Hierarchical reactivation of transcription during mitosis-to-G1 transition by Brn2 and Ascl1 in neural stem cells.

During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. In contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, respectively. Moreover, by using a mitotic-specific dominant-negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition.

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro.

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies-a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Transplantation of Human Skin-Derived Mesenchymal Stromal Cells Improves Locomotor Recovery After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

EGF-FGF2 stimulates the proliferation and improves the neuronal commitment of mouse epidermal neural crest stem cells (EPI-NCSCs).

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF2) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF2, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins ßIII-tubulin and nestin, as well as upregulation of the pan neuronal marker ßIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF-FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF-FGF2 in neuronal differentiation protocols.

JAK/STAT signaling promotes the emergence of unique cell states in ulcerative colitis.

The intestinal epithelium ensures uptake of vital nutrients and acts as a barrier between luminal contents and the underlying immune system. In inflammatory bowel diseases, such as ulcerative colitis (UC), this barrier is compromised, and patients experience debilitating symptoms. Here, we perform single-cell RNA profiling of epithelial cells and outline patterns of cell fate decisions in healthy individuals and UC patients. We demonstrate that patterns of hierarchical behavior are altered in UC patients and identify unique cellular states associated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation in ulcerated and non-ulcerated areas of the colonic epithelium. These transcriptional changes could be recapitulated in human colonic organoids, wherein cytokine-mediated activation of JAK/STAT led to the emergence of cell populations with augmented regenerative properties. Altogether, our findings indicate that intricate relationships between epithelial and cytokine signaling regulate cell fate during epithelial tissue regeneration in humans and have important implications for the understanding of UC biology.

Influence of vitamin D receptor signaling and vitamin D on colonic epithelial cell fate decisions in ulcerative colitis.

BACKGROUND AND AIMS: Epidemiological studies have shown that subnormal levels of vitamin D (25(OH)D) are associated with a more aggravated clinical course of ulcerative colitis (UC). Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor (VDR) signaling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25(OH)2D3)/VDR signaling in human organoids could influence the maintenance of the colonic epithelium. METHODS: Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterize the functional and transcriptional effects of 1,25(OH)2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. RESULTS: Our results suggest that 1,25(OH)2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25(OH)2D3/VDR signaling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. CONCLUSIONS: We identified an important role of vitamin D signaling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signaling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/ß-catenin to induce exit from quiescence.

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/ß-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional ß-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Benchmarking brain organoid recapitulation of fetal corticogenesis.

Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients' genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols. We evaluated the relevance of identified gene modules for neurodevelopmental disorders and psychiatric conditions finding significant enrichment of disease risk genes especially in modules related to neuronal maturation and synapsis development. The longitudinal transcriptomic analysis of CBO revealed a two-step differentiation composed of a fast-evolving phase, corresponding to the appearance of the main cell populations of the cortex, followed by a slow-evolving one characterized by milder transcriptional changes. Finally, we observed heterochronicity of differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as a resource to query for modeling human corticogenesis and the neuropsychiatric outcomes of its alterations.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Simultaneous disruption of PRC2 and enhancer function underlies histone H3.3-K27M oncogenic activity in human hindbrain neural stem cells.

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.

Regional identity of human neural stem cells determines oncogenic responses to histone H3.3 mutants.

Point mutations within the histone H3.3 are frequent in aggressive childhood brain tumors known as pediatric high-grade gliomas (pHGGs). Intriguingly, distinct mutations arise in discrete anatomical regions: H3.3-G34R within the forebrain and H3.3-K27M preferentially within the hindbrain. The reasons for this contrasting etiology are unknown. By engineering human fetal neural stem cell cultures from distinct brain regions, we demonstrate here that cell-intrinsic regional identity provides differential responsiveness to each mutant that mirrors the origins of pHGGs. Focusing on H3.3-G34R, we find that the oncohistone supports proliferation of forebrain cells while inducing a cytostatic response in the hindbrain. Mechanistically, H3.3-G34R does not impose widespread transcriptional or epigenetic changes but instead impairs recruitment of ZMYND11, a transcriptional repressor of highly expressed genes. We therefore propose that H3.3-G34R promotes tumorigenesis by focally stabilizing the expression of key progenitor genes, thereby locking initiating forebrain cells into their pre-existing immature state.

Genome Editing in Human Neural Stem and Progenitor Cells.

Experimental tools for precise manipulation of mammalian genomes enable reverse genetic approaches to explore biology and disease. Powerful genome editing technologies built upon designer nucleases, such as CRISPR/Cas9, have recently emerged. Parallel progress has been made in methodologies for the expansion and differentiation of human pluripotent and tissue stem cells. Together these innovations provide a remarkable new toolbox for human cellular genetics and are opening up vast opportunities for discoveries and applications across the breadth of life sciences research. In this chapter, we review the emergence of genome editing technologies and how these are being deployed in studies of human neurobiology, neurological disease, and neuro-oncology. We focus our discussion on CRISPR/Cas9 and its application in studies of human neural stem and progenitor cells.

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein.

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.