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Recently our group demonstrated that acellular tissue engineered vessels (A-TEVs) comprised of small intestinal submucosa (SIS) immobilized with heparin and vascular endothelial growth factor (VEGF) could be implanted into the arterial system of a pre-clinical ovine animal model, where they endothelialized within one month and remained patent. Here we report that immobilized VEGF captures blood circulating monocytes (MC) with high specificity under a range of shear stresses. Adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. In-vivo, newly recruited cells on the vascular lumen express MC markers and at later times they co-express MC and EC-specific proteins and maintain graft patency. This novel finding indicates that the highly prevalent circulating MC contribute directly to the endothelialization of acellular vascular grafts under the right chemical and biomechanical cues. Acellular tissue engineered vessels functionalised with VEGF are coated with a layer of endothelial cells after in vivo implantation, but the source of the cells are unknown. Here the authors provide evidence that monocytes expressing VEGF receptors can transdifferentiate into endothelial cells via a macrophage intermediate.

A constitutive mutation in a cancer-susceptibility gene can have implications for clinical treatment and genetic counseling of family members. This study involving 1120 children and adolescents showed that 95 (8.5%) had such a mutation. The frequency of germline mutations in cancer-predisposition genes in children and adolescents with cancer and the implications of such mutations are largely unknown. Previous studies have relied mainly on candidate-gene approaches, which are, by design, limited. To better determine the contribution of germline predisposition mutations to childhood cancer, we used next-generation sequencing, including whole-genome and whole-exome sequencing, to analyze the genomes of 1120 children and adolescents with cancer. We describe the prevalence and spectrum of germline variants among 565 cancer-associated genes, with an emphasis on the analysis of 60 genes that have been associated with autosomal dominant cancer-predisposition syndromes. We . . .

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage. Atoh7 is essential for retinal ganglion cell (RGC) genesis. Here the authors perform scRNAseq on embryonic mouse retina and conclude that Atoh7 defines a transitional state and that other factors also participate in establishing the RGC lineage.

To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing. Clinical oncology is rapidly adopting next-generation sequencing technology for nucleotide variant and indel detection. Here the authors present a three-platform approach (whole-genome, whole-exome, and whole-transcriptome) in pediatric patients for the detection of diverse types of germline and somatic variants.

A common feature shared by systemic fungal pathogens of environmental origin, such as Cryptococcus neoformans , is their ability to adapt to mammalian core body temperature. In C. neoformans , this adaptation is accompanied by Ccr4-mediated decay of ribosomal protein mRNAs. Here we use the related, but thermo-intolerant species Cryptococcus amylolentus to demonstrate that this response contributes to host-temperature adaptation and pathogenicity of cryptococci. In a C. neoformans ccr4 Δ mutant, stabilized ribosomal protein mRNAs are retained in the translating pool, and stress-induced transcriptomic changes are reduced in comparison with the wild type strain, likely due to ineffective translation of transcription factors. In addition, the mutant displays increased exposure of cell wall glucans, and recognition by Dectin-1 results in increased phagocytosis by lung macrophages, linking mRNA decay to adaptation and immune evasion. The fungal pathogen Cryptococcus neoformans can adapt to mammalian core body temperature. Here, Bloom et al. show that Ccr4-mediated decay of ribosomal protein mRNAs is important for thermotolerance and immune evasion by promoting masking of cell wall glucans.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse–human chimeric embryos. Here, we describe a protocol for generating mouse–human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3–4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1–4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals. The authors provide a protocol for generating mouse–human chimeric embryos by injecting naive human pluripotent stem cells converted from the primed state.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX–histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma. In most cancers, mutations that lead to oncogene activation and tumor suppressor inactivation synergize to promote tumorigenesis. However, in neuroblastomas, MYCN amplification and ATRX mutations are mutually exclusive and incompatible.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

Background Single-cell genome sequencing provides high-resolution details of the clonal genomic modifications that occur during cancer initiation, progression, and ongoing evolution as patients undergo treatment. One limitation of current single-cell sequencing strategies is a suboptimal capacity to detect all classes of single-nucleotide and structural variants in the same cells. Results Here we present a new approach for determining comprehensive variant profiles of single cells using a microfluidic amplicon-based strategy to detect structural variant breakpoint sequences instead of using relative read depth to infer copy number changes. This method can reconstruct the clonal architecture and mutational history of a malignancy using all classes and sizes of somatic variants, providing more complete details of the temporal changes in mutational classes and processes that led to the development of a malignant neoplasm. Using this approach, we interrogated cells from a patient with leukemia, determining that processes producing structural variation preceded single nucleotide changes in the development of that malignancy. Conclusions All classes and sizes of genomic variants can be efficiently detected in single cancer cells using our new method, enabling the ordering of distinct classes of mutations during tumor evolution.