In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

JRC.I.5-Systems Toxicolog

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.EU/FP7/ESNATSDFGDoerenkamp-Zbinden Foundatio

Molecular characterization and investigation of the differentiation potential of human mesenchymal stem cells

Τα βλαστικά κύτταρα μονοπωλούν το ενδιαφέρον περισσότερο από κάθε άλλο πεδίο στη Βιολογία τα τελευταία χρόνια και αποτελούν σημαντικά εργαλεία στην ανεύρεση δυνητικών θεραπειών με εφαρμογή στα εκφυλιστικά νοσήματα και στην Αναγεννητική Ιατρική. Μια κατηγορία βλαστικών κυττάρων είναι τα μεσεγχυματικά βλαστικά κύτταρα (MSCs). Τα MSCs αποτελούν ένα πληθυσμό προγονικών κυττάρων με δυνατότητα διαφοροποίησης σε επιμέρους ιστούς της μεσεγχυματικής σειράς και με ικανότητα αναγέννησης και in vivo αποκατάστασης του ομόλογου ιστού. Oι κύριες πηγές MSCs είναι ο μυελός των οστών (ΒΜ) και το αίμα ομφαλίου λώρου (UCB) που παρουσιάζουν όμως αρκετά προβλήματα και περιορισμούς και για αυτό το λόγο είναι αναγκαία η ανεύρεση εναλλακτικών πηγών αξιοποιήσιμων MSCs. Μια τέτοια πηγή, χωρίς την παρουσία ηθικών ζητημάτων, είναι και το αμνιακό υγρό (AF). Πιο συγκεκριμένα, στην παρούσα διδακτορική διατριβή απομονώθηκαν εμβρυϊκά MSCs από αμνιακό υγρό (AF-MSCs) δευτέρου τριμήνου κύησης και πραγματοποιήθηκε μοριακός και φαινοτυπικός χαρακτηρισμός τους. Αποδείχθηκε ότι τα κύτταρα εξέφραζαν το μεταγραφικό παράγοντα OCT-4, την πρωτεΐνη NANOG και τον επιφανειακό δείκτη SSEA-4, που αποτελούν χαρακτηριστικούς εμβρυϊκούς δείκτες ενώ ο ανοσοφαινοτυπικός χαρακτηρισμός των MSCs με κυτταρομετρία ροής απέδειξε ότι εξέφραζαν όλους τους μεσεγχυματικούς δείκτες Τέλος, όταν τα AF-MSCs καλλιεργήθηκαν υπό κατάλληλες συνθήκες με μέσα που περιείχαν τους απαραίτητους επαγωγικούς παράγοντες, αποδείχθηκε ότι μπορούσαν να διαφοροποιηθούν σε κύτταρα, προερχόμενα και από τα τρία βλαστικά δέρματα. Πιο συγκεκριμένα, τα AF-MSCs μπορούσαν να διαφοροποιηθούν προς λιποκύτταρα, οστεοκύτταρα, νευρικά κύτταρα και ηπατοκύτταρα. Ακολούθως, μελετήθηκε η ικανότητα απο- και δια-διαφοροποίησης των AF-MSCs. Πιο συγκεκριμένα, τα AF-MSCs μετά από καλλιέργειά τους με κατάλληλα πρωτόκολλα που προωθούν τη διαφοροποίησή τους προς λιποκύτταρα απο-διαφοροποιήθηκαν προς κύτταρα με προγονικό φαινότυπο (DAF-MSCs). Στη συνέχεια, πραγματοποιήθηκε συγκριτική μελέτη των τριών παραπάνω κυτταρικών τύπων ως προς την έκφραση των εμβρυϊκών δεικτών OCT-4, SOX-2 και NANOG, ως προς τη μεταβολική τους ενεργότητα και ως προς το πρωτεομικό τους πρότυπο. Αποδείχθηκε ότι τα AF-MSCs και DAF-MSCs είχαν παρόμοιο γονιδιακό και πρωτεομικό πρότυπο ενώ εμφάνιζαν χαμηλότερη μεταβολική ενεργότητα σε σχέση με τα πλήρως διαφοροποιημένα λιποκύτταρα. Ακολούθως, τα λιποκύτταρα, προερχόμενα από ΑF-MSCs, δια-διαφοροποιήθηκαν προς ηπατοκύτταρα, όπου και διαπιστώθηκε ότι η διαδικασία δεν πραγματοποιήθηκε απ’ευθείας αλλά μέσω μετάβασης των λιποκυττάρων προς προγονικούς κυτταρικούς τύπους που ονομάστηκαν TRAF-MSCs (trans-differentiated AF-MSCs). Τέλος, έγινε συγκριτική μελέτη των TRAF-MSCs, των AF-MSCs και των DAF-MSCs και αποδείχθηκε ότι είχαν παρόμοιο φαινότυπο, εξέφραζαν τους εμβυϊκούς δείκτες OCT-4, SOX-2 και ΝΑΝΟG και διέθεταν παρόμοια μεταβολική ενεργότητα και κλωνογονική ικανότητα. Τέλος, η συστηματική ανάλυση του πρωτεομικού τους προτύπου έδειξε πολλές ομοιότητες και αποδείχθηκε ότι και οι τρεις κυτταρικές κατηγορίες εκκρίνουν αυξητικούς παράγοντες που σχετίζονται με την κυτταρική διαφοροποίηση και την ιστική αναγέννηση. Στη συνέχεια διερευνήθηκαν οι θεραπευτικές ιδιότητες των AF-MSCs στην οξεία ηπατική ανεπάρκεια (OHA), δεδομένου ότι τα MSCs δεν δημιουργούν τερατώματα in vivo και εμφανίζουν ανοσοκατασταλτικές ιδιότητες κατά τη μεταμόσχευσή τους σε ζωϊκά μοντέλα. Ειδικότερα, κατασκευάστηκε λεντιϊκός φορέας με το μόριο φθορισμού GFP για την επιτυχή διαμόλυνση των AF-MSCs και στη συνέχεια χορηγήθηκαν ΑF-MSCs ή ηπατικά προγονικά κύτταρα (ΗΠΚ) από ΑF-MSCs σε ζωϊκά πρότυπα που έπασχαν από οξεία ηπατική ανεπάρκεια. Ιδιαιτέρως, διαπιστώθηκε ότι τα AF-MSCs μπορούσαν να εποικίσουν το κατεστραμμένο ήπαρ και να συμβάλλουν στη βελτίωση του φαινοτύπου. Επιπλέον, μελετήθηκε και η παρακρινική δράση των κυττάρων αυτών στη θεραπεία της ΟΗΑ όπου και αποδείχθηκε ότι εκκρίναν αντι-φλεγμονώδεις κυτταροκίνες, όπως είναι η ιντερλευκίνη-10 (IL-10), καθώς και αυξητικούς παράγοντες που έχουν ρόλο στην ιστική επιδιόρθωση, γεγονός που συνέβαλλε στη βελτίωση της ηπατικής βλάβης. Συνεπώς, τα AF-MSCs αποτελούν μία εναλλακτική πηγή MSCs που χαρακτηρίζονται από ευρύ δυναμικό διαφοροποίησης και πλαστικότητας ενώ ταυτόχρονα εμφανίζουν θεραπευτική δράση σε ασθένειες, όπως είναι η ΟΗΑ, ιδιότητες που τα καθιστούν ιδιαίτερα σημαντικά εργαλεία σε εφαρμογές της Αναγεννητικής Ιατρικής

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells are able to differentiate into various cell types that can be applied in human based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. Here we describe a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. We defined which signalling pathways are regulated and/or activated upon neuronal differentiation. Providing this information is critical in order to be able to apply the cell model for the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept we used rotenone, an inhibitor of mitochondrial respiratory complex I, to assess the activation of Nrf2 signaling pathway, a key regulator of the Antioxidant-Response-Element-(ARE)-driven cellular defense mechanism against oxidative stress.JRC.F.3-Chemicals Safety and Alternative Method

Nrf2 pathway activation upon rotenone treatment in human iPSC-derived neural stem cells undergoing differentiation towards neurons and astrocytes

Activation of Nrf2/ARE signaling pathway occurs ubiquitously in most cell types upon induction of oxidative stress. Rotenone, an inhibitor of mitochondrial complex I, can be used to trigger oxidative stress, stimulate the activation of Nrf2 pathway in neuronal and astrocytic cells and assess neurotoxicity. We have previously demonstrated that an acute treatment with rotenone can induce Nrf2 activation, which leads to astrocyte activation and dopaminergic (DA) neuronal cell death in a mixed neuronal/astrocytic cell model derived from human induced pluripotent stem cells (hiPSCs). In this study, we characterized the effects of a repeated dose treatment with rotenone (14 days) on hiPSC-derived neural stem cells (NSCs) undergoing differentiation, assessing the expression and the activation of the Nrf2 pathway. Our results show that Nrf2 signaling increases during NSC differentiation. Moreover, we observed that rotenone treatment induced a progressive activation of Nrf2 signaling together with a transient induction of astrocyte reactivity, a reduction of neurite length leading to neuronal cell death, in particular of DA neurons. Altogether these data indicate that hiPSC-NSC models are relevant test systems for the evaluation of Nrf2 pathway activation upon induced oxidative stress, allowing further understanding of the molecular mechanisms underlying exposure to (developmental) neurotoxicants.JRC.F.3-Chemicals Safety and Alternative Method

Regenerative toxicology: the role of stem cells in the development of chronic toxicities

Human stem cell lines and their derivatives have been widely discussed as cellular models in predictive toxicology. Huge efforts are currently undertaken to investigate the suitability of stem cells derivatives as unlimited cellular source for the development of in vitro toxicity tests that qualify for a better understanding of the mode of action of hazardous chemicals. Nevertheless, the contribution of stem cells and precursors to the development of chronic toxicities and carcinogenesis have not received so much attention so far and remains a gap in the current research strategy on stem cells as tools for toxicity testing in vitro. The current review describes selected examples of toxicants that are targeting stem cells/progenitor cells which result in the development of long term toxicities.JRC.I.5-Systems Toxicolog

SCR&Tox: Stem Cells for Relevant efficient extended and normalised Toxicology

Neurotoxicity is one of the most challenging fields for the development of in vitro testing systems. In the last years, alternative in vitro testing strategies for chemical risk assessment have been designed, according to the current REACH legislation, to reduce the number of animal required for testing. However, to date in vitro assays for neurotoxicity have not been formally validated yet [1]. This is mostly due to the extreme complexity of the nervous system in which many different cell types are organized in a well orchestrated and functional network difficult to reproduce in vitro, but also to the lack of in vitro systems and methods capable to fully cover some of the endpoints of the in vivo tests such as the neurobehavioral and neurocognitive aspects as well as the motor functionality. Nowadays, many cellular models are available for the nervous system, including primary cultures of fetal and adult neurons and glial cells, tumor-derived cell lines, hippocampal brain slices and neural progenitor cells, but all of this models suffer from considerable drawbacks, e.g. non-human origin, limited access or non-physiological transformed cell types [2]. Human pluripotent stem cells (hPSCs) are considered as a powerful tool for drug screening and the development of new in vitro testing strategies. Indeed, these cells can be indefinitely expanded and efficiently differentiated into neuronal derivatives, including different regionalized neuronal subtypes, glial cells and peripheral neurons [3]. In this context, the European project ESNATS has taken the first step toward the design of developmental neurotoxicity tests based on the use of hPSCs, particularly developing battery of tests covering different aspects of neural teratogenicity [4-6]. Many of these hPSCs-based models are very well characterized on the molecular basis, but to date there are few data indicating how they reflect the functionality of the in vivo central nervous system (CNS)/ peripheral nervous system (PNS) and clearly none of these systems can completely resemble the complex physiology of the entire nervous system. Consequently, the main problem in the development of novel test strategies relies on the fact that the mechanisms underlying neurotoxicity are too extensive to be covered with a single model and a small set of endpoints. Therefore, in vitro neurotoxicity tests should include different cellular models and multiple levels of evaluation, ranging from cytotoxicity and cell physiology to neuronal specific cell function endpoints. Moreover, the obtained data, in order to be considered reliable and predictive, should be compared across diverse in vitro models, extrapolated and further aligned to in vivo available data sets, in order to bridge the gap between in vivo and in vitro neurotoxicity.JRC.I.5-Systems Toxicolog

Changes in miRNA Expression Profiling during Neuronal Differentiation and Methyl Mercury-Induced Toxicity in Human in Vitro Models

MicroRNAs (miRNAs) are implicated in the epigenetic regulation of several brain developmental processes, such as neurogenesis, neuronal differentiation, neurite outgrowth, and synaptic plasticity. The main aim of this study was to evaluate whether miRNA expression profiling could be a useful approach to detect in vitro developmental neurotoxicity. For this purpose, we assessed the changes in miRNA expression caused by methyl mercury chloride (MeHgCl), a well-known developmental neurotoxicant, comparing carcinoma pluripotent stem cells (NT-2) with human embryonic stem cells (H9), both analyzed during the early stage of neural progenitor commitment into neuronal lineage. The data indicate the activation of two distinct miRNA signatures, one activated upon neuronal differentiation and another upon MeHgCl-induced toxicity. Particularly, exposure to MeHgCl elicited, in both neural models, the down-regulation of the same six out of the ten most up-regulated neuronal pathways, as shown by the up-regulation of the corresponding miRNAs and further assessment of gene ontology (GO) term and pathway enrichment analysis. Importantly, some of these common miRNA-targeted pathways defined in both cell lines are known to play a role in critical developmental processes, specific for neuronal differentiation, such as axon guidance and neurotrophin-regulated signaling. The obtained results indicate that miRNAs expression profiling could be a promising tool to assess developmental neurotoxicity pathway perturbation, contributing towards improved predictive human toxicity testing