Dimitra Zagoura

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure.

Background There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. Methods AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. Results Both HPL cells and AF-MSCs were incorporated into CCl4-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. Conclusions Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver.

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.

Sox2 Suppression by miR‐21 Governs Human Mesenchymal Stem Cell Properties

MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle‐shaped (SS) and round‐shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR‐21 was expressed at higher levels in RS‐AF‐MSCs and BM‐MSCs compared with SS‐AF‐MSCs. We further demonstrated for the first time a direct interaction between miR‐21 and the pluripotency marker Sox2. The induction of miR‐21 strongly inhibited Sox2 expression in SS‐AF‐MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR‐21 inhibition in RS‐AF‐MSCs and BM‐MSCs, which led to an enhanced proliferation rate. Finally, miR‐21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS‐AF‐MSCs. Therefore, these findings suggest that miR‐21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.

Human Amniotic Fluid-Derived Mesenchymal Stem Cells As Therapeutic Vehicles: A Novel Approach For the Treatment of Bladder Cancer

Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNβ) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNβ, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNβ-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.

AF-MSCs fate can be regulated by culture conditions

Human mesenchymal stem cells (hMSCs) represent a population of multipotent adherent cells able to differentiate into many lineages. In our previous studies, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF) and characterized them based on their phenotype, pluripotency and proteomic profile. In the present study, we investigated the plasticity of these cells based on their differentiation, dedifferentiation and transdifferentiation potential in vitro. To this end, adipocyte-like cells (AL cells) derived from AF-MSCs can regain, under certain culture conditions, a more primitive phenotype through the process of dedifferentiation. Dedifferentiated AL cells derived from AF-MSCs (DAF-MSCs), gradually lost the expression of adipogenic markers and obtained similar morphology and differentiation potential to AF-MSCs, together with regaining the pluripotency marker expression. Moreover, a comparative proteomic analysis of AF-MSCs, AL cells and DAF-MSCs revealed 31 differentially expressed proteins among the three cell populations. Proteins, such as vimentin, galectin-1 and prohibitin that have a significant role in stem cell regulatory mechanisms, were expressed in higher levels in AF-MSCs and DAF-MSCs compared with AL cells. We next investigated whether AL cells could transdifferentiate into hepatocyte-like cells (HL cells) directly or through a dedifferentiation step. AL cells were cultured in hepatogenic medium and 4 days later they obtained a phenotype similar to AF-MSCs, and were termed as transdifferentiated AF-MSCs (TRAF-MSCs). This finding, together with the increase in pluripotency marker expression, indicated the adaption of a more primitive phenotype before transdifferentiation. Additionally, we observed that AF-, DAF- and TRAF-MSCs displayed similar clonogenic potential, secretome and proteome profile. Considering the easy access to this fetal cell source, the plasticity of AF-MSCs and their potential to dedifferentiate and transdifferentiate, AF may provide a valuable tool for cell therapy and tissue engineering applications.

Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells

ABSTRACT Human induced pluripotent stem cells (hiPSCs) are considered as a powerful tool for drug and chemical screening and development of new in vitro testing strategies in the field of toxicology, including neurotoxicity evaluation. These cells are able to expand and efficiently differentiate into different types of neuronal and glial cells as well as peripheral neurons. These human cells‐based neuronal models serve as test systems for mechanistic studies on different pathways involved in neurotoxicity. One of the well‐known mechanisms that are activated by chemically‐induced oxidative stress is the Nrf2 signaling pathway. Therefore, in the current study, we evaluated whether Nrf2 signaling machinery is expressed in human induced pluripotent stem cells (hiPSCs)‐derived mixed neuronal/glial culture and if so whether it becomes activated by rotenone‐induced oxidative stress mediated by complex I inhibition of mitochondrial respiration. Rotenone was found to induce the activation of Nrf2 signaling particularly at the highest tested concentration (100 nM), as shown by Nrf2 nuclear translocation and the up‐regulation of the Nrf2‐downstream antioxidant enzymes, NQO1 and SRXN1. Interestingly, exposure to rotenone also increased the number of astroglial cells in which Nrf2 activation may play an important role in neuroprotection. Moreover, rotenone caused cell death of dopaminergic neurons since a decreased percentage of tyrosine hydroxylase (TH+) cells was observed. The obtained results suggest that hiPSC‐derived mixed neuronal/glial culture could be a valuable in vitro human model for the establishment of neuronal specific assays in order to link Nrf2 pathway activation (biomarker of oxidative stress) with additional neuronal specific readouts that could be applied to in vitro neurotoxicity evaluation. HIGHLIGHTSOxidative stress is a common key event in developmental and adult neurotoxicity.hiPSC‐derived mixed culture is a reliable model for neurotoxicity testing.Rotenone causes oxidative stress and leads to the activation of the Nrf2 pathway.Rotenone activates Nrf2 and increases astrocytes in hiPSC‐derived neurons and glia.Rotenone decreases the number of dopaminergic neurons derived from hiPSCs.

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

&NA; 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 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. HighlightshiPSC‐derived NSCs respond to Nrf2 activation upon sub‐chronic rotenone exposure.Rotenone‐induced oxidative stress activates Nrf2/ARE signaling pathway.Nrf2 signaling increases during NSC differentiation into neurons/astrocytes.Rotenone causes astrocyte reactivity, neurite reduction and DA neuronal cell death.

Role of environmental chemicals, processed food derivatives, and nutrients in the induction of carcinogenesis

In recent years it has been hypothesized that cancer stem cells (CSCs) are the actual driving force of tumor formation, highlighting the need to specifically target CSCs to successfully eradicate cancer growth and recurrence. Particularly, the deregulation of physiological signaling pathways controlling stem cell proliferation, self-renewal, differentiation, and metabolism is currently considered as one of the leading determinants of cancer formation. Given their peculiar, slow-dividing phenotype and their ability to respond to multiple microenvironmental stimuli, stem cells appear to be more susceptible to genetic and epigenetic carcinogens, possibly undergoing mutations resulting in tumor formation. In particular, some animal-derived bioactive nutrients and metabolites known to affect the hormonal milieu, and also chemicals derived from food processing and cooking, have been described as possible carcinogenic factors. Here, we review most recent literature in this field, highlighting how some environmental toxicants, some specific nutrients and their secondary products can induce carcinogenesis, possibly impacting stem cells and their niches, thus causing tumor growth.

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

Human pluripotent stem cells can differentiate into various cell types that can be applied to 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. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.