- Email: [email protected]
The “Neurosphere-Assay” as a tool to study specific disturbances of FGF-2 function during neurodevelopment in vitro
Abstracts / Toxicology Letters 258S (2016) S62–S324
fibroblasts so that no exogenous collagen is required for skin formation. In the dermal tissue compartment collagen type I and III rich extracellular matrix components are expressed and show high structural similarities to the native dermis. The epidermis separates in proliferating keratinocytes (Ki67, EGFR) in the basal layer and differentiating cells (Keratin 10, filaggrin) in the suprabasal layers. The cells at the periphery are terminally differentiated and determine the cornified layer with a penetration barrier function. The exposure to TX-100 (1%) demonstrated resistance similar to established full thickness skin models (ET50 between 4.0 and 8.7 h). The miniaturized skin model reduces the required primary cells amounts up to 100-fold and allows for up-scaling and implementation into automated processes. Moreover the reliable manufacturing process enables efficient production and increase reproducibility of assay readouts. http://dx.doi.org/10.1016/j.toxlet.2016.06.1596 P08-054 High-throughput microfluidic platform for culture of 3D kidney tissue models M.K. Vormann ∗ , S.J. Trietsch, R.V. Vught, J. Joore, P. Vulto, H. Lanz Mimetas B.V., United States Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) model in Mimetas’ OrganoPlatesTM [1,2] to predict kidney toxicity. The OrganoPlateTM is a microfluidic platform, enabling highthroughput culture of boundary tissues in miniaturized organ models. In OrganoPlatesTM , extracellular matrix (ECM) gels can be freely patterned in microchambers by using PhaseGuidesTM . PhaseGuidesTM (capillary pressure barriers) define channels within microchambers that can be used for extracellular matrix deposition or medium perfusion. The microfluidic channel dimensions not only allow solid tissue and barrier formation, but also perfused tubular epithelial vessel structures can be grown in the medium perfusion channel. The goal of developing a perfused RPTC model is to reconstruct viable and leak-tight boundaries for performing cytotoxicity and metabolism assays, as well as transport and efficacy studies. The proximal tubule cell line LLC-PK1 was grown against an ECM gel in a three channel OrganoPlateTM , yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. The LLC-PK1 3D tubule stained positive for ZO-1 (tight junctions) and acetylated tubulin (polarization marker). Interestingly, cilia pointed in the direction of the lumen of tubules. The tightness of LLC-PK1 boundaries was assessed by diffusion of FITC-dextran dye added to the lumen of the tubule. Boundaries of LLC-PK1 showed leak-tight barriers and were maintained for several days. Furthermore, first experiments on primary human RPTEC resulted in partly leak tight tubules. http://dx.doi.org/10.1016/j.toxlet.2016.06.1597
S157
P08-055 3D networks of iPSC-derived neurons and glia for high-throughput neurotoxicity screening N. Wevers ∗ , S.J. Trietsch, R.V. Vught, J. Joore, P. Vulto, H. Lanz, K. Wilschut Mimetas B.V., United States The assessment of neurotoxicity remains a major scientific challenge due to the complexity of the central nervous system. Current strategies to evaluate toxicity of drugs and chemicals are predominantly based on ex vivo or in vivo animal studies. These models have limited predictability for neurotoxicity in humans and are not amenable to high-throughput testing. In order to overcome these limitations we are developing a neurotoxicity model based on iPSC-derived neurons in OrganoPlatesTM (1, 2). This microfluidic platform enables high-throughput screening of miniaturized organ models. A mixed population of human iPSC-derived neurons consisting of GABAergic and glutamatergic neurons with supporting astrocytes was cultured in 3D, closely representing the physiology of the human brain. As a part of the validation, proper network formation was observed by neuron-specific immunostainings and neuronal electrophysiology was analyzed by a calcium sensitive dye indicating spontaneous neuronal firing. Additionally, we investigated the dose-response neurotoxic effects of methylmercury and endosulfan on neuronal viability. The OrganoPlateTM platform enables real time analysis of neurotoxic effects of compounds in high-throughput. This iPSC-derived neuronal model can be used to refine animal experiments and has the potential to better predict adverse effect in humans and hence to improve clinical development success. http://dx.doi.org/10.1016/j.toxlet.2016.06.1598 P08-056 The “Neurosphere-Assay” as a tool to study specific disturbances of FGF-2 function during neurodevelopment in vitro M. Barenys ∗ , E. Kefalakes, E. Fritsche IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany Fibroblast Growth Factor 2 (FGF-2) is implied in numerous complex functions of embryonic and fetal neurodevelopment modulating for example proliferation, migration and differentiation of neural progenitor cells (NPCs). In this study, we adapted the protocols of the “Neurosphere-Assay” to assess specific disturbances of compounds on FGF-2 functions during neurodevelopment. FGF-2 induced concentration-dependent effects in migration, differentiation to neurons and proliferation in human and rodent neurospheres, being the rodent ones more sensitive to its effects. These concentration-dependent effects observed in vitro mimic the known distinct cellular responses occurring within the ventricularpial FGF-2 gradient in the developing brain in vivo, e.g. creating niches of proliferation or promoting neuronal differentiation. Besides, FGF-2 effects in the model were proved to be specific, as another growth factor from the same family, FGF-8, did not induce any of those concentration-dependent responses. By mechanistical studies, we unravelled the underlying signalling pathway guiding FGF-2-dependent migration of human NPCs and in addition, we identified a drug inhibiting FGF-2-dependent migration of human NPC in vitro.
S158
Abstracts / Toxicology Letters 258S (2016) S62–S324
This adapted “Neurosphere Assay” is an alternative in vitro approach to identify compounds disturbing FGF-2 signalling during brain development and thus can support neurodevelomental risk assessment by performing inter-species mode-of-action analyses. http://dx.doi.org/10.1016/j.toxlet.2016.06.1599 P08-057 Cellular and molecular evidence on the protection of embryonic stem cell-derived cardiomyocytes from phosphine-induced oxidative stress, mitochondrial dysfunction and apoptosis by mesenchymal stem cells derived microvesicles M. Rezvanfar ∗ , M. Baeeri, Z. Afsartala, M. Hodjat, A. Baghaei, M. Navaei Nigjeh, M. Rahimifard, M. Abdollahi Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran Embryonic stem cell (ESC)-derived beating cardiomyocytes is recently applied as an appropriate validated model for in vitro developmental toxicity assessment. Aluminum phosphide (AlP) is a widely used fumigant and organophosphorous rodenticide. In this regard, this study aimed to assess AlP-induced cardiotoxicity by using ESC-derived cardiomyocytes. The results revealed that AlP-cardiotoxicity was reduced by co-treatment with secreted microvesicles (MVs) from mesenchymal stem cells (MSCs). As a result, MVs significantly attenuated the oxidative stress parameters caused by AlP toxicity. Moreover, secreted MVs increased the activity of cytochrome oxidase, which in turn increased ATP production. Furthermore, flow cytometric assays and caspase activity indicated that MVs prohibited AlP-induced apoptosis in cardiomyocytes. Although the exact causal factor is not clear, we propose that MSC-derived MVs harbor a wide variety of MSC trophic factors (expressed at both mRNA and protein levels) which make it as a new therapeutic approach for reproductive/developmental toxicity of environmental toxicants. http://dx.doi.org/10.1016/j.toxlet.2016.06.1600 P08-058 Mesenchymal stem cell-derived microvesicles can prevent chemotherapy induced sperm DNA damage through suppression of free radicals toxic stress M. Rezvanfar 1,∗ , A. Mokarizadeh 2 , M. Ghasemzadeh Hasankolaei 3 , M. Baeeri 1 , M. Hodjat 1 , M. Abdollahi 1 1
Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran 2 Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran 3 Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Iran The reproductive system is highly susceptible to the toxic effects of cancer therapy. Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. Since, a variety of evidence has indicated that mesenchymal stem cells (MSCs)-derived microvesicles (MVs) are involved in regen-
eration of injured endogenous cells via shuttling MSCs trophic molecules, the present study investigated the molecular, cellular and biochemical mechanisms underlying possible protective effect of MVs as an emerging therapeutic agents in CIS-induced spermatotoxicity. A single IP dose of CIS (7 mg/kg) and protective dose of MVs (100 g/mouse/day) were administered alone or in combination and after 10 days, sperm characteristics, sperm DNA integrity and chromatin quality and spermatogenic disorders were assessed. In addition, the level of lipid peroxidation (LPO) and antioxidant enzymes and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. The CIS administration caused a significant increase in testicular and serum LPO and ONOO levels, along with a significant decrease in enzymatic antioxidants levels. Moreover, CIS significantly decreased serum T concentration and impaired sperm quality concomitant with increased DNA damage and decreased chromatin quality. Application of MVs preserved the serum T, sperm quality, and spermatogenesis and prevented CIS-induced free radicals’ toxic stress and DNA damage. This is the first report which determined successful application of MSCs-derived MVs as a novel protective strategy in CIS-induced spermatotoxicity. http://dx.doi.org/10.1016/j.toxlet.2016.06.1601 P08-059 A novel standardized in vitro islet model system for efficacy and toxicity testing in pancreatic -cells B. Yesildag ∗ , A. Neelakandhan, S. Messner, W. Moritz InSphero AG, Schlieren, Switzerland The lack of in vitro models that reliably assess pancreatic islet function for high-throughput toxicity and efficacy testing remains a significant challenge in drug development. To address this unmet need, we have developed a standardized 3D islet microtissue model generated from dispersed islets and cultured in 96-well-plates in a single microtissues per well format. We observed robust glucose-dependent insulin secretion from islet microtissues across donors, with stable glucose responsiveness, viability and size during long-term culture, as well as homogenous size distribution, tissue architecture and cellular composition allowing high-throughput data acquisition with low intra-assay variability. To evaluate our model for compound risk assessment, human and rat islet microtissues from individual donors were used to assess chronic effects of marketed drugs previously associated with pancreatic -cell toxicity. Olanzapine (an antipsychotic drug), Tunicamycin (an ER stress-inducing antibiotic), Tacrolimus and Rapamycin (immunosuppressive agents), were evaluated in a dose-dependent manner for their influence on illet ATP and insulin content, chronic, basal and glucose-stimulated insulin secretion following 14 days of compound exposure. In human islet microtissues, Tunicamycin treatment resulted in decreased ATP (>1 g/ml) and insulin content (>0.1 g/ml), impaired chronic, basal and glucose-stimulated insulin secretion (>0.1 g/ml). Olanzapine decreased insulin content (>10 M) and impaired chronic insulin secretion (>10 M). Tacrolimus and Rapamycin suppressed chronic (>10 nM and 0.1 nM), basal (>0.01 nM and 0.1 nM) and glucose stimulated insulin secretion (>10 nM and 1 nM). Rat islet microtissues displayed similar trends in compound sensitivity. Robust long-term functionality of islet microtissues makes them an ideal model for in vitro assessment of drug-induced -cell injury. http://dx.doi.org/10.1016/j.toxlet.2016.06.1602
fibroblasts so that no exogenous collagen is required for skin formation. In the dermal tissue compartment collagen type I and III rich extracellular matrix components are expressed and show high structural similarities to the native dermis. The epidermis separates in proliferating keratinocytes (Ki67, EGFR) in the basal layer and differentiating cells (Keratin 10, filaggrin) in the suprabasal layers. The cells at the periphery are terminally differentiated and determine the cornified layer with a penetration barrier function. The exposure to TX-100 (1%) demonstrated resistance similar to established full thickness skin models (ET50 between 4.0 and 8.7 h). The miniaturized skin model reduces the required primary cells amounts up to 100-fold and allows for up-scaling and implementation into automated processes. Moreover the reliable manufacturing process enables efficient production and increase reproducibility of assay readouts. http://dx.doi.org/10.1016/j.toxlet.2016.06.1596 P08-054 High-throughput microfluidic platform for culture of 3D kidney tissue models M.K. Vormann ∗ , S.J. Trietsch, R.V. Vught, J. Joore, P. Vulto, H. Lanz Mimetas B.V., United States Drug toxicity remains a major issue in drug discovery and stresses the need for better predictive models. Here, we describe the development of a perfused renal proximal tubule cell (RPTC) model in Mimetas’ OrganoPlatesTM [1,2] to predict kidney toxicity. The OrganoPlateTM is a microfluidic platform, enabling highthroughput culture of boundary tissues in miniaturized organ models. In OrganoPlatesTM , extracellular matrix (ECM) gels can be freely patterned in microchambers by using PhaseGuidesTM . PhaseGuidesTM (capillary pressure barriers) define channels within microchambers that can be used for extracellular matrix deposition or medium perfusion. The microfluidic channel dimensions not only allow solid tissue and barrier formation, but also perfused tubular epithelial vessel structures can be grown in the medium perfusion channel. The goal of developing a perfused RPTC model is to reconstruct viable and leak-tight boundaries for performing cytotoxicity and metabolism assays, as well as transport and efficacy studies. The proximal tubule cell line LLC-PK1 was grown against an ECM gel in a three channel OrganoPlateTM , yielding access to both the apical and basal side. Confocal imaging revealed that the cells formed a tubular structure. The LLC-PK1 3D tubule stained positive for ZO-1 (tight junctions) and acetylated tubulin (polarization marker). Interestingly, cilia pointed in the direction of the lumen of tubules. The tightness of LLC-PK1 boundaries was assessed by diffusion of FITC-dextran dye added to the lumen of the tubule. Boundaries of LLC-PK1 showed leak-tight barriers and were maintained for several days. Furthermore, first experiments on primary human RPTEC resulted in partly leak tight tubules. http://dx.doi.org/10.1016/j.toxlet.2016.06.1597
S157
P08-055 3D networks of iPSC-derived neurons and glia for high-throughput neurotoxicity screening N. Wevers ∗ , S.J. Trietsch, R.V. Vught, J. Joore, P. Vulto, H. Lanz, K. Wilschut Mimetas B.V., United States The assessment of neurotoxicity remains a major scientific challenge due to the complexity of the central nervous system. Current strategies to evaluate toxicity of drugs and chemicals are predominantly based on ex vivo or in vivo animal studies. These models have limited predictability for neurotoxicity in humans and are not amenable to high-throughput testing. In order to overcome these limitations we are developing a neurotoxicity model based on iPSC-derived neurons in OrganoPlatesTM (1, 2). This microfluidic platform enables high-throughput screening of miniaturized organ models. A mixed population of human iPSC-derived neurons consisting of GABAergic and glutamatergic neurons with supporting astrocytes was cultured in 3D, closely representing the physiology of the human brain. As a part of the validation, proper network formation was observed by neuron-specific immunostainings and neuronal electrophysiology was analyzed by a calcium sensitive dye indicating spontaneous neuronal firing. Additionally, we investigated the dose-response neurotoxic effects of methylmercury and endosulfan on neuronal viability. The OrganoPlateTM platform enables real time analysis of neurotoxic effects of compounds in high-throughput. This iPSC-derived neuronal model can be used to refine animal experiments and has the potential to better predict adverse effect in humans and hence to improve clinical development success. http://dx.doi.org/10.1016/j.toxlet.2016.06.1598 P08-056 The “Neurosphere-Assay” as a tool to study specific disturbances of FGF-2 function during neurodevelopment in vitro M. Barenys ∗ , E. Kefalakes, E. Fritsche IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany Fibroblast Growth Factor 2 (FGF-2) is implied in numerous complex functions of embryonic and fetal neurodevelopment modulating for example proliferation, migration and differentiation of neural progenitor cells (NPCs). In this study, we adapted the protocols of the “Neurosphere-Assay” to assess specific disturbances of compounds on FGF-2 functions during neurodevelopment. FGF-2 induced concentration-dependent effects in migration, differentiation to neurons and proliferation in human and rodent neurospheres, being the rodent ones more sensitive to its effects. These concentration-dependent effects observed in vitro mimic the known distinct cellular responses occurring within the ventricularpial FGF-2 gradient in the developing brain in vivo, e.g. creating niches of proliferation or promoting neuronal differentiation. Besides, FGF-2 effects in the model were proved to be specific, as another growth factor from the same family, FGF-8, did not induce any of those concentration-dependent responses. By mechanistical studies, we unravelled the underlying signalling pathway guiding FGF-2-dependent migration of human NPCs and in addition, we identified a drug inhibiting FGF-2-dependent migration of human NPC in vitro.
S158
Abstracts / Toxicology Letters 258S (2016) S62–S324
This adapted “Neurosphere Assay” is an alternative in vitro approach to identify compounds disturbing FGF-2 signalling during brain development and thus can support neurodevelomental risk assessment by performing inter-species mode-of-action analyses. http://dx.doi.org/10.1016/j.toxlet.2016.06.1599 P08-057 Cellular and molecular evidence on the protection of embryonic stem cell-derived cardiomyocytes from phosphine-induced oxidative stress, mitochondrial dysfunction and apoptosis by mesenchymal stem cells derived microvesicles M. Rezvanfar ∗ , M. Baeeri, Z. Afsartala, M. Hodjat, A. Baghaei, M. Navaei Nigjeh, M. Rahimifard, M. Abdollahi Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran Embryonic stem cell (ESC)-derived beating cardiomyocytes is recently applied as an appropriate validated model for in vitro developmental toxicity assessment. Aluminum phosphide (AlP) is a widely used fumigant and organophosphorous rodenticide. In this regard, this study aimed to assess AlP-induced cardiotoxicity by using ESC-derived cardiomyocytes. The results revealed that AlP-cardiotoxicity was reduced by co-treatment with secreted microvesicles (MVs) from mesenchymal stem cells (MSCs). As a result, MVs significantly attenuated the oxidative stress parameters caused by AlP toxicity. Moreover, secreted MVs increased the activity of cytochrome oxidase, which in turn increased ATP production. Furthermore, flow cytometric assays and caspase activity indicated that MVs prohibited AlP-induced apoptosis in cardiomyocytes. Although the exact causal factor is not clear, we propose that MSC-derived MVs harbor a wide variety of MSC trophic factors (expressed at both mRNA and protein levels) which make it as a new therapeutic approach for reproductive/developmental toxicity of environmental toxicants. http://dx.doi.org/10.1016/j.toxlet.2016.06.1600 P08-058 Mesenchymal stem cell-derived microvesicles can prevent chemotherapy induced sperm DNA damage through suppression of free radicals toxic stress M. Rezvanfar 1,∗ , A. Mokarizadeh 2 , M. Ghasemzadeh Hasankolaei 3 , M. Baeeri 1 , M. Hodjat 1 , M. Abdollahi 1 1
Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran 2 Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran 3 Infertility and Reproductive Health Research Center, Babol University of Medical Sciences, Babol, Iran The reproductive system is highly susceptible to the toxic effects of cancer therapy. Cisplatin (CIS), an anticancer alkylating agent, induces DNA adducts and effectively cross links the DNA strands and so affects spermatozoa as a male reproductive toxicant. Since, a variety of evidence has indicated that mesenchymal stem cells (MSCs)-derived microvesicles (MVs) are involved in regen-
eration of injured endogenous cells via shuttling MSCs trophic molecules, the present study investigated the molecular, cellular and biochemical mechanisms underlying possible protective effect of MVs as an emerging therapeutic agents in CIS-induced spermatotoxicity. A single IP dose of CIS (7 mg/kg) and protective dose of MVs (100 g/mouse/day) were administered alone or in combination and after 10 days, sperm characteristics, sperm DNA integrity and chromatin quality and spermatogenic disorders were assessed. In addition, the level of lipid peroxidation (LPO) and antioxidant enzymes and peroxynitrite (ONOO) as a marker of nitrosative stress (NS) and testosterone (T) concentration as a biomarker of testicular function were measured in the blood and testes. The CIS administration caused a significant increase in testicular and serum LPO and ONOO levels, along with a significant decrease in enzymatic antioxidants levels. Moreover, CIS significantly decreased serum T concentration and impaired sperm quality concomitant with increased DNA damage and decreased chromatin quality. Application of MVs preserved the serum T, sperm quality, and spermatogenesis and prevented CIS-induced free radicals’ toxic stress and DNA damage. This is the first report which determined successful application of MSCs-derived MVs as a novel protective strategy in CIS-induced spermatotoxicity. http://dx.doi.org/10.1016/j.toxlet.2016.06.1601 P08-059 A novel standardized in vitro islet model system for efficacy and toxicity testing in pancreatic -cells B. Yesildag ∗ , A. Neelakandhan, S. Messner, W. Moritz InSphero AG, Schlieren, Switzerland The lack of in vitro models that reliably assess pancreatic islet function for high-throughput toxicity and efficacy testing remains a significant challenge in drug development. To address this unmet need, we have developed a standardized 3D islet microtissue model generated from dispersed islets and cultured in 96-well-plates in a single microtissues per well format. We observed robust glucose-dependent insulin secretion from islet microtissues across donors, with stable glucose responsiveness, viability and size during long-term culture, as well as homogenous size distribution, tissue architecture and cellular composition allowing high-throughput data acquisition with low intra-assay variability. To evaluate our model for compound risk assessment, human and rat islet microtissues from individual donors were used to assess chronic effects of marketed drugs previously associated with pancreatic -cell toxicity. Olanzapine (an antipsychotic drug), Tunicamycin (an ER stress-inducing antibiotic), Tacrolimus and Rapamycin (immunosuppressive agents), were evaluated in a dose-dependent manner for their influence on illet ATP and insulin content, chronic, basal and glucose-stimulated insulin secretion following 14 days of compound exposure. In human islet microtissues, Tunicamycin treatment resulted in decreased ATP (>1 g/ml) and insulin content (>0.1 g/ml), impaired chronic, basal and glucose-stimulated insulin secretion (>0.1 g/ml). Olanzapine decreased insulin content (>10 M) and impaired chronic insulin secretion (>10 M). Tacrolimus and Rapamycin suppressed chronic (>10 nM and 0.1 nM), basal (>0.01 nM and 0.1 nM) and glucose stimulated insulin secretion (>10 nM and 1 nM). Rat islet microtissues displayed similar trends in compound sensitivity. Robust long-term functionality of islet microtissues makes them an ideal model for in vitro assessment of drug-induced -cell injury. http://dx.doi.org/10.1016/j.toxlet.2016.06.1602