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

Stem cells and differentiation - a synoptic review of patents granted since 2009

Introduction: Innovations in human pluripotent stem cell research and their application in therapeutics have seen a giant leap in the past decade. Patent applications related to human pluripotent stem cell generation, culture and differentiation show an ever-increasing trend worldwide with hundreds of patents being applied for every year. With the turn of the second decade in stem cell patenting, a review of the latest patents issued will be significant. Areas covered: The growing need in healthcare sector has revolutionized stem cell application in clinical therapeutics by extending in unprecedented dimensions. With the potential of being able to differentiate into any desired adult cell lineage, human pluripotent stem cells find a wide range of applicability in clinical as well as cosmetic therapy. Moreover, the recent innovation of isolating a disease-specific pluripotent stem cell has opened new horizons to stem cell application in cell therapy. This review gives an overview of significant international patents granted on innovations in human pluripotent stem cell differentiation methodologies between 2009 and 2014. Expert opinion: The discovery of human pluripotent stem cells and their immense potential in clinical therapeutics has increasingly channeled scientific research in their orientation. Although being widely used to fathom human physiology, the trend in stem cell application is slowly shifting toward disease-modeling, drug safety evaluation and toxicity-testing. And in order to probe those unexplored realms of stem cell applications, a unified approach from the scientific community is imperative

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a gold standard cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action

Unique Metabolic Features of Stem Cells, Cardiomyocytes, and Their Progenitors

Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration. This metabolic switch of differentiating stem cells is required to cover the energy demands of the different organ-specific cell types. Among other metabolic signatures, amino acid and carbohydrate metabolism is most prominent in undifferentiated embryonic stem cells, whereas the fatty acid metabolic signature is unique in cardiomyocytes derived from embryonic stem cells. Identifying the specific metabolic pathways involved in pluripotency and differentiation is critical for further progress in the field of developmental biology and regenerative medicine. The recently generated knowledge on metabolic key processes may help to generate mature stem cell-derived somatic cells for therapeutic applications without the requirement of genetic manipulation. In the present review, the literature about metabolic features of stem cells and their cardiovascular cell derivatives as well as the specific metabolic gene signatures differentiating between stem and differentiated cells are summarized and discussed

High-efficient serum-free differentiation of endothelial cells from human iPS cells

Introduction Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. Method Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3 ',5 '-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. Result This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 +/- 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). Conclusion The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs

Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives

Abstract The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs. A transcriptome study performed at  differentiated short hairpin RNA (shRNA)-mediated Mageb16 knockdown (KD) ESCs and scrambled control (SCR) ESCs until a period of 22 days, revealed that Mageb16 KD ESCs mainly differentiated towards cells expressing mesodermal and cardiovascular lineage - gene markers. Gene markers of other mesoderm-oriented biological processes such as adipogenesis, osteogenesis, limb morphogenesis and spermatogenesis were also significantly enriched in the differentiated Mageb16 KD ESCs. The expression levels of contractile genes were higher in differentiated Mageb16 KD ESCs when compared to differentiated SCR and wild ESCs, suggesting a higher cardiomyogenic potential of Mageb16 depleted ESCs. Further analysis indicates  that regulative epigenetic networks and nucleocytoplasmic modifications induced by the depletion of Mageb16, may play a probable role in differentiation

Neuronal-specific deficiency of the splicing factor Tra2b causes apoptosis in neurogenic areas of the developing mouse brain.

Alternative splicing (AS) increases the informational content of the genome and is more prevalent in the brain than in any other tissue. The splicing factor Tra2b (Sfrs10) can modulate splicing inclusion of exons by specifically detecting GAA-rich binding motifs and its absence causes early embryonic lethality in mice. TRA2B has been shown to be involved in splicing processes of Nasp (nuclear autoantigenic sperm protein), MAPT (microtubule associated protein tau) and SMN (survival motor neuron), and is therefore implicated in spermatogenesis and neurological diseases like Alzheimer's disease, dementia, Parkinson's disease and spinal muscular atrophy. Here we generated a neuronal-specific Tra2b knock-out mouse that lacks Tra2b expression in neuronal and glial precursor cells by using the Nestin-Cre. Neuronal-specific Tra2b knock-out mice die immediately after birth and show severe abnormalities in cortical development, which are caused by massive apoptotic events in the ventricular layers of the cortex, demonstrating a pivotal role of Tra2b for the developing central nervous system. Using whole brain RNA on exon arrays we identified differentially expressed alternative exons of Tubulinδ1 and Shugoshin-like2 as in vivo targets of Tra2b. Most interestingly, we found increased expression of the cyclin dependent kinase inhibitor 1a (p21) which we could functionally link to neuronal precursor cells in the affected brain regions. We provide further evidence that the absence of Tra2b causes p21 upregulation and ultimately cell death in NSC34 neuronal-like cells. These findings demonstrate that Tra2b regulates splicing events essential for maintaining neuronal viability during development. Apoptotic events triggered via p21 might not be restricted to the developing brain but could possibly be generalized to the whole organism and explain early embryonic lethality in Tra2b-depleted mice

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

According to the new paradigm shift in toxicity testing, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Moreover, pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to be used in these mechanistic studies. In the present study, we compared the neuronal differentiation propensity of hESCs and hiPSCs aiming to develop tolls for mechanistic neurotoxicity testing. The gene expression and signaling pathway analyses demonstrate the activation of a similar neuronal signature in the two cellular models, particularly indicating that the neuronal survival related cAMP responsive element binding protein (CREB) pathway gets activated upon differentiation. Furthermore, analysis of CREB pathway inhibition, using 2-naphthol-AS-E-phosphate, shows a decrease in neuronal cells as well as an inhibition of neurite outgrowth, synaptogenesis and impairment of electrical activity. These data indicate that inhibition of the CREB pathway can be related to relevant endpoints for neurotoxicity testing in our in vitro cell model, and, as such, qualify the use of this cellular model for mechanism-based toxicity testingJRC.I.5-Systems Toxicolog

<i>p21</i> is upregulated as a response to <i>Tra2b</i> depletion in the mouse brain and in neural stem cells.

<p>(<b>A,B</b>) Semi-quantitative RT-PCR using whole brain RNA of neuronal specific <i>Tra2b</i> KO mice and controls. <i>p21</i> is significantly upregulated by 1.5-fold in KO mice as compared to HET or control mice. <i>p21</i> expression is indifferent between controls and HET mice. <i>p21</i> expression was normalized to <i>Hprt</i>. (<b>C–G</b>) NSC34 neural stem cells were transfected with siRNAs specific to <i>Tra2b</i> or scrambled siRNAs. siRNA treatment but not scr-treatment effectively reduced Tra2b protein and mRNA levels after 24 h, 48 h and 72 h after transfection (<b>C–E</b>). Tra2b function was strongly reduced as the <i>Nasp</i> transcript showed a significantly lower inclusion of the T-exon at 24 h, 48 h and 72 hours after transfection (<b>F</b>). 48 hours after transfection <i>p21</i> expression was found slightly but significantly increased on RNA level (<b>G</b>) but not on protein level (<b>D</b>). 72 hours after transfection p21 was massively and highly significantly upregulated on RNA and protein level by +2.2-fold (<b>D,G</b>). a.u., arbitrary units; nt, non-treated; scr, scrambled siRNA; si, siRNA against <i>Tra2b</i>; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p

Mouse whole exon array analysis reveals <i>Tubd1</i> exon4 and <i>Sgol2</i> exon4 as <i>in vivo</i> targets of <i>Tra2b</i>.

<p>Whole brain RNA of 4 CTRL animals and 4 KO animals was analyzed on mouse exon array. (<b>A</b>) The inclusion ratio (PSI, percent splicing inclusion) of each identified exon is defined as [PSI_KO]/[PSI_CTRL]. PSI distribution reached from ∼0.2 until ∼4.0. (<b>B</b>) Initial filtering strategies comprised exclusion of PSIs between 0.66 and 1.5 (grey bars) as well as restriction to p-values smaller than 0.05 which yielded a total of 1,006 exons. Exons associated with transcripts identified as being transcriptionally up- or downregulated were excluded from analysis. Ranking of those was further refined using large PSI values and considering presence of putative Tra2b binding sites (AGAA-motifs). Thereby, exons had to contain at least a single AGAA-site and a AGAA-frequency higher than 1.5. (<b>C,D,G,H</b>) Semi-quantitative RT-PCT on whole brain RNA was carried out using isoform specific primers for <i>Sgol2</i> FL (<b>C</b>), <i>Sgol2</i> Δ4 (<b>D</b>), <i>Tubd1</i> FL (<b>G</b>) and <i>Tubd1</i> Δ4 (<b>H</b>) confirming splicing events identified on the microarray. All isoform expression levels were densitometrically measured and normalized against <i>Hprt</i> (<b>E,F,I</b>). The <i>Tubd1</i> Δ4 isoform could not be detected using whole brain RNA, as skipping of exon4 introduces numerous premature termination codons leading to nonsense-mediated decay of the transcript (<b>H</b>). Treatment of wt and <i>Tra2b</i>-depleted murine embryonic fibroblasts with emetine successfully inhibited NMD and the <i>Tubd1</i> Δ4 isoform was detectable in <i>Tra2b</i>-depleted cells only (<b>J</b>). FL, full length; Δ4, transcript lacking exon 4, (−) PCR negative control; a. u., arbitrary units; error bars show the s.e.m.; significance levels are *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test).</p