Exclusion of HDAC1/2 complexes by oncogenic nuclear condensates.

Nuclear condensates have been shown to regulate cell fate control, but its role in oncogenic transformation remains largely unknown. Here we show acquisition of oncogenic potential by nuclear condensate remodeling. The proto-oncogene SS18 and its oncogenic fusion SS18-SSX1 can both form condensates, but with drastically different properties and impact on 3D genome architecture. The oncogenic condensates, not wild type ones, readily exclude HDAC1 and 2 complexes, thus, allowing aberrant accumulation of H3K27ac on chromatin loci, leading to oncogenic expression of key target genes. These results provide the first case for condensate remodeling as a transforming event to generate oncogene and such condensates can be targeted for therapy. One sentence summary: Expulsion of HDACs complexes leads to oncogenic transformation.

Human umbilical cord mesenchymal stem cells-derived exosomes exert anti-inflammatory effects on osteoarthritis chondrocytes.

Inflammation of chondrocytes plays a critical role in the occurrence and development of osteoarthritis (OA). Recent evidence indicated exosomes derived from mesenchymal stem cells (MSCs-Exos) exhibit excellent anti-inflammatory ability in many troublesome inflammatory diseases including OA. In the present study, we aimed to explore the role of human umbilical cord-derived MSCs-Exos (hUC-MSCs-Exos) in treating the inflammation of chondrocytes and its related mechanisms. Ultracentrifugation was applied to isolate hUC-MSCs-Exos from the culture supernatant of hUC-MSCs. Two OA-like in vitro inflammation models of human articular chondrocytes induced with interleukin 1ß (IL-1ß) and co-incubation with macrophage utilizing transwell cell culture inserts were both used to evaluate the anti-inflammatory effects of hUC-MSCs-Exos. The mRNA sequencing of chondrocytes after treatment and microRNA (miRNA) sequencing of hUC-MSCs-Exos were detected and analyzed for possible mechanism analysis. The results of the study confirmed that hUC-MSCs-Exos had a reversed effect of IL-1ß on chondrocytes in the expression of collagen type II alpha 1 (COL2A1) and matrix metalloproteinase 13 (MMP13). The addition of hUC-MSCs-Exos to M1 macrophages in the upper chamber showed down-regulation of IL-1ß and tumor necrosis factor α (TNF-α), up-regulation of IL-10 and arginase1 (ARG1), and reversed the gene and protein expression of COL2A1 and MMP13 of the chondrocytes seeded in the lower chamber. The results of this study confirmed the anti-inflammatory effects of hUC-MSCs-Exos in the human articular chondrocytes inflammation model. hUC-MSCs-Exos may be used as a potential cell-free treatment strategy for chondrocyte inflammation in OA.

Research Progress on the Role and Mechanism of IL-37 in Liver Diseases.

Cytokines are important components of the immune system that can predict or influence the development of liver diseases. IL-37, a new member of the IL-1 cytokine family, exerts potent anti-inflammatory and immunosuppressive effects inside and outside cells. IL-37 expression differs before and after liver lesions, suggesting that it is associated with liver disease; however, its mechanism of action remains unclear. This article mainly reviews the biological characteristics of IL-37, which inhibits hepatitis, liver injury, and liver fibrosis by inhibiting inflammation, and inhibits the development of hepatocellular carcinoma (HCC) by regulating the immune microenvironment. Based on additional evidence, combining IL-37 with liver disease markers for diagnosis and treatment can achieve more significant effects, suggesting that IL-37 can be developed into a powerful tool for the clinical adjuvant treatment of liver diseases, especially HCC.

Inverted U Waves-Red Flags in Electrocardiograms.

This case report presents the electrocardiogram findings of a patient in their 50s with intermittent compression-like pain in the chest and a history of hypertension and diabetes.

ZBTB7A regulates primed-to-naïve transition of pluripotent stem cells via recognition of the PNT-associated sequence by zinc fingers 1-2 and recognition of γ-globin -200 gene element by zinc fingers 1-4.

ZBTB7A, a transcription factor containing a tandem array of four Cys2-His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all four ZFs (ZF1-4) are involved in binding to γ-globin -200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naïve transition (PNT) of pluripotent stem cells through binding to a 12-bp consensus sequence ([AAGGACCCAGAT], referred to as PNT-associated sequence). Here, we report a crystal structure of ZBTB7A ZF1-3 in complex with the PNT-associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ-globin -200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1-2 remarkably reduce their binding affinities for the PNT-associated sequence in vitro and cannot restore epiblast stem cells to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT-associated sequence but recognizes γ-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.

Biodegradable double-network GelMA-ACNM hydrogel microneedles for transdermal drug delivery.

As a minimally invasive drug delivery platform, microneedles (MNs) overcome many drawbacks of the conventional transdermal drug delivery systems, therefore are favorable in biomedical applications. Microneedles with a combined burst and sustained release profile and maintained therapeutic molecular bioactivity could further broaden its applications as therapeutics. Here, we developed a double-network microneedles (DN MNs) based on gelatin methacrylate and acellular neural matrix (GelMA-ACNM). ACNM could function as an early drug release matrix, whereas the addition of GelMA facilitates sustained drug release. In particular, the double-network microneedles comprising GelMA-ACNM hydrogel has distinctive biological features in maintaining drug activity to meet the needs of application in treating different diseases. In this study, we prepared the double-network microneedles and evaluated its morphology, mechanical properties, drug release properties and biocompatibility, which shows great potential for delivery of therapeutic molecules that needs different release profiles in transdermal treatment.

Niacin exacerbates ß cell lipotoxicity in diet-induced obesity mice through upregulation of GPR109A and PPARγ2: Inhibition by incretin drugs.

The widely used lipid-lowering drug niacin was reported to increase blood glucose in diabetes. How does niacin regulate ß Cell function in diabetic patients remains unclear. This study aimed to investigate the effect of niacin on ß cell lipotoxicity in vitro and in vivo. Niacin treatment sensitized the palmitate-induced cytotoxicity and apoptosis in INS-1 cells. In addition, palmitate significantly increased the niacin receptor GPR109A and PPARγ2 levels, which could be further boosted by niacin co-treatment, creating a vicious cycle. In contrast, knocking down of GPR109A could reverse both PPARγ2 expression and niacin toxicity in the INS-1 cells. Interestingly, we found that GLP-1 receptor agonist exendin-4 showed similar inhibitive effects on the GPR109A/PPARγ2 axis and was able to reverse niacin induced lipotoxicity in INS-1 cells. In diet-induced obesity (DIO) mouse model, niacin treatment resulted in elevated blood glucose, impaired glucose tolerance and insulin secretion, accompanied by the change of islets morphology and the decrease of ß cell mass. The combination of niacin and DPP-4 inhibitor sitagliptin can improve glucose tolerance, insulin secretion and islet morphology and ß cell mass, even better than sitagliptin alone. Our results show that niacin increased ß cell lipotoxicity partially through upregulation of GPR109A and PPARγ2, which can be alleviated by incretin drugs. We provide a new mechanism of niacin toxicity, and suggest that the combination of niacin and incretin may have better blood glucose and lipid control effect in clinical practice.

CircRNA/miRNA/mRNA axis participates in the progression of partial bladder outlet obstruction.

BACKGROUND: More and more evidence showed that circRNA/miRNA/mRNA axis played a vital role in the pathogenesis of some diseases. However, the role of circRNA/miRNA/mRNA axis in partial bladder outlet obstruction (pBOO) remains unknown. Our study aimed to explore the complex regulatory mechanism of circRNA/miRNA/mRNA axis in pBOO. METHODS: The pBOO rat model was established, and the bladder tissues were collected for mRNA sequencing. The differentially expressed mRNAs were analyzed by high-throughput sequencing, and the GO and KEGG analysis of the differentially expressed mRNAs were performed. Competing endogenous RNAs (ceRNAs) analysis identified the potential regulation function of circRNA/miRNA/mRNA axis in pBOO. qRT-PCR detected the expression of circRNA/miRNA/mRNA. miRanda software was performed to predict the relationship between circRNA and miRNA, miRNA and mRNA. RESULTS: Compared with the sham group, a total of 571 mRNAs were differentially expressed in the pBOO group, of which 286 were up-regulated and 285 were down-regulated. GO analysis showed that the mRNAs were mainly involved in cellular process, single-organism process, and cell, etc. KEGG analysis showed that the enriched signaling pathways were metabolic pathways, cell adhesion molecules (CAMs), and HTLV-I infection, etc. Based on the previous transcriptome data and differentially expressed circRNAs, we drew the ceRNA network regulation diagram. qRT-PCR results confirmed that chr3:113195876|113197193/rno-miR-30c-1-3p/Gata4, chr1:126188351|126195625/rno-miR-153-5p/Diaph3, and chr9:81258380|81275269/rno-miR-135b-5p/Pigr axis may have ceRNA function. miRanda confirmed there have the binding sites of circRNA/miRNA/mRNA axis. CONCLUSIONS: CircRNA/miRNA/mRNA axis was involved in the progression of pBOO. Our research on the circRNA/miRNA/mRNA axis revealed new pathogenesis and treatment strategies for pBOO.

Exosomes derived from umbilical cord mesenchymal stem cells protect cartilage and regulate the polarization of macrophages in osteoarthritis.

Background: Osteoarthritis (OA) is one of the most common joint diseases and a major global public health concern. Mesenchymal stem cells (MSCs) have been widely used for the treatment of OA owing to their paracrine secretion of trophic factors, a phenomenon in which exosomes may play a major role. Here, we investigate the potential of exosomes from human umbilical cord-derived MSCs (hUC-MSCs-Exos) in alleviating OA. Methods: The hUC-MSCs-Exos were harvested from hUC-MSC-conditioned medium using ultracentrifugation. Rats with surgically-induced OA were intra-articularly injected with hUC-MSCs-Exos. The effect of hUC-MSCs-Exos in repairing osteoarticular cartilage was assessed using hematoxylin and eosin (HE) staining, safranin-O and fast green staining and immunohistochemistry. The in vitro experiments were further carried out to verify the therapeutic effect. The effects of hUC-MSCs-Exos on the proliferation and migration of human chondrocytes were evaluated using the cell counting kit-8, EdU-555 cell proliferation kit, and transwell assays. Annexin V-FITC/PI staining were used to evaluate the effect of exosomes on chondrocyte apoptosis. An in vitro model of human articular chondrocytes treated with interleukin 1 beta (IL-1ß) was used to evaluate the effects of exosomes, analyses involved using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting. The role of hUC-MSCs-Exos in macrophage polarization was examined in the monocyte cell line, Tohoku Hospital Pediatrics-1 (THP-1) by qRT-PCR and immunofluorescence. Results: The results showed that hUC-MSCs-Exos prevented severe damage to the knee articular cartilage in the rat OA model. We confirmed the high efficacy of hUC-MSCs-Exos in promoting chondrocyte proliferation and migration and inhibiting chondrocyte apoptosis. Additionally, hUC-MSCs-Exos could reverse IL-1ß-induced injury of chondrocytes and regulate the polarization of macrophages in vitro. Conclusions: There is potential for hUC-MSCs-Exos to be used as a treatment strategy for OA.

JMJD3 and UTX determine fidelity and lineage specification of human neural progenitor cells.

Neurogenesis, a highly orchestrated process, entails the transition from a pluripotent to neural state and involves neural progenitor cells (NPCs) and neuronal/glial subtypes. However, the precise epigenetic mechanisms underlying fate decision remain poorly understood. Here, we delete KDM6s (JMJD3 and/or UTX), the H3K27me3 demethylases, in human embryonic stem cells (hESCs) and show that their deletion does not impede NPC generation from hESCs. However, KDM6-deficient NPCs exhibit poor proliferation and a failure to differentiate into neurons and glia. Mechanistically, both JMJD3 and UTX are found to be enriched in gene loci essential for neural development in hNPCs, and KDM6 impairment leads to H3K27me3 accumulation and blockade of DNA accessibility at these genes. Interestingly, forced expression of neuron-specific chromatin remodelling BAF (nBAF) rescues the neuron/glia defect in KDM6-deficient NPCs despite H3K27me3 accumulation. Our findings uncover the differential requirement of KDM6s in specifying NPCs and neurons/glia and highlight the contribution of individual epigenetic regulators in fate decisions in a human development model.

Vitamin C-dependent lysine demethylase 6 (KDM6)-mediated demethylation promotes a chromatin state that supports the endothelial-to-hematopoietic transition.

Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.

Establishment of an ectodermal dysplasia related gene EDA Knockout human embryonic stem cell line (WAe001-A-22) by CRISPR-Cas9 technology.

EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.

BMI1 enables interspecies chimerism with human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) exhibit very limited contribution to interspecies chimeras. One explanation is that the conventional hPSCs are in a primed state and so unable  to form chimeras in pre-implantation embryos. Here, we show that the conventional hPSCs undergo rapid apoptosis when injected into mouse pre-implantation embryos. While, forced-expression of BMI1, a polycomb factor in hPSCs overcomes the apoptosis and enables hPSCs to integrate into mouse pre-implantation embryos and subsequently contribute to chimeras with both embryonic and extra-embryonic tissues. In addition, BMI1 also enables hPSCs to integrate into pre-implantation embryos of other species, such as rabbit and pig. Notably, BMI1 high expression and anti-apoptosis are also indicators for naïve hPSCs to form chimera in mouse embryos. Together, our findings reveal that the apoptosis is an initial barrier in interspecies chimerism using hPSCs and provide a rational to improve it.

Antagonism between the transcription factors NANOG and OTX2 specifies rostral or caudal cell fate during neural patterning transition.

During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3ß (GSK3ß) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans.

Establishment of a congenital tooth agenesis related gene MSX1 knockout human embryonic stem cell lines by CRISPR-Cas9 technology.

Human MSX1 gene is mapped to chromosome 4 and encodes a 303aa homeobox protein MSX1. MSX1 expression appears during early tooth development of vertebrate embryogenesis. Mutations in this protein are related to human tooth anomalie, cleft lip and palate and congenital ectodermal dysplasia syndrome. Most of the confirmed pathogenic mutations are located in exon2 encoded homeobox domain. Here, we report the establishment of MSX1 gene knockout human embryonic stem (hES) cell lines by CRISPR-Cas9 technology. These cell lines provide good materials for further studies of the roles MSX1 plays in human tooth development and congenital tooth agenesis.

PRC2 specifies ectoderm lineages and maintains pluripotency in primed but not naïve ESCs.

Polycomb repressive complex 2 and the epigenetic mark that it deposits, H3K27me3, are evolutionarily conserved and play critical roles in development and cancer. However, their roles in cell fate decisions in early embryonic development remain poorly understood. Here we report that knockout of polycomb repressive complex 2 genes in human embryonic stem cells causes pluripotency loss and spontaneous differentiation toward a meso-endoderm fate, owing to de-repression of BMP signalling. Moreover, human embryonic stem cells with deletion of EZH1 or EZH2 fail to differentiate into ectoderm lineages. We further show that polycomb repressive complex 2-deficient mouse embryonic stem cells also release Bmp4 but retain their pluripotency. However, when converted into a primed state, they undergo spontaneous differentiation similar to that of hESCs. In contrast, polycomb repressive complex 2 is dispensable for pluripotency when human embryonic stem cells are converted into the naive state. Our studies reveal both lineage- and pluripotent state-specific roles of polycomb repressive complex 2 in cell fate decisions.Polycomb repressive complex 2 (PRC2) plays an essential role in development by modifying chromatin but what this means at a cellular level is unclear. Here, the authors show that ablation of PRC2 genes in human embryonic stem cells and in mice results in changes in pluripotency and the primed state of cells.

A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes.

Reprogramming has been shown to involve EMT-MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT-MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFß signalling followed by a MET process. Pharmacological inhibition of TGFß signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT-METs in both differentiation and reprogramming.

Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells.

Dissociation-induced apoptosis is a striking phenomenon in human embryonic stem cells (hESCs), but not in naive mouse ESCs. Rho-associated kinase-dependent actin-myosin hyperactivation is an underlying mechanism that triggers apoptosis in dissociated hESCs; however, in this study, we show that the Ink4A-ARF-mediated senescence pathway is another mechanism to cause apoptosis in individualized hESCs. We show that P16INK4A and P14ARF are immediately induced in hESCs upon dissociation, but not in mouse ESCs. Overexpression of BMI1, a suppressor for Ink4A-ARF, greatly promotes survival and cloning efficiency of individualized hESCs mechanistically via direct binding the H3K27me3-marked Ink4A-ARF locus. Forced expression of BMI1 in hESCs does not reduce the actin-myosin activation that is triggered by dissociation, which indicates it is an independent pathway for hESC survival. Furthermore, dual inhibition of both Ink4A-ARF and actin-myosin hyperactivation enables successful passaging of hESCs via gelatin, a nonbioactive matrix. In sum, we provide an additional mechanism that underlies cell death in individualized hESCs that might help to fully understand the differential cell characteristics between naive and primed ESCs.-Wang, W., Zhu, Y., Huang, K., Shan, Y., Du, J., Dong, X., Ma, P., Wu, P., Zhang, J., Huang, W., Zhang, T., Liao, B., Yao, D., Pan, G., Liu, J. Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells.

Generation and Analysis of GATA2w/eGFP Human ESCs Reveal ITGB3/CD61 as a Reliable Marker for Defining Hemogenic Endothelial Cells during Hematopoiesis.

The transition from hemogenic endothelial cells (HECs) to hematopoietic stem/progenitor cells (HS/PCs), or endothelial to hematopoietic transition (EHT), is a critical step during hematopoiesis. However, little is known about the molecular determinants of HECs due to the challenge in defining HECs. We report here the generation of GATA2w/eGFP reporter in human embryonic stem cells (hESCs) to mark cells expressing GATA2, a critical gene for EHT. We show that during differentiation, functional HECs are almost exclusively GATA2/eGFP+. We then constructed a regulatory network for HEC determination and also identified a panel of positive or negative surface markers for discriminating HECs from non-hemogenic ECs. Among them, ITGB3 (CD61) precisely labeled HECs both in hESC differentiation and embryonic day 10 mouse embryos. These results not only identify a reliable marker for defining HECs, but also establish a robust platform for dissecting hematopoiesis in vitro, which might lead to the generation of HSCs in vitro.