A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroinalginate microcarriers using light sheet fluorescent microscopy

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device

Barrier-to-autointegration factor (BAF) involvement in prelamin a-related chromatin organization changes

Chromatin disorganization is one of the major alterations linked to prelamin A processing impairment. In this study we demonstrate that BAF is necessary to modulate prelamin A effects on chromatin structure. We show that when prelamin A and BAF cannot properly interact no prelamin A-dependent effects on chromatin occur; similar to what is observed in human Nestor Guillermo Progeria Syndrome cells harboring a BAF mutation, in HEK293 cells expressing a BAF mutant unable to bind prelamin A, or in siRNA mediated BAF-depleted HEK293 cells expressing prelamin A. BAF is necessary to induce histone trimethyl-H3K9 as well as HP1-alpha and LAP2-alpha nuclear relocalization in response to prelamin A accumulation. These findings are enforced by electron microscopy evaluations showing how the prelamin A-BAF interaction governs overall chromatin organization. Finally, we demonstrate that the LAP2-alpha nuclear localization defect observed in HGPS cells involves the progerin-BAF interaction, thus establishing a functional link between BAF and prelamin A pathological forms

Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells.

This study was designed to investigate the migratory behavior of adult human mesenchymal stem cells (MSC) and the underlying mechanism. Cell migration was assessed by transwell, wound healing and time-lapse in vivo motility assays. Pharmacological inhibitors were used to determine the potential mechanism responsible for cell migration and invasion. The tests that were implemented revealed that MSC were fairly migratory. Protein kinase B (AKT) was strongly activated at the basal level. Through our analyses we demonstrated that pharmacological inactivation of AKT2 but not AKT1 significantly decreased cell migration and invasion. Although preliminary, collectively our results indicate that AKT2 activation plays a critical role in enabling MSC migration

Handheld Co-Axial Bioprinting: Application to in situ surgical cartilage repair

Three-dimensional (3D) bioprinting is driving major innovations in the area of cartilage tissue engineering. Extrusion-based 3D bioprinting necessitates a phase change from a liquid bioink to a semi-solid crosslinked network achieved by a photo-initiated free radical polymerization reaction that is known to be cytotoxic. Therefore, the choice of the photocuring conditions has to be carefully addressed to generate a structure stiff enough to withstand the forces phisiologically applied on articular cartilage, while ensuring adequate cell survival for functional chondral repair. We recently developed a handheld 3D printer called Biopen . To progress towards translating this freeform biofabrication tool into clinical practice, we aimed to define the ideal bioprinting conditions that would deliver a scaffold with high cell viability and structural stiffness relevant for chondral repair. To fulfill those criteria, free radical cytotoxicity was confined by a co-axial Core/Shell separation. This system allowed the generation of Core/Shell GelMa/HAMa bioscaffolds with stiffness of 200KPa, achieved after only 10seconds of exposure to 700mW/cm2 of 365nm UV-A, containing \u3e90% viable stem cells that retained proliferative capacity. Overall, the Core/Shell handheld 3D bioprinting strategy enabled rapid generation of high modulus bioscaffolds with high cell viability, with potential for in situ surgical cartilage engineering

Morphogenesis of follicular epithelium in Drosophila melanogaster: function of von Hippel-Lindau tumour suppressor gene

During my PhD I have been involved in several projects regarding the morphogenesis of the follicular epithelium, such as the analysis of the pathways that correlate follicular epithelium patterning and eggshell genes expression. Moreover, I used the follicular epithelium as a model system to analyze the function of the Drosophila homolog of the human von Hippel-Lindau (d-VHL) during oogenesis, in order to gain insight into the role of h-VHL for the pathogenesis of VHL disease. h-VHL is implicated in a variety of processes and there is now a greater appreciation of HIF-independent h-VHL functions that are relevant to tumour development, including maintenance and organization of the primary cilium, maintenance of the differentiated phenotype in renal cells and regulation of epithelial-mesenchymal transition. However, the function of h-VHL gene during development has not been fully understood. It was previously shown that d-VHL down-regulates the motility of tubular epithelial cells (tracheal cells) during embryogenesis. Epithelial morphogenesis is important for organogenesis and pivotal for carcinogenesis, but mechanisms that control it are poorly understood. The Drosophila follicular epithelium is a genetically tractable model to understand these mechanisms in vivo. Therefore, to examine whether d-VHL has a role in epithelial morphogenesis and maintenance, I performed genetic and molecular analyses by using in vivo and in vitro approaches. From my analysis, I determined that d-VHL binds to and stabilizes microtubules. Loss of d-VHL depolymerizes the microtubule network during oogenesis, leading to a possible deregulation in the subcellular trafficking transport of polarity markers from Golgi apparatus to the different domains in which follicle cells are divided. The analysis carried out has allowed to establish a significant role of d-VHL in the maintenance of the follicular epithelium integrity

Evidence for a novel function of Awd in maintenance of genomic stability

The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. Here, we analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. We show that awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, our data demonstrate a novel function for awd in maintenance of genomic stability. Our results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd - induced genomic instability on tumour development and metastasis formation

Detection of mesenchymal stem cells senescence by prelamin A accumulation at the nuclear level

Background: Human mesenchymal stem cells (MSC), during in vitro expansion, undergo a progressive loss of proliferative potential that leads to the senescent state, associated with a reduction of their \u201cmedicinal\u201d properties. This may hampers their efficacy in the treatment of injured tissues. Quality controls on MSC-based cell therapy products should include an assessment of the senescent state. However, a reliable and specific marker is still missing. From studies on lamin-associated disorders, has emerged the correlation between defective lamin A maturation and cellular senescence. Findings: Primary cultured hMSC lines (n\ua0=\ua03), were analyzed by immunostaining at different life-span stages for the accumulation of prelamin A, along with other markers of cellular senescence. During culture, cells at the last stage of their life span displayed evident signs of senescence consistent with the positivity of SA-\u3b2-gal staining. We also observed a significant increase of prelamin A positive cells. Furthermore, we verified that the cells marked by prelamin A were also positive for p21Waf1 while negative for Ki67. Conclusions: Overall data support that the detection of prelamin A identifies senescent MSC, providing an easy and reliable tool to be use alone or in combination with known senescence markers to screen MSC before their use in clinical applications

In vitro quantitative analysis of mesenchymal stromal cells migration towards tumours

Analysis of in vitro migration of cell populations plays a key role in studying a wide range of dynamic cell behaviours. Cell migration is dependent on a multitude of signals ranging from growth factors to chemokines secreted by injured cells and/or respondent immune cells. In the last decade, several studies were addressed to investigate tropism of Mesenchymal Stromal Cells (MSC) for some types of cancers. Understanding the factors involved in regulating MSC migration towards tumours is essential to ultimately develop novel clinical strategies aiming at using MSC as vehicles to deliver antitumor drugs. However, the mechanism behind MSC migration is still in its infancy, and the main cause is the lack of quantitative methods to analyse the MSC motion. The analysis of objects motion relies on image-based tracking techniques and several methods have been recently proposed to track cells in vitro. However the vast majority of them are limited to fluorescently labelled cells. Very few tracking tools can analyze images taken using label-free microscopy techniques (e.g. phase contrast), that do not compromise the biological status of the cell and are the most common ways to observe living cells. However, to accurately track irregular-shaped, flat, and poor contrasted cells, such as MSC observed in phase-contrast, is really challenging. Furthermore, the majority of the tracking tools require relevant image processing skills, which strongly limits their practical usefulness within the biologist community. User-friendly and versatile open source software with track editing possibilities would boost live cell analysis research. In this work we introduced CellTracker, a user-friendly open-source software tool for tracking cells, including MSC. CellTracker is freely available at: http://celltracker.website/ and it works with Windows, Macintosh, and UNIX-based systems. By using CellTracker and \u3bc-Slide Chemotaxis2D (IBIDI, Madison, Wisconsin, USA) we performed experiments to analyse the motility of MSC when seeded near to cancer cells, simply seeding MSC in a chamber communicating to another one containing conditioned medium from MG-63 osteosarcoma cells. As negative and chemotaxis controls, we used respectively fresh culture medium and culture medium supplemented with serum. From the CellTracker analysis we could not detect any significant change in MSC motility towards the culture medium with or without serum, whereas a different behaviour was observed by using culture conditioned medium taken from flasks previously containing MG-63 cells. We can therefore argue that MSC migration towards tumour cells is mediated by the presence of specific proteins (e.g. chemokines) secreted by cancer cells in the culture medium. These preliminary results pave the way for future applications such as the possibility to develop hybrid disposable diagnostic devices where biological fluids (e.g. blood) sampled from the area of interest are analysed by using the MSC motility as a cancer sensor