Toxicity of ionizing radiation (IR) in a human induced pluripotent stem cell (hiPSC)-derived 3D early neurodevelopmental model.

Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1 Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48 h and 9 days post IR (pIR). All but one of the analyzed marker genes were reduced 48 h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.

A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles.

Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6(+) precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

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.

GFAP-independent inflammatory competence and trophic functions of astrocytes generated from murine embryonic stem cells.

The directed generation of pure astrocyte cultures from pluripotent stem cells has proven difficult. Generation of defined pluripotent-stem-cell derived astrocytes would allow new approaches to the investigation of plasticity and heterogeneity of astrocytes. We here describe a two-step differentiation scheme resulting in the generation of murine embryonic stem cell (mESC) derived astrocytes (MEDA), as characterized by the upregulation of 19 astrocyte-associated mRNAs, and positive staining of most cells for GFAP (glial fibrillary acidic protein), aquaporin-4 or glutamine synthetase. The MEDA cultures could be cryopreserved, and they neither contained neuronal, nor microglial cells. They also did not react to the microglial stimulus lipopolysaccharide, while inflammatory activation by a complete cytokine mix (CCM) or its individual components (TNF-α, IL1-ß, IFN-γ) was readily observed. MEDA, stimulated by CCM, became susceptible to CD95 ligand-induced apoptosis and produced NO and IL-6. This was preceded by NF-kB activation, and up-regulation of relevant mRNAs. Also GFAP-negative astrocytes were fully inflammation-competent. Neurotrophic support by MEDA was found to be independent of GFAP expression. In summary, we described here the generation and functional characterization of microglia-free murine astrocytes, displaying phenotypic heterogeneity as is commonly observed in brain astrocytes.

microRNA 184 regulates expression of NFAT1 in umbilical cord blood CD4+ T cells.

The reduced expression of nuclear factor of activated T cells-1 (NFAT1) protein in umbilical cord blood (UCB)-derived CD4+ T cells and the corresponding reduction in inflammatory cytokine secretion after stimulation in part underlies their phenotypic differences from adult blood (AB) CD4+ T cells. This muted response may contribute to the lower incidence and severity of high-grade acute graft-versus-host disease (aGVHD) exhibited by UCB grafts. Here we provide evidence that a specific microRNA, miR-184, inhibits NFAT1 protein expression elicited by UCB CD4+ T cells. Endogenous expression of miR-184 in UCB is 58.4-fold higher compared with AB CD4+ T cells, and miR-184 blocks production of NFAT1 protein through its complementary target sequence on the NFATc2 mRNA without transcript degradation. Furthermore, its negative effects on NFAT1 protein and downstream interleukin-2 (IL-2) transcription are reversed through antisense blocking in UCB and can be replicated via exogenous transfection of precursor miR-184 into AB CD4+ T cells. Our findings reveal a previously uncharacterized role for miR-184 in UCB CD4+ T cells and a novel function for microRNA in the early adaptive immune response.

Recipient-specific tolerance after HLA-mismatched umbilical cord blood stem cell transplantation.

BACKGROUND: Lower incidence and severity of acute graft versus host disease (GVHD) has been observed in leukemia patients receiving HLA-mismatched umbilical cord (UCB) transplants. However, despite the increased use of UCB in stem cell transplantation, the mechanisms underlying these favorable outcomes are not well delineated. METHODS: We analyzed antigen specific lymphocyte responses after transplant to determine whether the decreased allogeneic responsiveness of UCB lymphocytes is attributable to pan-unresponsiveness, lymphocyte repressive or recipient-specific tolerance. RESULTS: Circulating lymphocytes collected early (3 months) after UCB transplant demonstrate a less naïve phenotype compared with that in the infused graft. Additionally, after transplant, circulating peripheral blood UCB-derived lymphocytes produced normal levels of interferon-gamma and proliferated normally when stimulated with mitogen or third party alloantigen. In contrast, when stimulated with recipient antigen, circulating lymphocytes emerging posttransplant did not proliferate nor produce interferon-gamma. Moreover, analysis of interleukin-4 production revealed a Th2 response to recipient antigens. These data indicate early induction of immune tolerance of naïve UCB graft lymphocytes with skewing of transplant recipient-specific immune response towards Th2 cytokine profile. CONCLUSIONS: UCB graft lymphocyte immune naivety and observed early tolerance induction may contribute to the observed favorable GVHD incidence, despite infusion of HLA mismatch grafts in the unrelated allogeneic setting.

Reduced CTLA-4 protein and messenger RNA expression in umbilical cord blood T lymphocytes.

OBJECTIVE: A favorable incidence and severity of graft-vs-host disease is observed in patients transplanted with banked, unrelated, HLA-mismatched umbilical cord blood (UCB) grafts, while the incidence of malignant relapse remains low. CTLA-4 mediates negative T-cell signaling and may contribute to the development of allogeneic tolerance. In this study, we compared protein and mRNA expression of CTLA-4 in stimulated UCB and adult peripheral blood T cells. MATERIALS AND METHODS: T cells were isolated from UCB and adult peripheral blood and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Cells were immunostained and analyzed by flow cytometry for both surface and intracellular expression of CTLA-4 in the presence and absence of cyclosporin A, and kinetics of CTLA-4 expression compared. CTLA-4 mRNA expression was measured using quantitative real-time polymerase chain reaction. NFAT1 protein levels were measured by Western blot analysis. RESULTS: These studies demonstrate reduced surface and intracellular expression of CTLA-4 in stimulated UCB T cells compared to adult controls. Furthermore, reduced CTLA-4 protein expression in UCB T cells was noted to be in part transcriptionally regulated, as CTLA-4 mRNA levels also were significantly lower. Reduced CLTA-4 expression by UCB T cells followed the kinetics of delayed and reduced expression of the transcription factor NFAT1 by UCB T lymphocytes during primary stimulation. Moreover, cyclosporin A, which is known to modulate NFAT activation, reduced CTLA-4 protein expression in adult and UCB T cells. CONCLUSION: Reduced expression of the key regulatory proteins CTLA-4 and NFAT-1 may contribute to favorable UCB T lymphocyte allogeneic responses.

Metabolite profiling in posttraumatic stress disorder.

BACKGROUND: Traumatic stress does not only increase the risk for posttraumatic stress disorder (PTSD), but is also associated with adverse secondary physical health outcomes. Despite increasing efforts, we only begin to understand the underlying biomolecular processes. The hypothesis-free assessment of a wide range of metabolites (termed metabolite profiling) might contribute to the discovery of biological pathways underlying PTSD. METHODS: Here, we present the results of the first metabolite profiling study in PTSD, which investigated peripheral blood serum samples of 20 PTSD patients and 18 controls. We performed liquid chromatography (LC) coupled to Quadrupole/Time-Of-Flight (QTOF) mass spectrometry. Two complementary statistical approaches were used to identify metabolites associated with PTSD status including univariate analyses and Partial Least Squares Discriminant Analysis (PLS-DA). RESULTS: Thirteen metabolites displayed significant changes in PTSD, including four glycerophospholipids, and one metabolite involved in endocannabinoid signaling. A biomarker panel of 19 metabolites classifies PTSD with 85% accuracy, while classification accuracy from the glycerophospholipid with the highest differentiating ability already reached 82%. CONCLUSIONS: This study illustrates the feasibility and utility of metabolite profiling for PTSD and suggests lipid-derived and endocannabinoid signaling as potential biological pathways involved in trauma-associated pathophysiology.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Compound selection for in vitro modeling of developmental neurotoxicity.

Development of in vitro systems, such as those based on embryonic stem cell differentiation, depends on the selection of adequate test and training compounds. We recommend the use of two classes of positive controls, the "gold standard compounds" for which developmental neurotoxicity (DNT) has been proven in man, and the "pathway compounds" that are known to disrupt signalling pathways and key processes relevant for neuronal differentiation. We introduce the concept of toxicity endophenotypes (TEP) as changes in neuronal connectivity resulting from exposure to developmental toxicants. Thus, TEPs provide the scientific rationale for modeling DNT with simple in vitro models of key neurodevelopmental events. In this context, we discuss scientific and technical aspects of the test compound selection process. We suggest to include compounds with unspecific toxicity, besides negative control compounds, and we recommend tandem approaches to determine relative toxicities instead of absolute measures. Finally, we discuss how to avoid pitfalls by distinguishing between unspecific forms of cytotoxicity and specific developmental neurotoxicity. A compilation of compound lists corresponding to the above-discussed principles supplement this review.

In vitro immunogenicity of undifferentiated pluripotent stem cells (PSC) and derived lineages.

The observation that embryonic stem cells (ESCs) expressed reduced levels of major histocompatibility (MHC) class I genes, no MHC class II or costimulatory molecules suggested early on that pluripotent stem cells (PSCs) could be "immune-privileged" and were unable to induce immune reactions. However, soon it became apparent that in some instances, ESCs were recognized by immune cells but still could reduce an active and strong immune response. Similar results were obtained with other PSCs. Almost 10 years later, the exact mechanisms are still not well understood and seem to differ between the different human and rodent PSC lines (even between different murine cell lines). These differences could be due to differing experimental approaches, different derivation protocols (to obtain the PSC lines), species specificity, or genetic background of the cells lines. A better understanding of the immune regulatory mechanisms deployed by PSCs and early derivates may inform us on immune regulation and could be exploitable for regenerative medicine using allogeneic cells. As PSCs grow robustly in culture and can easily be gene-modified, one could envision the generation of cell lines that maintain these immune suppressive properties through terminal differentiation, thus generating universal donor cells.

Sensitivity of dopaminergic neuron differentiation from stem cells to chronic low-dose methylmercury exposure.

Perinatal exposure to low doses of methylmercury (MeHg) can cause adult neurological symptoms. Rather than leading to a net cell loss, the toxicant is assumed to alter the differentiation and neuronal functions such as catecholaminergic transmission. We used neuronally differentiating murine embryonic stem cells (mESC) to explore such subtle toxicity. The mixed neuronal cultures that formed within 20 days contained a small subpopulation of tyrosine hydroxylase (TH)-positive neurons with specific dopaminergic functions such as dopamine transport (DAT) activity. The last 6 days of differentiation were associated with the functional maturation of already preformed neuronal precursors. Exposure to MeHg during this period downregulated several neuronal transcripts, without affecting housekeeping genes or causing measurable cell loss. Profiling of mRNAs relevant for neurotransmitter systems showed that dopamine receptors were coordinately downregulated, whereas known counterregulatory systems such as galanin receptor 2 were upregulated. The chronic (6 days) exposure to MeHg, but not shorter incubation periods, attenuated the expression levels of endogenous neurotrophic factors required for the maturation of TH cells. Accordingly, the size of this cell population was diminished, and DAT activity as its signature function was lost. When mixed lineage kinase activity was blocked during MeHg exposure, DAT activity was restored, and the reduction of TH levels was prevented. Thus, transcriptional profiling in differentiating mESC identified a subpopulation of neurons affected by MeHg, and a pharmacological intervention was identified that specifically protected these cells.

Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing.

Developmental neurotoxicity (DNT) is a serious concern for environmental chemicals, as well as for food and drug constituents. Animal-based DNT models have relatively low sensitivity, and they are burdened by high work-load, cost and animal ethics. Murine embryonic stem cells (mESC) recapitulate several critical processes involved in the development of the nervous system if they are induced to differentiate into neural cells. They therefore represent an alternative toxicological model to predict human hazard. In this review, we discuss how mESC can be used for DNT assays. We have compiled a list of mRNA markers that define undifferentiated mESC (n = 42), neural stem cells (n = 73), astrocytes (n = 25) and the pattern of different neuronal and non-neuronal cell types generated (n = 57). We propose that transcriptional profiling can be used as a sensitive endpoint in toxicity assays to distinguish neural differentiation states during normal and disturbed development. Importantly, we believe that it can be scaled up to relatively high throughput whilst still providing rich information on disturbances affecting small cell subpopulations. Moreover, this approach can provide insight into underlying mechanisms and pathways of toxicity. We broadly discuss the methodological basis of marker lists and DNT assay design. The discussion is put in the context of a new generation of alternative assays (embryonic stem cell based DNT testing = ESDNT V2.0), that may later include human induced pluripotent stem cells, and that are not designed for 1:1 replacement of animal experiments, but are rather intended to improve human risk assessment by using independent scientific principles.

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.

Deficient IFN-gamma expression in umbilical cord blood (UCB) T cells can be rescued by IFN-gamma-mediated increase in NFATc2 expression.

Regulation of nuclear factor of activated T cells-c2 (NFATc2) gene expression is not clearly defined. We previously reported reduced NFATc2 protein expression in cord blood T lymphocytes. Here we show that NFATc2 expression in T cells is dependent in part on the presence of IFN-gamma during primary stimulation, as blocking of IFN-gamma blunted NFATc2 protein and mRNA upregulation. Conversely, addition of exogenous IFN-gamma during stimulation resulted in increased expression of NFATc2 in cord blood T lymphocytes. This correlated with rescue of deficient IFN-gamma expression by cord blood T cells. Rescue of IFN-gamma expression in cord blood T cells was dependent on the presence of antigen-presenting cells, as addition of IFN-gamma during stimulation of purified cord blood T cells did not result in an increase of IFN-gamma expression, and depletion of monocytes ablated the rescue of IFN-gamma expression. Our results point to impaired function in the antigen-presenting cell population of cord blood, playing a role in the hyporesponsiveness of T cells.

Reduced expression of NFAT-associated genes in UCB versus adult CD4+ T lymphocytes during primary stimulation.

The cellular and molecular mechanisms underlying the blunted allo-responsiveness of umbilical cord blood (UCB) T cells have not been fully elucidated. Protein expression of NFATc2 (nuclear factor of activated T cells c2), a critical transcription factor necessary for up-regulation of multiple cytokines known to amplify T-cell allogeneic responses, is reduced in UCB T cells. Affymetrix oligonucleotide microarrays were used to compare gene expression of primary purified CD4+ UCB T cells to adult peripheral blood CD4+ T cells (AB) at baseline, 6, and 16 hours of primary stimulation. NFAT-regulated genes exhibited lower expression in UCB CD4+ T cells including the following: granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 3 (IL-3), IL-4, IL-5, IL-13, IL-2 receptor alpha (IL-2Ralpha; CD25), CD40L, and macrophage inflammatory protein 1 alpha (MIP-1alpha). Transcription factors involved in the NFAT pathway including C/EBPbeta, JunB, and Fosl1 (Fra-1), as well as Th1- and Th2-related transcription factors STAT4 (signal transducers and activators of transcription 4), T-bet, and c-maf showed reduced expression in UCB compared with AB during primary stimulation. Reduced cytokine, chemokine, and receptor expression was also found in UCB. Gene array data were confirmed using RNase protection assays, flow cytometry, and quantitative multiplexed cytokine measurements. Reduced global expression of NFAT-associated genes, as well as cytokines and chemokines, in UCB CD4+ T cells may contribute to the decreased graft-versus-host disease (GVHD) observed after UCB transplantation.

Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer.

Allogeneic transplantation with umbilical cord blood (UCB) is limited in adult recipients by a low CD34(+) cell dose. Clinical trials incorporating cytokine-based UCB in vitro expansion have not demonstrated significant shortening of hematologic recovery despite substantial increases in CD34(+) cell dose, suggesting loss of stem cell function. To sustain stem cell function during cytokine-based in vitro expansion, a feeder layer of human mesenchymal stem cells (MSCs) was incorporated in an attempt to mimic the stem cell niche in the marrow microenvironment. UCB expansion on MSCs resulted in a 7.7-fold increase in total LTC-IC output and a 3.8-fold increase of total early CD34(+) progenitors (CD38(-)/HLA-DR(-)). Importantly, early CD34(+)/CD38(-)/HLA-DR(-) progenitors from cultures expanded on MSCs demonstrated higher cytoplasmic expression of the cell-cycle inhibitor, p21(cip1/waf1), and the antiapoptotic protein, BCL-2, compared with UCB expanded in cytokines alone, suggesting improved maintenance of stem cell function in the presence of MSCs. Moreover, the presence of MSCs did not elicit UCB lymphocyte activation. Taken together, these results strongly suggest that the addition of MSCs as a feeder layer provides improved conditions for expansion of early UCB CD34(+)/CD38(-)/HLA-DR(-) hematopoietic progenitors and may serve to inhibit their differentiation and rates of apoptosis during short-term in vitro expansion.