A novel mouse model of diffuse midline glioma initiated in neonatal oligodendrocyte progenitor cells highlights cell-of-origin dependent effects of H3K27M.

Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.

Optic vesicle morphogenesis requires primary cilia.

Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b-/- eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.

Bidirectional developmental potential in reprogrammed cells with acquired pluripotency.

We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency.

Emergence of dorsal-ventral polarity in ESC-derived retinal tissue.

We previously demonstrated that mouse embryonic stem cell (mESC)-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro Here, we demonstrate that mESC-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 and Vax2 were expressed in a mutually exclusive manner, as seen in vivo Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of mESC-derived retina, we examined the effects of patterning factors, and found that endogenous BMP signaling plays a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

Stem cells and genome editing: approaches to tissue regeneration and regenerative medicine.

Understanding the basis of regeneration of each tissue and organ, and incorporating this knowledge into clinical treatments for degenerative tissues and organs in patients, are major goals for researchers in regenerative biology. Here we provide an overview of current work, from high-regeneration animal models, to stem cell-based culture models, transplantation technologies, large-animal chimeric models, and programmable nuclease-based genome-editing technologies. Three-dimensional culture generating organoids, which represents intact tissue/organ identity including cell fate and morphology are getting more general approaches in the fields by taking advantage of embryonic stem cells, induced pluripotent stem cells and adult stem cells. The organoid culture system potentially has profound impact on the field of regenerative medicine. We also emphasize that the large animal model, in particular pig model would be a hope to manufacture humanized organs in in vivo empty (vacant) niche, which now potentially allows not only appropriate cell fate identity but nearly the same property as human organs in size. Therefore, integrative and collaborative researches across different fields might be critical to the aims needed in clinical trial.

Self-organizing optic-cup morphogenesis in three-dimensional culture.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

Self-formation of functional adenohypophysis in three-dimensional culture.

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke's pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke's-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma.

Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Machine learning-based estimation of spatial gene expression pattern during ESC-derived retinal organoid development.

Organoids, which can reproduce the complex tissue structures found in embryos, are revolutionizing basic research and regenerative medicine. In order to use organoids for research and medicine, it is necessary to assess the composition and arrangement of cell types within the organoid, i.e., spatial gene expression. However, current methods are invasive and require gene editing and immunostaining. In this study, we developed a non-invasive estimation method of spatial gene expression patterns using machine learning. A deep learning model with an encoder-decoder architecture was trained on paired datasets of phase-contrast and fluorescence images, and was applied to a retinal organoid derived from mouse embryonic stem cells, focusing on the master gene Rax (also called Rx), crucial for eye field development. This method successfully estimated spatially plausible fluorescent patterns with appropriate intensities, enabling the non-invasive, quantitative estimation of spatial gene expression patterns within each tissue. Thus, this method could lead to new avenues for evaluating spatial gene expression patterns across a wide range of biology and medicine fields.

Supramolecular Nanofibers Block SARS-CoV-2 Entry into Human Host Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on its spike protein binding to angiotensin-converting enzyme 2 (ACE2) on host cells to initiate cellular entry. Blocking the interactions between the spike protein and ACE2 offers promising therapeutic opportunities to prevent infection. We report here on peptide amphiphile supramolecular nanofibers that display a sequence from ACE2 in order to promote interactions with the SARS-CoV-2 spike receptor binding domain. We demonstrate that displaying this sequence on the surface of supramolecular assemblies preserves its α-helical conformation and blocks the entry of a pseudovirus and its two variants into human host cells. We also found that the chemical stability of the bioactive structures was enhanced in the supramolecular environment relative to the unassembled peptide molecules. These findings reveal unique advantages of supramolecular peptide therapies to prevent viral infections and more broadly for other targets as well.

Lactate-dependent transcriptional regulation controls mammalian eye morphogenesis.

Mammalian retinal metabolism favors aerobic glycolysis. However, the role of glycolytic metabolism in retinal morphogenesis remains unknown. We report that aerobic glycolysis is necessary for the early stages of retinal development. Taking advantage of an unbiased approach that combines the use of eye organoids and single-cell RNA sequencing, we identify specific glucose transporters and glycolytic genes in retinal progenitors. Next, we determine that the optic vesicle territory of mouse embryos displays elevated levels of glycolytic activity. At the functional level, we show that removal of Glucose transporter 1 and Lactate dehydrogenase A gene activity from developing retinal progenitors arrests eye morphogenesis. Surprisingly, we uncover that lactate-mediated upregulation of key eye-field transcription factors is controlled by the epigenetic modification of histone H3 acetylation through histone deacetylase activity. Our results identify an unexpected bioenergetic independent role of lactate as a signaling molecule necessary for mammalian eye morphogenesis.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues.

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Fifteen-year trends and differences in mortality rates across sex, age, and race/ethnicity in patients with brainstem tumors.

BACKGROUND: Localization of tumors to the brainstem carries a poor prognosis, however, risk factors are poorly understood. We examined secular trends in mortality from brainstem tumors in the United States by age, sex, and race/ethnicity. METHODS: We extracted age-adjusted incidence-based mortality rates of brainstem tumors from the Surveillance, Epidemiology, and End Results (SEER) database between 2004 and 2018. Trends in age-adjusted mortality rate (AAMR) were compared by sex and race/ethnicity among the younger age group (0-14 years) and the older age group (>15 years), respectively. Average AAMRs in each 5-year age group were compared by sex. RESULTS: This study included 2039 brainstem tumor-related deaths between 2004 and 2018. Trends in AAMRs were constant during the study period in both age groups, with 3 times higher AAMR in the younger age group compared to the older age group. Males had a significantly higher AAMR in the older age group, while no racial differences were observed. Intriguingly, AAMRs peaked in patients 5-9 years of age (0.57 per 100 000) and in patients 80-84 years of age (0.31 per 100 000), with lower rates among middle-aged individuals. Among 5-9 years of age, the average AAMR for females was significantly higher than that of males (P = .017), whereas the reverse trend was seen among those 50-79 years of age. CONCLUSIONS: Overall trends in AAMRs for brainstem tumors were constant during the study period with significant differences by age and sex. Identifying the biological mechanisms of demographic differences in AAMR may help understand this fatal pathology.

Therapeutic targeting of transcriptional elongation in diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG. METHODS: Short hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival. RESULTS: Depletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs. CONCLUSIONS: SEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

Non-receptor tyrosine kinase CSK-1 controls pharyngeal muscle organization in Caenorhabditis elegans.

C-terminal Src kinase (Csk) is a non-receptor type of tyrosine kinase, and serves as an essential negative regulator of Src family tyrosine kinases (SFKs) in vertebrates. However, analyses of Csk and SFKs from primitive animals suggest that the Csk-mediated mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members. We examined in vivo roles of CSK-1, a Caenorhabditis elegans orthologue of Csk, by generating animals lacking csk-1 function. Although some csk-1 mutants died during embryogenesis, the majority of mutants died during the first stage of larval development. In csk-1 mutants, the function of pharyngeal muscles, the major site of CSK-1 expression, was severely damaged. The pumping of pharyngeal grinder cells became arrhythmic, causing disabled feeding. Electron microscopy showed that pharyngeal muscle filaments were disorientated in the csk-1 mutants. These indicate that CSK-1 is crucial for proper organization of pharyngeal muscles. However, the growth arrest phenotype in csk-1 mutants could not be suppressed by src-1 and/or src-2 mutation, and SRC-1 was not significantly activated in the csk-1 mutants. These results suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require C. elegans SFKs.

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Self-patterning of rostral-caudal neuroectoderm requires dual role of Fgf signaling for localized Wnt antagonism.

The neuroectoderm is patterned along a rostral-caudal axis in response to localized factors in the embryo, but exactly how these factors act as positional information for this patterning is not yet fully understood. Here, using the self-organizing properties of mouse embryonic stem cell (ESC), we report that ESC-derived neuroectoderm self-generates a Six3+ rostral and a Irx3+ caudal bipolarized patterning. In this instance, localized Fgf signaling performs dual roles, as it regulates Six3+ rostral polarization at an earlier stage and promotes Wnt signaling at a later stage. The Wnt signaling components are differentially expressed in the polarized tissues, leading to genome-wide Irx3+ caudal-polarization signals. Surprisingly, differentially expressed Wnt agonists and antagonists have essential roles in orchestrating the formation of a balanced rostral-caudal neuroectoderm pattern. Together, our findings provide key processes for dynamic self-patterning and evidence that a temporally and locally regulated interaction between Fgf and Wnt signaling controls self-patterning in ESC-derived neuroectoderm.