Msps governs acentrosomal microtubule assembly and reactivation of quiescent neural stem cells

The ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (NSCs) extend a primary cellular protrusion from the cell body prior to their reactivation. However, the structure and function of this protrusion are not well established. Here, we show that in the protrusion of quiescent NSCs, microtubules are predominantly acentrosomal and oriented plus‐end‐out toward the tip of the primary protrusion. We have identified Mini Spindles (Msps)/XMAP215 as a key microtubule regulator in quiescent NSCs that governs NSC reactivation via regulating acentrosomal microtubule growth and orientation. We show that quiescent NSCs form membrane contact with the neuropil and E‐cadherin, a cell adhesion molecule, localizes to these NSC‐neuropil junctions. Msps and a plus‐end directed motor protein Kinesin‐2 promote NSC cell cycle re‐entry and target E‐cadherin to NSC‐neuropil contact during NSC reactivation. Together, this work establishes acentrosomal microtubule organization in the primary protrusion of quiescent NSCs and the Msps‐Kinesin‐2 pathway that governs NSC reactivation, in part, by targeting E‐cad to NSC‐neuropil contact sites.     

Toll‐7 promotes tumour growth and invasion in Drosophila

Objectives Drosophila melanogaster has become an excellent model organism to explore the genetic mechanisms underlying tumour progression. Here, by using well‐established Drosophila tumour models, we identified Toll‐7 as a novel regulator of tumour growth and invasion.Materials and methodsTransgenic flies and genetic epistasis analysis were used. All flies were raised on a standard cornmeal and agar medium at 25°C unless otherwise indicated. Immunostaining and RT‐qPCR were performed by standard procedures. Images were taken by OLYMPUS BX51 microscope and Zeiss LSM 880 confocal microscope. Adobe Photoshop 2020 and Zeiss Zen were used to analyse the images. All results were presented in Scatter plots or Column bar graphs created by GraphPad Prism 8.0.ResultsLoss of Toll‐7 suppresses Ras^V12/lgl ^−/−‐induced tumour growth and invasion, as well as cell polarity disruption‐induced invasive cell migration, whereas expression of a constitutively active allele of Toll‐7 is sufficient to promote tumorous growth and cell migration. In addition, the Egr‐JNK signalling is necessary and sufficient for Toll‐7‐induced invasive cell migration. Mechanistically, Toll‐7 facilitates the endocytosis of Egr, which is known to activate JNK in the early endosomes. Moreover, Toll‐7 activates the EGFR‐Ras signalling, which cooperates with the Egr‐JNK signalling to promote Yki‐mediated cell proliferation and tissue overgrowth. Finally, Toll‐7 is necessary and sufficient for the proper maintenance of EGFR protein level.ConclusionsOur findings characterized Toll‐7 as a proto‐oncogene that promotes tumour growth and invasion in Drosophila, which shed light on the pro‐tumour function of mammalian Toll‐like receptors (TLRs).     

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron‐enriched genes

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron‐specific ChIP‐seq and RNA‐seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron‐specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B‐dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.     

Neural G0: a quiescent‐like state found in neuroepithelial‐derived cells and glioma

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

Fine‐tuning of dual‐SMAD inhibition to differentiate human pluripotent stem cells into neural crest stem cells

ObjectivesThe derivation of neural crest stem cells (NCSCs) from human pluripotent stem cells (hPSCs) has been commonly induced by WNT activation in combination with dual‐SMAD inhibition.In this study, by fine‐tuning BMP signalling in the conventional dual‐SMAD inhibition, we sought to generate large numbers of NCSCs without WNT activation.Materials and methodsIn the absence of WNT activation, we modulated the level of BMP signalling in the dual‐SMAD inhibition system to identify conditions that efficiently drove the differentiation of hPSCs into NCSCs. We isolated two NCSC populations separately and characterized them in terms of global gene expression profiles and differentiation ability.ResultsOur modified dual‐SMAD inhibition containing a lower dose of BMP inhibitor than that of the conventional dual‐SMAD inhibition drove hPSCs into mainly NCSCs, which consisted of HNK⁺p75high and HNK⁺p75low cell populations. We showed that the p75high population formed spherical cell clumps, while the p75low cell population generated a 2D monolayer.We detected substantial differences in gene expression profiles between the two cell groups and showed that both p75high and p75low cells differentiated into mesenchymal stem cells (MSCs), while only p75high cells had the ability to become peripheral neurons.ConclusionsThis study will provide a framework for the generation and isolation of NCSC populations for effective cell therapy for peripheral neuropathies and MSC‐based cell therapy.     

miR‐221 promotes keratinocyte proliferation and migration by targeting SOCS7 and is regulated by YB‐1

Proliferation and migration of keratinocytes are vital processes for the successful epithelization specifically after wounding. MiR‐221 has been identified to play a potential role in promoting wound regeneration by inducing blood vessel formation. However, little is known about the role of miR‐221 in the keratinocyte proliferation and migration during wound healing. An in vivo mice wound‐healing model was generated; the expression levels of miR‐221 were assessed by qRT‐PCR and fluorescence in situ hybridization. Initially, we found that miR‐221 was upregulated in the proliferative phase of wound healing. Further, in an in vivo wound‐healing mice model, targeted delivery of miR‐221 mimics accelerated wound healing. Contrastingly, inhibition of miR‐221 delayed healing. Additionally, we observed that overexpression of miR‐221 promoted cell proliferation and migration, while inhibition of miR‐221 had the opposite effects. Moreover, we identified SOCS7 as a direct target of miR‐221 in keratinocytes and overexpression of SOCS7 reversed the effects of miR‐221 in HaCaT keratinocytes. Finally, we identified that YB‐1 regulates the expression of miR‐221 in HaCaT keratinocytes. Overall, our experiments suggest that miR‐221 is regulated by YB‐1 in HaCaT keratinocytes and acts on SOCS7, thereby playing an important role in HaCaT keratinocyte proliferation and migration during wound healing.     

Stromal cell‐derived factor‐1/Exendin‐4 cotherapy facilitates the proliferation,migration and osteogenic differentiation of human periodontal ligament stem cells in vitro and promotes periodontal bone regeneration in vivo

ObjectivesStromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo.MethodsCell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo.ResultsSDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4⁺ cells and CD90⁺/CD34^‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo.ConclusionsSDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.     

ClC‐c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila

ObjectivesAdult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified.Materials and MethodsFlies were used to generate tissue‐specific gene knockdown and gene knockout. qRT‐PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA‐seq was used to screen downstream mechanisms.ResultsHere, we report a member of the chloride channel family, ClC‐c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, we found that the ISC loss induced by the depletion of ClC‐c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway.ConclusionOur findings reveal an ISC‐specific function of ClC‐c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis.     

Adipose‐derived stem cell spheroids are superior to single‐cell suspensions to improve fat autograft long‐term survival

Autologous fat transplantation is a widely used procedure for surgical reconstruction of tissues. The resorption rate of this transplantation remains high and unpredictable, reinforcing the need of adjuvant treatments that increase the long‐term stability of grafts. Adipose‐derived stem cells (ASC) introduced as single cells in fat has been shown clinically to reduce the resorption of fat grafts. On the other hand, the formulation of ASC into cell spheroids results in the enhancement of their regenerative potential. In this study, we developed a novel method to produce highly homogeneous ASC spheroids and characterized their features and efficacy on fat transplantation. Spheroids conserved ASC markers and multipotency. A regenerative gene expression profile was maintained, and genes linked to autophagy were upregulated whereas proliferation was decreased. Their secreted proteome was enriched in comparison with single‐cell ASC suspension. Addition of spheroids to fat graft in an animal model of transplantation resulted in a better graft long‐term stability when compared to single ASC suspension. In conclusion, we provide a novel method to manufacture homogenous ASC spheroids. These ASC spheroids are superior to ASC in single‐cell suspension to improve the stability of fat transplants, reinforcing their potential in reconstructive surgery.     

Histone chaperone CAF‐1 promotes HIV‐1 latency by leading the formation of phase‐separated suppressive nuclear bodies

HIV‐1 latency is a major obstacle to achieving a functional cure for AIDS. Reactivation of HIV‐1‐infected cells followed by their elimination via immune surveillance is one proposed strategy for eradicating the viral reservoir. However, current latency‐reversing agents (LRAs) show high toxicity and low efficiency, and new targets are needed to develop more promising LRAs. Here, we found that the histone chaperone CAF‐1 (chromatin assembly factor 1) is enriched on the HIV‐1 long terminal repeat (LTR) and forms nuclear bodies with liquid–liquid phase separation (LLPS) properties. CAF‐1 recruits epigenetic modifiers and histone chaperones to the nuclear bodies to establish and maintain HIV‐1 latency in different latency models and primary CD4⁺ T cells. Three disordered regions of the CHAF1A subunit are important for phase‐separated CAF‐1 nuclear body formation and play a key role in maintaining HIV‐1 latency. Disruption of phase‐separated CAF‐1 bodies could be a potential strategy to reactivate latent HIV‐1.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Androgen receptor suppresses vasculogenic mimicry in hepatocellular carcinoma via circRNA7/miRNA7‐5p/VE‐cadherin/Notch4 signalling

Androgen receptor (AR) can suppress hepatocellular carcinoma (HCC) invasion and metastasis at an advanced stage. Vasculogenic mimicry (VM), a new vascularization pattern by which tumour tissues nourish themselves, is correlated with tumour progression and metastasis. Here, we investigated the effect of AR on the formation of VM and its mechanism in HCC. The results suggested that AR could down‐regulate circular RNA (circRNA) 7, up‐regulate micro RNA (miRNA) 7‐5p, and suppress the formation of VM in HCC Small hairpin circR7 (ShcircR7) could reverse the impact on VM and expression of VE‐cadherin and Notch4 increased by small interfering AR (shAR) in HCC, while inhibition of miR‐7‐5p blocked the formation of VM and expression of VE‐cadherin and Notch4 decreased by AR overexpression (oeAR) in HCC. Mechanism dissection demonstrated that AR could directly target the circR7 host gene promoter to suppress circR7, and miR‐7‐5p might directly target the VE‐cadherin and Notch4 3′UTR to suppress their expression in HCC. In addition, knockdown of Notch4 and/or VE‐cadherin revealed that shVE‐cadherin or shNotch4 alone could partially reverse the formation of HCC VM, while shVE‐cadherin and shNotch4 together could completely suppress the formation of HCC VM. Those results indicate that AR could suppress the formation of HCC VM by down‐regulating circRNA7/miRNA7‐5p/VE‐Cadherin/Notch4 signals in HCC, which will help in the design of novel therapies against HCC.     

Insights into neural stem cell biology from flies

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.     

B‐cell capacity for differentiation changes with age

BackgroundAge‐related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin.AimsTo compare the intrinsic B‐cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T‐cell help or activation via TLR engagement.MethodsB cells were isolated from healthy individuals, in younger (30–38 years) and older (60–64 years) donors. An in vitro model system of B‐cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T‐dependent (TD: CD40/BCR stimulation) or T‐independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR.ResultsTI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B‐cell‐executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age‐related differences in B‐cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient.ConclusionAltogether, B‐cell differentiation into PC appeared similar between age groups when provided with T‐cell help, in contrast to TI differentiation, where multiple age‐related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.     

Novel long non‐coding RNA CYB561‐5 promotes aerobic glycolysis and tumorigenesis by interacting with basigin in non‐small cell lung cancer

Abnormally expressed long non‐coding RNAs (lncRNAs) have been recognized as potential diagnostic biomarkers or therapeutic targets in non‐small cell lung cancer (NSCLC). The role of the novel lnc‐CYB561‐5 in NSCLC and its specific biological activity remain unknown. In this study, lncRNAs highly expressed in NSCLC tissue samples compared with paired adjacent normal tissue samples and atypical adenomatous hyperplasia were identified by RNA‐seq analysis. Lnc‐CYB561‐5 is highly expressed in human NSCLC and is associated with a poor prognosis in lung adenocarcinoma. In vivo, downregulation of lnc‐CYB561‐5 significantly decreases tumour growth and metastasis. In vitro, lnc‐CYB561‐5 knockdown treatment inhibits cell migration, invasion and proliferation ability, as well as glycolysis rates. In addition, RNA pulldown and RNA immunoprecipitation (RIP) assays show that basigin (Bsg) protein interacts with lnc‐CYB561‐5. Overall, this study demonstrates that lnc‐CYB561‐5 is an oncogene in NSCLC, which is involved in the regulation of cell proliferation and metastasis. Lnc‐CYB561‐5 interacts with Bsg to promote the expression of Hk2 and Pfk1 and further lead to metabolic reprogramming of NSCLC cells.