Using Genome Editing to Engineer Universal Platelets

Genome editing technologies such as Zinc Finger nucleases, TALENS and CRISPR/Cas9 have recently emerged as tools with the potential to revolutionise cellular therapy. This is particularly exciting for the field of regenerative medicine, where the large-scale, quality controlled editing of large numbers of cells could generate essential cellular products ready to move towards the clinic. This review details recent progress towards generating HLA Class-I null platelets using genome editing technologies for beta-2-microglobulin deletion, generating a universally transfusable cellular product. In addition, we discuss various methods for megakaryocyte (MK) production from human pluripotent stem cells and subsequent platelet production from the MKs. As well as simply producing platelets, differentiating MK cultures can enable us to understand megakaryopoeisis in vivo and take steps towards ameliorating bleeding disorders or deficiencies in MK maturation in patients. Thus by intersecting both these areas of research, we can produce optimised differentiation systems for the production of universal platelets, thus offering a stable supply of platelets for difficult-to-match patients and providing areas with transmissible disease concerns or an unpredictable supply of platelets with a steady supply of quality controlled platelet units.UK Regenerative Medicine Platform and the Pluripotent and Engineered Cell Hub. Research in the laboratory is supported by core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institut

The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) plays crucial roles in brain function. Numerous studies report alterations in BDNF levels in human serum in various neurological conditions, including mood disorders such as depression. However, little is known about BDNF levels in the blood during pregnancy. We asked whether maternal depression and/or anxiety during pregnancy were associated with altered serum BDNF levels in mothers (n = 251) and their new-born infants (n = 212). As prenatal exposure to maternal mood disorders significantly increases the risk of neurological conditions in later life, we also examined the possibility of placental BDNF transfer by developing a new mouse model. We found no association between maternal symptoms of depression and either maternal or infant cord blood serum BDNF. However, maternal symptoms of anxiety correlated with significantly raised maternal serum BDNF exclusively in mothers of boys (r = 0.281; P = 0.005; n = 99). Serum BDNF was significantly lower in male infants than female infants but neither correlated with maternal anxiety symptoms. Consistent with this observation, we found no evidence for BDNF transfer across the placenta. We conclude that the placenta protects the developing fetus from maternal changes in serum BDNF that could otherwise have adverse consequences for fetal development

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnβ, Il1β, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal

Interferon-γ signaling in human iPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes.

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-γ (IFN-γ), in offspring's brains play a central role. IFN-γ activates an antiviral cellular state, limiting viral entry and replication. Moreover, IFN-γ is implicated in brain development. We tested the hypothesis that IFN-γ signaling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. Transient IFN-γ treatment of neural progenitors derived from human induced pluripotent stem cells increased neurite outgrowth. RNA sequencing analysis revealed that major histocompatibility complex class I (MHCI) genes were persistently up-regulated through neuronal differentiation-an effect that was mediated by IFN-γ-induced promyelocytic leukemia protein (PML) nuclear bodies. Critically, IFN-γ-induced neurite outgrowth required both PML and MHCI. We also found evidence that IFN-γ disproportionately altered the expression of genes associated with schizophrenia and autism, suggesting convergence between genetic and environmental risk factors. Together, these data implicate IFN-γ signaling in neurodevelopmental disorder etiology

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology

CRLF3 plays a key role in the final stage of platelet genesis and is a potential therapeutic target for thrombocythemia.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3-/- preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia

Glyphosate targets fish monoaminergic systems leading to oxidative stress and anxiety

Artículo científico indizadoGlyphosate is the active ingredient of some of the most highly produced and used herbicides worldwide. The intensive applications of glyphosate-based herbicides and its half-life in water lead to its presence in many aquatic ecosystems. Whereas recent studies have reported neurotoxic effects of glyphosate including autism- related effects, most of them used extremely high (mg/L to g/L) concentrations, so it is still unclear if chronic, low environmentally relevant concentrations of this compound (ng/L to μg/L) can induce neurotoxicity. In this study we analyzed the neurotoxicity of glyphosate in adult zebrafish after waterborne exposure to environmentally relevant concentrations (0.3 and 3 μg/L) for two weeks. Our data showed that exposed fish presented a significant impairment of exploratory and social behaviors consistent with increased anxiety. The anterior brain of the exposed fish presented a significant increase in dopamine and serotonin levels, as well as in the DOPAC/dopamine and homovanillic acid/dopamine turnover ratios. Moreover, the expression of genes involved in the dopaminergic system, as th1, th2, comtb, and scl6a3 was downregulated. Finally, the brain of exposed fish presented a significant increase in the catalase and superoxide dismutase activities, with a concomitant decrease of glutathione stores. These changes in the antioxidant defense system are consistent with the observed increase in oxidative stress, reflected by the increase in the levels of lipid peroxidation in the brain. The presented results show that current glyphosate concentrations commonly found in many aquatic ecosystems may have detrimental consequences on fish survival by decreasing exploration of the environment or altering social interactions. Furthermore, as zebrafish is also a vertebrate model widely used in human neurobehavioral studies, these results are relevant not only for environmental risk assessment, but also for understanding the risk of chronic low-dose exposures on human health.This work was supported by the Spanish Government with FEDER Funds (CTM2017-83242-R; D.R.) and the network of recognized research groups by the Catalan Government (2017 SGR_902). J.B. was supported by a Spanish fellowship PRE2018-083513. Mention of spe- cific products or trade names does not indicate endorsement by the US federal government

Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays

As a response to insect attack, maize (Zea mays) has inducible defenses that involve large changes in gene expression and metabolism. Piercing/sucking insects such as corn leaf aphid (Rhopalosiphum maidis) cause direct damage by acquiring phloem nutrients as well as indirect damage through the transmission of plant viruses. To elucidate the metabolic processes and gene expression changes involved in maize responses to aphid attack, leaves of inbred line B73 were infested with corn leaf aphids for 2 to 96 h. Analysis of infested maize leaves showed two distinct response phases, with the most significant transcriptional and metabolic changes occurring in the first few hours after the initiation of aphid feeding. After 4 d, both gene expression and metabolite profiles of aphid-infested maize reverted to being more similar to those of control plants. Although there was a predominant effect of salicylic acid regulation, gene expression changes also indicated prolonged induction of oxylipins, although not necessarily jasmonic acid, in aphid-infested maize. The role of specific metabolic pathways was confirmed using Dissociator transposon insertions in maize inbred line W22. Mutations in three benzoxazinoid biosynthesis genes, Bx1, Bx2, and Bx6, increased aphid reproduction. In contrast, progeny production was greatly decreased by a transposon insertion in the single W22 homolog of the previously uncharacterized B73 terpene synthases TPS2 and TPS3. Together, these results show that maize leaves shift to implementation of physical and chemical defenses within hours after the initiation of aphid feeding and that the production of specific metabolites can have major effects in maize-aphid interactions.Peer reviewe