for the production of functional and transplantable tissue substitutes in Tissue Engineering. In particular, the Centre is developing a bioprinting system based on the microfluidic extrusion of cell-laden hydrogel fibres. Different hydrogels with tuneable chemical and mechanical properties are used as supporting extracellular matrix to recreate a 3D, instructive environment for cultured cells.
The flow cytometry laboratory at CLNS-IIT@Sapienza is equipped with two laser-based instruments which main purpose is to characterize and isolate a variety of cell population both of human and mouse origin. The technology of these instruments allows simultaneous multi-parametric analysis of thousands of cells for second for rapid analysis of complex cell populations. This is achieved by the use of different labelled antibodies or dyes to target specific molecules express at the surface or in the cytoplasm of the cell, giving a screening able to describe in detail a complex sample or to identify low frequency subset of cells.
Through these instruments, a variety of fluorochromes attached to different antibodies can screen the molecular components of a cell first to characterize it (flow cytometer) and then to specifically select for isolation (cell sorter).
The BD LSRFortessa cell analyzer is a 5 laser cytometer (488nm, UV 355nm, V 405nm, 561nm and 640nm) and 18 fluorescence detectors running a BD FACSDiva software on XP.
The characteristics of the cell analyzer are:
- to identify cells using antibodies for specific surface and intracellular markers;
- to discriminate transfetted cells using specific probes (i.e. GFP, RFP etc);
- to study cell proliferation (CFSE…) apoptosis and/or cell death (Annexin-V, 7-AAD). The BD FACSAriaIII is a 4 laser cell sorter (488nm, Near UV 375nm, 561 nm and 640nm) and 10 fluorescence detectors running a BD FACSDiva software on XP.
The details of the cell sorter are:
- up to 4 population cell sorting;
- single cell isolation;
- it can sort into tubes (1,5ml, 5ml, 15ml), multiwell plates (6, 24, 48, 96 and 384 wells), chamber slides (2 or 8 wells) and standard or frosted-end slides.
Imaging at IIT@Sapienza includes both commercial and state-of-the-art facilities, aimed at providing a complete panorama of techniques to tackle biomedical research activities. These include:
- Single and two-photon laser-scanning confocal microscopy.
- Spinningdisk confocal microscopy.
- Non-linear microscopy for CARS and SRS imaging.
- nanoIR microscopy.
- nanoRaman microscopy.
- Structured light microscopy.
The main purpose of the lab is to design micro and nanofluidics systems for biomedical applications. We are presently working on the development of a blood vessel model on chip and on the design of nanopore based sensors for single molecule protein and peptide analysis. In addition, we are involved in some fundamental science research lines on cavitation, microswimmers and multiphase flows at microscale.
The lab is equipped with a micro-PIV set-up for measurement of the flow velocity field at microscale (pulsed laser, inverted microscope, dual-frame camera), a high-speed camera and a set of pumps for manipulating small volume of fluids. The lab runs also in numerical simulations ranging from atomistic model to continuum fluid dynamics.
A cell culture facility supports the research of the two main projects A1 and A2 by providing the appropriate equipment for the mammalian cell culture work. The purpose of the facility is to provide a common platform to carry out cell based experiments and to maintain and to store experimental cell lines. A flow laminar hood with a stereoscope dedicated to cell reprogramming is dedicated to mouse and human pluripotent stem cells.