Chiara Scanarotti

A Comparative Study of Proliferation and Hepatic Differentiation of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells possess a lot of stem cell characteristics, so they may be considered a source of stem cell population. On the basis of that, we have investigated the hepatic potential of adipose-derived stem cells, obtained from liposuction, following two differentiation protocols. In the first procedure, medium was supplemented with epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF) and nicotinamide; the second involved the addition of factors such as dexametasone, EGF, insulin-transferrin-sodium selenite, HGF, dimethyl sulfoxide and oncostatin. In parallel, we carried out our study in the Hep G2 cell line, as human hepatic differentiated in vitro model. Immunocytochemical analysis and RT-PCR were performed using hepatic markers to evaluate cell differentiation. DNA content, MTT test and carboxyl fluorescein succinimidyl ester staining were carried out to evaluate cell proliferation. We reported the evidence of basal hepatic marker in undifferentiated adipose-derived stem cells, which confirmed their multipotency. A strong expression of albumin and α-fetoprotein was observed in hepatic-induced adipose-derived stem cells following both differentiation procedures. Morphological aspects of the two types of hepatic adipose-derived stem cells were alike. Proliferation index suggested that the first differentiation procedure promoted better growth than the second. These preliminary findings suggest adipose-derived stem cells may be induced into hepatic lineage, and the most significant difference between the two standard differentiation procedures concerns proliferation rate. This aspect is to be considered when adipose-derived stem cells are employed in research and clinical studies.

In vitro polydeoxyribonucleotide effects on human pre-adipocytes

Abstract.  Objectives: Adipose tissue is the most abundant and accessible source of adult stem cells. Human processed lipoaspirate contains pre‐adipocytes that possess one of the a characteristic pathways of multipotent adult stem cells and are able to differentiate in vitro into mesenchymal and also neurogenic lineages. Because stem cells have great potential for use in tissue repair and regeneration, it would be significant to be able to obtain large amounts of these cells in vitro. As demonstrated previously, purine nucleosides and nucleotides mixtures can act as mitogens for several cell types. The aim of this study was to evaluate the effects of polydeoxyribonucleotides (PDRN), at appropriate concentrations, on human pre‐adipocytes grown in a controlled medium, also using different passages, so as to investigate the relationship between the effect of this compound and cellular senescence, which is the phenomenon when normal diploid cells lose the ability to divide further. Materials and methods: Human pre‐adipocytes were obtained by liposuction. Cells from different culture passages (P6 and P16) were treated with PDRN at different experimental times. Cell number was evaluated for each sample by direct counting after trypan blue treatment. DNA assay and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide test were also carried out in all cases. Results and Conclusions: PDRN seemed to promote proliferation of human pre‐adipocytes at both passages, but cell population growth increased in pre‐adipocyte at P16, after 9 days as compared to control. Our data suggest that PDRN could act as a pre‐adipocyte growth stimulator.

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimers disease and motor neuron disease). A stem cell population (i.e. pre‐adipocytes) is present in the adipose stromal compartment. Pre‐adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre‐adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre‐adipocytes, in parallel with neural morphology. The passage of the choline‐acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic‐like phenotype from pre‐adipocytes.

Neurogenic-committed human pre-adipocytes express CYP1A isoforms

Stem cell models offer an opportunity both for therapeutic use and for the assessment of alternative in vitro models. Human lipoaspirate is a source of adult stem cells (pre-adipocytes), which are able to differentiate into various phenotypes, such as neurogenic lineage. Here, we analyse the suitability of these in vitro models in screening exogenous compounds, such as environmental pollutants, that may affect adipose cells and neurogenic development. To evaluate neurogenic differentiation, we analysed expression of cholinergic system and acetylcholinesterase immunoreactivity. Heterocyclic derivatives of polycyclic aromatic hydrocarbons (PAHs) are often significant components of environmental contaminants. As they contain inducers of cytochrome P450 1A1 (CYP1A1), we explored the activity of CYP1A1-related enzymes, i.e. 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase (ECOD and EROD) in both cell systems in basal conditions and after exposure to non-cytotoxic doses of beta-naphthoflavone (BNF), a well-known PAH-type inducer. Both cell models showed basal and inducible levels of ECOD. Analysis of CYP1A1 protein expression and EROD-related enzyme activity confirmed the inducibility of the CYP1A1 isoform by BNF. These results demonstrate that mesenchymal adult stem cells can constitute innovative models. We therefore propose the use of pre-adipocytes and their neurogenic derivates to evaluate the cytotoxic/biological effects of unintended exposure to contaminants.

Mechanisms for reduced pulmonary diffusing capacity in haematopoietic stem-cell transplantation recipients

Lung diffusing capacity for CO (DLCO) is compromised in haematopoietic stem-cell transplantation (HSCT) recipients. We derived alveolar-capillary membrane conductance (DM,CO) and pulmonary capillary volume (VC) from DLCO and diffusing capacity for NO (DLNO). Forty patients were studied before and 6 weeks after HSCT. Before HSCT, DLNO and DLCO were significantly lower than in 30 healthy controls. DM,CO was ∼40% lower in patients than in controls (p<0.001), whereas VC did not differ significantly. After HSCT, DLNO and DM,CO further decreased, the latter by ∼22% from before HSCT (p<0.01) while VC did not change significantly. Lung density, serum CRP and reactive oxygen metabolites were significantly increased, with the latter being correlated (R2=0.71, p<0.001) with the decrement in DLNO. We conclude that DLNO and, to a lesser extent, DLCO are compromised before HSCT mainly due to a DM,CO reduction. A further reduction of DM,CO without VC loss occurs after HSCT, possibly related to development of oedema, or interstitial fibrosis, or both.

A comparative study of leukaemia inhibitory factor and interleukin-1α intracellular content in a human keratinocyte cell line after exposure to cosmetic fragrances and sodium dodecyl sulphate

According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.

Dietary supplementation of coenzyme Q10 plus multivitamins to hamper the ROS mediated cisplatin ototoxicity in humans: A pilot study

Oxidative stress exerts major role in the pathogenesis of side effects of many antineoplastic drugs, including ototoxicity of cisplatin. In particular, increased levels of reactive oxygen species (ROS) represent one of the molecular mechanisms underlying the apoptosis of different types of hearing cells. Antioxidants and ROS scavengers may thus represent potential therapeutic options to prevent platinum-associated ototoxicity. The aim of this preliminary case-control study was to explore the efficacy of a dietary antioxidant supplement, in order to hamper the occurrences of ototoxicity in patients undergoing cisplatin chemotherapy. As results, a significant protection against cochlear toxic damage was demonstrated in patients who took the antioxidant supplement, which furthermore prevented the occurrence of hearing disorders and tinnitus. These clinical evidences were corroborated by the oxidative status of patients. After cisplatin chemotherapy, the plasma derivatives of reactive oxygen metabolites (d-ROMs) content rapidly increased in control patients, but it was maintained in those under dietary supplementation, likely because of a higher anti-ROMs potential. Indeed, an increment in rapid anti-ROMs was detected in supplemented patients, though no differences were highlighted in terms of slow anti-ROMs. In conclusion, in this preliminary report we demonstrated the feasibility of a dietary antioxidant supplementation in order to prevent the cisplatin induced hearing damage.

Alternative approach to animal testing and cell cultures, according to European laws

ALTEX 34(3), 2017 441 such as skin/eye irritation and partially for skin sensitization. Some non-validated alternative approaches, such as read-across and weight-of-evidence approaches, are also accepted in the EU when sufficiently justified. The OECD launched a program on the development of Adverse Outcome Pathways (AOP) in 2012. AOP is an analytical construct that starts from a mechanistic/ cellular/in vitro sub-profiling to reach the effects observed in populations, and describes sequential events at different levels of biological organization that lead to adverse health or ecotoxicological effects. Dr Stefania Vernazza (LARF-DIMES, University of Genoa) provided an overview on in vitro 3D alternative methods for evaluation of the human risk related to exposure to chemical compounds. International organizations, such as ECVAM and OECD, cooperate on the development of regulations on reducing the use of laboratory animals through the validation of in vitro toxicity tests. Some OECD test guidelines (439, 431 and 492) accept data from in vitro 3D models to classify a substance as irritant/corrosive for skin or eye without the need to perform animal tests. 3D tissues for these applications are commercially available. Prof. Anna Maria Bassi (LARF-DIMES, University of Genoa) explained the concept of a reliable predictive toxicology. Most of the failures in drug discovery are a result of the low predictivity of preclinical animal models for human diseases such as autism syndrome or asthma. There is the pressing need for new toxicity testing models based on human biology. In this regard, Dr Bassi spotlighted the development of a mini-brain by Thomas Hartung’s team (CAAT, Johns Hopkins University) to improve research and drug development for neurodegenerative diseases. Mini-brains derive from patient cells and so can reproduce epigenetic interactions. The second part of the lecture was focused on AOPs, which provide a comprehensive knowledge of human disease and link chemical properties of a toxicant, its macro-molecular interactions, cellular/organ/organism responses and adverse outcomes in the population. This complex of data is critical to identify exposure biomarkers and toxicity pathways. The OECD’s AOP website invites project proposals, offers stakeholder summaries, proposal templates and guidance on developing AOPs. The AOP program foresees a biannual update for new project proposals and new information on ongoing projects. Dr Giorgio Mattei (Research Centre “E. Piaggio”, University of Pisa, Italy) presented an update on innovative technologies for dynamic in vitro 3D models. He explained the characteristics of biomimetic in vitro models resulting from combinations of cell culture systems, advanced materials design, tissue engiOn September 29-30, 2016, the 8th edition of the training course on “Alternative Approach to Animal Testing and Cell Cultures, According to European Laws”, was held in Genoa, chaired by Prof. Anna Maria Bassi and organized and hosted by the team of Laboratory Analysis and Research in Physiopathology (LARF), Department of Experimental Medicine, University of Genoa, Italy. The course was focused on practical work and demonstration/lessons by specialist(s) aiming to supply basic knowledge or improve existing knowledge on alternative methods. Each edition of the course provides an update on innovative in vitro models with a particular emphasis on 3D models. Substantial practical time is included to allow participants to gain hands-on experience. Twenty-four participants, from experts of in vitro methods to undergraduates, from all over Italy took part. The course was opened by Prof. Giovanni Murialdo and Prof. Adriana Voci, coordinators of the degree courses in Medicine and Surgery, and Biological Sciences, respectively, at the University of Genoa. Both stressed the need for such events to ensure that research in Italy for the study of risks to human and animal health following exposure to chemical compounds (drugs, pollutants, food additives, etc.) is in step with new technologies. Prof. Rosagemma Ciliberti (DISSAL, University of Genoa) and Susanna Penco (LARF-DIMES, University of Genoa) highlighted ethical issues of animal-based research. According to Italian Law 413/1993 on “Rules on conscientious objection to animal experimentation”, physicians, researchers, students and healthcare providers can choose not to take part in activities that involve animal experimentation. Conscientious objection to animal experimentation arose from the acknowledgement of animals as conscious beings. Dr Chiara Scanarotti (LARF-DIMES, University of Genoa) gave an update on the development and validation of several well-standardized and reproducible alternative methods for toxicity testing of chemical compounds, underlining how the use of human instead of animal cells improves the accuracy and relevance of the in vitro tests. The lecture highlighted 3D models, spheroids, body-on-a-chip and the latest advancements in stem cell research. Dr Costanza Rovida (CAAT Europe – University of Konstanz, Germany) presented a lecture on the implementation of alternative methods. Full replacement of in vivo tests is a lengthy process as they are mandatory for the registration and risk assessment of chemicals (drugs, additives, etc.) and the use of alternative methods requires formal validation before acceptance. To date, in vitro tests are accepted for some endpoints, Alternative Approach to Animal Testing and Cell Cultures, According to European Laws

Giving meaning to alternative methods to animal testing

669 for their ability to determine corneal permeability. Jan Markus (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia) explained the use of a reconstructed 3D human small intestine model (EpiIntestinal, MatTek Corporation) to predict drug toxicity and absorption/metabolism patterns. The EpiIntestinal model aims to improve the prediction of drug absorption and toxicity over models that are not organor species-specific, rely on cell lines or lack 3D architecture and functionality. It allows bidirectional drug penetration through the intestinal wall and expresses proteins involved in active drug transport and metabolism at physiological levels, which makes it ideal for modelling complex drug absorption profiles. Comparative studies revealed that the absorption of drug in EpiIntestinal mimics the in vivo profile more closely than the currently used Caco-2 model. EpiIntestinal was also able to predict toxicity with much higher specificity and sensitivity than an animal model. Sonia Scarfì (Centro3R, University of Genova) reported on comparing silica-induced fibrosis in early metazoans and human cells. Maria Grazia Cascone (Centro3R, University of Pisa) gave an overview on scaffolds used for tissue engineering, which serve as an artificial extracellular matrix (ECM) that hosts the cells and improves their survival, proliferation, and differentiation, enabling the formation of new tissue. In particular, protein/polysaccharide-based porous scaffolds for use as ECM substitutes in cardiac tissue engineering were discussed. These are based on blends of collagen or gelatin with alginate, produced by freeze-drying and ionic and chemical crosslinking. Tommaso Sbrana (IvTech s.r.l.) focused on 3-dimensional microfluidic in vitro models, which aim to reproduce the in vivo structure and environment of tissues to improve prediction of their response to stimuli or chemicals. This includes the exchange of nutrients and oxygen by means of microfluidics. Lauri Paasonen (UPM-Kymmene Oyj, Finland) covered the use of nanofibrillar cellulose (NFC) hydrogel (GrowDex) derived from wood as a matrix for 3D cell culture applications. NFC is biocompatible with human cells and tissues and allows free diffusion of small molecules, such as nutrients and oxygen. The 3D cell structures can be collected for detailed downstream analysis after cellulase treatment, which degrades GrowDex to soluble glucose with no observed adverse impact on cells. The shear-thinning property of GrowDex permits its use with automated dispensing systems at ambient temperature in high throughput screening-related 3D cell culture applications. Laura Pastorino (Centro3R, University of Genova) focused on the design of a 3D in vitro model of neuronal cultures to mimic the natural cell environment and enable physiological cell-cell interaction, gene expression, synaptogenesis and neurophysiological The 4th advanced theoretical-training course “Give meaning to alternative methods to animal experimentation” was held in Genoa on November 29-30, 2018, hosted by the Italian Centro3R (Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research) and LARF-DIMES (Laboratory for Analysis and Research in Physiopathology – Department of Experimental Medicine, Genova, Italy). The course, chaired by Prof. Arti Ahluwalia (CentroPiaggio, University of Pisa, Italy) and Prof. Anna Maria Bassi (LARF-DIMES), Director and Vice Director of Centro3R, respectively, with the valuable support of members of the Centro3R Operative Units of the Universities of Pisa and Genoa, and of the LARF Research Team, provided an update on new in vitro approaches. The welcome addresses were given by Prof. Michela Tonetti, Director of DIMES, and Prof. Nicola Traverso (DIMES), who stressed the relevance of new in vitro approaches and technologies in toxicological research as well the role of universities in teaching and training future researchers. Arti Ahluwalia (Centro3R, University of Pisa) introduced the course by stating that the role of universities is to provide a nurturing and liberal environment for teaching and research and therefore they represent an ideal place for the implementation and the promotion of the 3Rs. The Italian Interuniversity Center for the Promotion of the 3Rs Principles in Teaching and Research (Centro 3R) was recently established to promote rational and scientific thinking in experimental science through a multidisciplinary teaching and research approach, which includes all 3 Rs. Specifically, the Centro 3R will develop courses and credits on animal care and welfare and advanced in vitro methods, integrating them into bachelor’s and master’s curricula. In the research field, the Center is committed to the development of innovative 3Rs methods as well as knowledge and resource sharing. Silvia Letasiova (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia) spoke on the development and validation of the EpiDerm in vitro skin irritation protocol for the evaluation of medical device extracts. EpiDerm can identify irritant polymer samples either in saline or in sesame oil device extracts. The use of the reconstructed tissue models as replacements for the rabbit intracutaneous test are being implemented into the ISO 10993 standards for the evaluation of medical device biocompatibility. Daniela Monti (Centro3R, University of Pisa) gave an overview on 3Rs approaches for predicting drug-induced eye irritation. The in vivo Draize eye test in rabbits is highly criticized for limited predictability, high costs, and ethical reasons. In vitro methods based on reconstituted corneal epithelium have been developed to assess eye irritation and are now also being evaluated Meeting Report

From cells to QSAR: Alternative predictive models in toxicology

167 She also described the outcome of the recent EU project Anim. al.SEE (http://www.inemm.cnr.it/animalsee/), as an example of the recent progresses achieved in this field. Laura Gribaldo, from JRC (Ispra, Italy), described the potential of toxicogenomic approaches for evidence-based prediction of risk. Toxicogenomic combines the information obtained by transcriptomic (genes), proteomic (proteins) and metabolomic (metabolites) approaches to understand adverse effects of chemical substances and their mechanisms of action. Toxicogenomic approaches are also useful to identify candidate biomarkers of exposure and disease and to build adverse outcome pathways (AOPs). These AOPs can be combined with cell culture data, in silico cheminformatics data and exposure modelling into a network of integrated testing and assessment strategies to predict safety in humans (Bell et al., 2016). Fabiola Pizzo of the Mario Negri Pharmacological Research Institute (Milan, Italy) further expounded the concepts of read-across, the technique for predicting endpoint information for one substance by using data from the same endpoint for other substances. She explained how in silico approaches used to evaluate the potential toxicity of chemicals can be particularly useful when dealing with a very large number of substances. Increased computational power, new algorithms, better molecular descriptors, non-linear modelling, together with the introduction of a large number of properties and of results from complex biological endpoints have strengthened the Quantitative Structure Activity Relationship (QSAR) approaches. The computational platform VEGA (http://www.vega-qsar.eu) for QSAR analysis and ToxRead (http://www.toxgate.eu) for readacross evaluations were introduced. She also stressed that further contributions, e.g., from toxicity databases and in vitro data are needed to improve in silico methods and that results from several computational models should be compared. Chantra Eskes (SECAM, Switzerland) talked about challenges to the implementation of alternative methods in regulatory toxicology. She described the recent updates in the Organisation for Economic Cooperation and Development (OECD) guidelines for the validation of new methods for hazard assessment and for integrated approaches for testing and assessment (IATA) in the field of skin corrosion and irritation and eye damage/irritation. These guidelines were strongly driven by the EU Cosmetics Directive (EU 2003/15/EC) placing a ban on animal testing in cosmetics production, and the REACH Reg.1907/2006 imposing the revision of toxicity data for all chemicals produced in amounts greater that 1 ton/year, but also The Italian Association for In Vitro Toxicology CELLTOX in collaboration with the Laboratory of Analysis and research in Physiopathology (LARF), Department of Experimental Medicine (DIMES) of the University of Genova, in April 2016 organized a three-day course on alternative predictive models in toxicology. The course was also supported by ESTIV, The European Society of Toxicology In Vitro. Thomas Hartung, Professor of Evidence Based Toxicology and Director of the Center for Alternatives to Animal Testing (CAAT) at the Johns Hopkins University in Baltimore (US) opened the course with an introductory lecture on Cell “culture” in the 21st century. Playing on the double sense of culture as a way to maintain cells in vitro and culture in the sense of “the behaviours and beliefs characteristic of a particular group”, he described the scientific and ethical advantages as well as the weaknesses and critical points of in vitro cell cultures, as opposed to animal models, for modern toxicology. The recent advent of differentiated cell models derived from stem cells and the use of 3D preparations for organotypic cultures, together with the application of good cell culture practices and integrated testing strategies, will improve the predictive value of cell cultures in toxicological research (Pamies et al., 2017). As an example, he described the novel mini-brains obtained from human donors’ skin fibroblasts reprogrammed into induced pluripotent stem cells, then differentiated into neural stem cells and grown in 3D culture to form identical spheroid micro-physiological minibrains that can be used to study CNS mechanisms, for developmental and neurotoxicity studies, for drug development and to study brain diseases (Pamies et al., 2016). He also introduced the read-across approaches to predict potential toxicity of new chemical substances by the use of novel screening tools built by mining the existing toxicological data (Patlewicz et al., 2014; Ball et al., 2016). Margherita Ferro, one of the founders of CELLTOX, gave an overview of her personal experience on in vitro toxicological research since the mid 1980’s, emphasizing the importance of the first studies on hepatic metabolism of xenobiotics using competent hepatoma cell lines, and the later development of other in vitro cell models for toxicological studies (Ferro et al., 1987). Flavia Zucco, founder and first President of CELLTOX, introduced the ethical and philosophical principles underlying the choice of experimental models, alternative to the use of animals, and described the initial difficulties encountered in the pioneering years by scientists supporting the principles of the 3Rs. Meeting report